Table of contents :

malformations regressive processes progressive processes hypertrophy hyperplasia dysplasia cancer / neoplasm / tumor

defensive processes immune response innate immune response inflammation / flogosis (post-flogistic) repair adaptive immune response primary adaptive immune response secondary adaptive immune response immunopathological processes hypersensitivity reactions autoimmune reactions transplant complications transplant rejection graft-versus-host disease (GvHD) prognosis web resources

The classification of disease according to kind(s) of process evoked by the aetiological factor(s) is mainly used by pathologists because every kind of process may arise from a limited set of aetiological factors and because it often allows to produce a diagnosis and a prognosis.

• congenital malformation (see also multisystem birth defects) : a morphologic defect of an organ or larger region of the body, resulting from an intrinsically abnormal developmental process.
• malposition / allotopia / displacement / dystopia / heterotopia : abnormal or anomalous position of an organ or part
• ectopia : malposition, especially if congenital.
• chorista : defective development due to, or characterized by, displacement of the primordium.
• choristoma : a mass of tissue that is normal histologically, but not for the site in which it is located
• dermoid : a congenital choristoma consisting of skin and its dermal appendages
• coloboma : an absence or defect of tissue, particularly, a defect of ocular tissue, usually due to malclosure of the fetal intraocular fissure, or sometimes from trauma or disease.
• hamartia : a defect in tissue combination during development.
• hamartoma : a benign tumor-like nodule composed of an overgrowth of mature cells and tissues that normally occur in the affected part, but with disorganization and often with one element predominating.
• nevus :
• nevus cell / nevocyte : a polymorphic, melanin-containing cell variously postulated to be a normal mature melanocyte or a modified one, or to be derived from a Schwann cell or an embryonal nevoblast. Such cells occur in aggregations, or nests (theques), in the epidermis and reach the dermis by a kind of centripetal extrusion (abtropfung), and are the main constituents of nevocytic nevi.
• phakomatoses
• congenital epulis / epulis of newborn
• hamartomatosis : the development of multiple hamartomas.
• fistula : an abnormal passage or communication, usually between two internal organs, or leading from an organ to the surface of the body. Such passages may also be created experimentally to obtain body secretions for physiologic study.
• blind or incomplete fistula : a fistula that is open at one end only; it may open only upon the cutaneous surface of the body (external blind fistula) or on an internal mucous surface (internal blind fistula)
• complete fistula : an abnormal passage in the body, each end of which opens on a mucous surface or on the cutaneous surface of the body.
• external fistula : an abnormal communication between a hollow organ and the external surface of the body.
• internal fistula : an abnormal communication between two internal organs.
• parietal fistula : an abnormal passage in the body wall, ending blindly or communicating with an internal organ or body cavity.
• parapedesis : passage of body substances into channels not normally conveying them, as of bile pigments into the blood capillaries
• environmental stress : organisms limit protein synthesis by storing inactive ribosomes that are rapidly reactivated when conditions improve. As a measure for molecular motion, temperature is one of the most important environmental factors for life as it directly influences structural and hence functional properties of cellular components. Sress proteins includes the HSPs, the glucose-regulated proteins (GRPs) and ubiquitin. The stress proteins may be grouped by molecular weight ranging from the large 110 kDa HSP110 to ubiquitin at 8 kDa. These proteins serve as cellular chaperones, participating in protein synthesis and transport through the various cellular compartments. Because these proteins have unique cellular localizations, the chaperone function of the stress proteins often involves a transfer of peptides between stress proteins as the peptide is moved between cellular compartments. For example, HSP70 is a cytosolic and nuclear chaperone, which is critical for the transfer of cellular peptides in the mitochondrion through a hand-off that involves mitochondrial HSP60 at the inner mitochondrial membrane. Similarly, cytosolic proteins are transferred from HSP70 to gp96 as they move into the endoplasmic reticulum. The central role of the stress proteins in the transfer of peptides through the cell may be responsible for the recently recognized importance of the stress proteins in the modulation of the immune system^ref. This importance in immune regulation is best addressed using the danger model of the immune response. This model states that the basic function of all cells of the organism is appropriately timed death "from natural causes". This type of cell death, or apoptosis, generates no stress signals. If, on the other hand, a cell is "murdered" by an infectious agent or dies an untimely death due to necrosis or ischemia, the cell undergoes a stress response with the liberation of stress protein-peptide complexes into the extracellular environment upon cell lysis. Not only do they serve as a "danger signal" to alert the immune system to the death of a cell under stress, but their role as protein carriers allows the immune effector cells to survey the peptides released by this stressed cell and to activate against new or unrecognized peptides carried by the stress protein^ref
• cold shock : in response to temperature downshift, a number of changes occur in cellular physiology such as, (i) decrease in membrane fluidity, (ii) stabilization of secondary structures of nucleic acids leading to reduced efficiency of mRNA translation and transcription, (iii) inefficient folding of some proteins, and (iv) hampered ribosome function. Cold-shock response and adaptation has been quite extensively studied in Escherichia coli and Bacillus subtilis. A number of cold shock proteins (CSPs) are induced to counteract these harmful effects of temperature downshift. Mesophiles like Escherichia coli, which are adapted to living at both warm temperatures inside animals and cooler ambient temperatures, respond to low temperatures (10-15°C) by adjusting membrane lipid composition and increasing the production of proteins that act as "RNA chaperones" required for transcription and translation and proteins that facilitate ribosomal assembly. During this response, bulk transcription and translation slow or come to an almost complete stop, while a set of about 26 cold-shock genes is preferentially and transiently expressed. This protein set, however, is different from that expressed under heat shock conditions and predominantly comprises proteins such as helicases, nucleases, and ribosome-associated components that directly or indirectly interact with the biological information molecules DNA and RNA. Interestingly, in contrast to the heat shock response, to date no cold-specific sigma factor has been identified. Rather, it appears that the cold shock response is organized as a complex stimulon in which post-transcriptional events play an important role. At least some of the proteins encoded by these genes are essential for survival in the cold, but none plays an exclusive role in cold adaptation, not even the "major cold-shock protein" CspA and none is induced de novo. The majority of these proteins binds nucleic acids and are involved in fundamental functions (DNA packaging, transcription, RNA degradation, translation, ribosome assembly, etc.). Although cold-induced activation of specific promoters has been implicated in upregulating some cold-shock genes, post-transcriptional mechanisms play a major role in cold adaptation; cold stress-induced changes of the RNA degradosome determine a drastic stabilization of the cold-shock transcripts and cold shock-induced modifications of the translational apparatus determine their preferential translation in the cold. This preferential translation at low temperature is due to cis elements present in the 5' untranslated region of at least some cold-shock mRNAs and to trans-acting factors whose levels are increased substantially by cold stress. Protein CspA and the three translation initiation factors (IF3 in particular), whose stoichiometry relative to the ribosomes is more than doubled during the acclimation period, are among the trans elements found to selectively stimulate cold-shock mRNA translation in the cold^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8}. Protein Y, an Escherichia coli stress protein, fills the tRNA- and mRNA-binding channel of the small ribosomal subunit to stabilize intact ribosomes. Protein Y inhibits translation initiation during cold shock but not at normal temperatures by competing with conserved translation initiation factors that, in bacteria, are required for ribosomal subunit dissociation. The mechanism used by protein Y to reduce translation initiation during stress and quickly release ribosomes for renewed translation initiation may therefore occur widely in nature^ref. In contrast, yeast, which are adapted to cooler temperatures, show a relatively minor cold shock response after temperature shifts from 30° to 10°C but respond with a dramatic increase in the synthesis of trehalose and a heat shock protein when exposed to freezing or near-freezing temperatures. Progression through the cell cycle is temperature sensitive, but the relationship is not straightforward. In culture, many types of mammalian cells fail to undergo the G₂/M transition after cooling from 37°C to 16-20°C (moderate hypothermia). However, progression through G₁ and S is not blocked at these temperatures, nor is progression through mitosis in cells cooled after they have become committed to the division process. Thus, at least one pathway is present during G₂-but not during G₁, S or mitosis-that is selectively disrupted at or below a critical temperature. As a result, a prolonged (24-48 hr) exposure to moderate hypothermia can be used to enrich cultures for G₂ cells. A brief (1 hr) exposure to severe hypothermia (4-10°) is also reported to induce a high degree of mitotic synchrony (up to 80%) in some mammalian cultures. Although the mechanism behind this synchronization remains vague, it may involve a cell cycle checkpoint, triggered in response to the cold shock, that transiently inhibits the G₁/S transition^ref. We live on a cold planet where > 80% of the biosphere is permanently < 5°C, and yet comparatively little is known about the genetics and physiology of the microorganisms inhabiting these environments. Based on molecular probe and sequencing studies, it is clear that Archaea are numerically abundant in diverse low-temperature environments throughout the globe. In addition, non-low-temperature-adapted Archaea are commonly exposed to sudden decreases in temperature, as are other microorganisms, animals, and plants. Considering their ubiquity in nature, it is perhaps surprising to find that there is such a lack of knowledge regarding low-temperature adaptation mechanisms in Archaea, particularly in comparison to what is known about archaeal thermophiles and hyperthermophiles and responses to heat shock^ref.
• heat shock : since its discovery in 1962 by Ritossa, the heat shock response has been extensively studied by a number of investigators to understand the molecular mechanism underlying the cellular response to heat stress. The most well characterized heat shock response is induction of the heat shock protein (HSPs) that function as molecular chaperones and exert cell cycle regulatory and anti-apoptotic activities. While most investigators have focused their studies on the toxic effects of heat stress in organisms such as severe heat stress-induced cell cycle arrest and apoptosis, the cellular response to fever-ranged mild heat stress has been rather underestimated. However, the cellular response to mild heat stress is likely to be more important in a physiological sense than that to severe heat stress because the body temperature of homeothermic animals increases by only 1-2°C during febrile diseases. Mild heat stress does have some beneficial role in organisms via positively regulating cell proliferation and differentiation, and immune response in mammalian cells^ref. In Escherichia coli the heat shock response is, controlled by the sigma factor s32, which acts as a master regulator that specifically directs RNA polymerase to transcribe from the HSP promotors. Heat shock factor Hsf in nonvertebrate animals and homologous heat shock factor Hsf1 in vertebrate animals are key transcriptional regulators of the stress protein response. Hsf/Hsf1 is constitutively present in cells but is, typically, only active during periods during which cells are experiencing a physical or chemical proteotoxic stress. It has become increasingly clear that regulation of Hsf/Hsf1 activity occurs at multiple levels: the oligomeric status of Hsf/Hsf1, its DNA-binding ability, posttranslational modification, transcriptional competence, nuclear/ subnuclear localization, as well as its interactions with regulatory cofactors or other transcription factors all appear to be carefully controlled^ref. The existence of HSP receptors on antigen-presenting cells (APCs) was hypothesized in 1994. The first such receptor, CD91 / LRP / a₂-macroglobulin R, was identified and characterized in 2000. The pace of attribution has quickened since and during 2002-2004 alone, 6 putative HSP receptors have been identified. These include CD40L, LOX-1, CD36 / thrombospondin receptor / p95^{gpIV / gpIIIb}, TLR2, TLR4 and SR-A^ref. During the heat shock response in mouse cells, a small noncoding RNA polymerase III transcript from short interspersed elements (SINE) that are abundant in the mouse genome and historically considered to be "junk DNA", B2 RNA, associates with RNA polymerase II and represses transcription of specific mRNA genes. These studies define a unique transcriptional regulatory mechanism involving an RNA regulator and reveal how mRNA transcription is repressed upon heat shock. Moreover, B2 RNA is transcribed'^ref. arimoclomol (source : CytRx Corp.) is a coinducer of HSPs. The heat-shock transcription factor 1 (HSF1) has an important role in the heat-shock response in vertebrates by inducing the expression of heat-shock proteins (HSPs) and other cytoprotective proteins. HSF1 is present in unstressed cells in an inactive monomeric form and becomes activated by heat and other stress stimuli. HSF1 activation involves trimerization and acquisition of a site-specific DNA-binding activity, which is negatively regulated by interaction with certain HSPs. Here we show that HSF1 activation by heat shock is an active process that is mediated by a ribonucleoprotein complex containing translation elongation factor eEF1A and heat shock RNA-1 (HSR1). HSR1 is constitutively expressed in human and rodent cells and its homologues are functionally interchangeable. Both HSR1 and eEF1A are required for HSF1 activation in vitro; antisense oligonucleotides or short interfering (si)RNA against HSR1 impair the heat-shock response in vivo, rendering cells thermosensitive. The central role of HSR1 during heat shock implies that targeting this RNA could serve as a new therapeutic model for cancer, inflammation and other conditions associated with HSF1 deregulation^ref.
• hyperoxia
• hypoxia
• other perturbations
• regressive processes
• progressive processes

• hypertrophy : the enlargement or overgrowth of an organ or part due to an increase in size of its constituent cells

Aetiology :
• adaptive hypertrophy : increase in size in response to changed conditions, as, for example, increased thickness of the walls of a hollow organ when the outflow is obstructed.
• functional hypertrophy : hypertrophy of an organ or part caused by its increased activity.
• compensatory hypertrophy : that which results from an increased workload due to some physical defect, as occurs in one kidney when the other is absent or destroyed by disease.
• complementary hypertrophy : increase in size of the remaining part of an organ to take the place of a portion which has been lost.
• vicarious hypertrophy : hypertrophy of an organ in consequence of the failure of another organ of allied function
• concentric hypertrophy : hypertrophy of a hollow organ in which there is increased thickness of the walls with no enlargement in external size, with diminished capacity.
• eccentric hypertrophy : hypertrophy of a hollow organ in which there is dilatation of its cavity and enlargement of its external size.
• false hypertrophy : enlargement due to an increase in only one constituent element of an organ or part, commonly the stroma.
• true hypertrophy : enlargement due to an increase of all the component elements of an organ or part.
• numeric hypertrophy : that which is due to an increased number of structural elements.
• physiologic hypertrophy : temporary increase in the size of an organ produced by physiologic activity, as in the female breast during pregnancy and lactation.
• simple hypertrophy : that which is due to a simple increase of the number of structural elements.
• unilateral hypertrophy : overgrowth of one side of the entire body or of a portion of one side, as of the face.
• xenoma (tumor) / xenom : a symbiotic complex formed by hypertrophying host cells and multiplying intra-cellular parasites, e.g. certain micro-sporidians.
• hyperplasia : abnormal multiplication or increase in the number of normal cells in normal arrangement in a tissue.
• inflammatory hyperplasia : hyperplasia brought about by inflammation
• Swiss-cheese hyperplasia : hyperplasia of a tissue which on section shows openings as in Swiss cheese
• adenosis : the abnormal development or formation of gland tissue
• seroma : a tumor-like collection of serum in the tissues
• cancer (neoplasm)
• pseudotumor / pseudoneoplasm / false tumor : an enlargement that resembles a tumor; it may result from inflammation, accumulation of fluid, or other causes, and may or may not regress spontaneously
• inflammatory pseudotumor (IPT) : a general term for a rare space-occupying tumorlike mass of unknown etiology representing an inflammatory reaction, occurring in a variety of organs and composed of granulation tissue with leukocyte infiltration (histopathologically by aggregates of inflammatory lymphocytes, plasma cells (plasma cell granuloma), neutrophils, and fibroblasts (inflammatory myofibroblastic tumor (IMT))). It can mimic malignancy on clinicoradiological and pathological examination. IPT is a quasineoplastic lesion that most commonly involves the lung and the orbit (expecially conjunctiva) (rarely simultaneously^ref), but it has been reported to occur in nearly every site in the body (heart^ref, gastrointestinal tract (multifocal omental mesenteric IPT presumed to result following an Entamoeba histolytica infestation^ref), adrenal gland, iliopsoas muscle, CNS, and spleen). Sporadic cases have been reported in the trunk, genitourinary tract, and extremities as well as in the head and neck (the areas most commonly involved are the orbit and paranasal sinuses^ref, but they have been also described in the nasal septum^ref,  larynx, pterygomaxilar space, tonsils, ears, gingiva and other periodontal tissues). Of critical importance is this entity's correct histopathologic diagnosis that differentiate it from malignant neoplasms such as spindle cell carcinoma and fibrosarcoma, benign tumors such as neurofibroma, and other pseudoneoplastic lesions such as nodular fasciitis. Correct diagnosis is followed by wide local excision to prevent recurrence; however, treatment must be tailored to the location of tumor and the condition of the patient. Because IPT mimic malignant tumors both clinically and radiologically, the radiologist should be familiar with this entity and help avoid unnecessary radical surgery when possible. Diagnostic accuracy of cytology for IPT is low (42%). IPT shows hypercellular smears (on FNA) with an admixture of various cell types including inflammatory cells with predominance of plasma cells, fibroblastic proliferation, granulation tissue formation, and atypical-appearing histiocytes with enlarged nuclei and intranuclear inclusions. Fibroblastic proliferation with mitoses may mimic mesenchymal neoplasms. Cytomorphology is nonspecific and IPT usually is a diagnosis of exclusion
• calcifying fibrous pseudotumor (CFP) is one of the most unusual benign tumors of childhood and is located mostly in soft tissues, pleura^ref, and peritoneum, and rarely lung^ref. Microscopically, the lesion is mostly composed of dense collagenous tissue, with sparse benign spindle cells, a rich inflammatory infiltrate and scattered calcifications, sometimes laminated. IHC, spindle cells are vimentin⁺, SMA^-, desmin^-, S100 protein^-, CD34^-, CD99^- and Bcl2^-.
• mycobacterial pseudotumor (MP) is a rare pathologic presentation of both Mycobacterium tuberculosis and non-tuberculous mycobacterial disease, hitherto reported to occur only in immunosuppressed patients with or without HIV-1 infection. This lesion shares close pathologic resemblance to certain mesenchymal neoplasms, particularly Kaposi's sarcoma (KS), from which it must be properly differentiated due to distinct prognosis and therapy. It can obliterate the lumen of the appendix vermiformis^ref
• emperipolesis is random passage of one cell through the cytoplasm of another one^ref with no physiologic consequence for either of them.
• emperipolesis is particularly frequent in megakayocytes (MK), 5% of which are normally found engulfed with other cell types, most frequently neutrophils or red blood cells (Zucker-Franklin D. Atlas of Blood Cells Function and Pathology. Bologna, Italy: Edi Ermes; 2003). Increased frequency of neutrophil emperipolesis within the MKs is observed in the majority of patients who experience extreme thrombocytosis, whether myeloproliferative or reactive^ref, and is not necessarily associated with marrow fibrosis. The MK abnormalities observed in patients with idiopathic myelofibrosis also include increased emperipolesis of neutrophils within MKs^{ref1, ref2}. The significance, however, of this observation is not clear, because emperipolesis of neutrophils within MKs increases in other stress-related conditions not necessarily associated with marrow fibrosis, such as recovery after sublethal irradiation^ref, aging^ref, and altered MK maturation associated with the gunmetal mutation^ref.
• histiocytes showed emperipolesis (i.e. intact lymphocytes within the cells) (which should be differentiated by lymphohistiocytosis)
• various CNS diseases (oligodendrocytes within astrocytes) showing destruction of myelin or inflammation of the white matter, including multiple sclerosis, cerebral infarct, chronic metabolic disorders and CJD^ref.
• emperipolesis in pleural mesothelial cells in a patient with chronic lymphocytic leukemia^ref.
• defensive processes
• innate immune response
• hemostasis
• inflammation or flogosis : a localized protective response elicited by injury or destruction of tissues, which serves to destroy, dilute, or wall off (sequester) both the injurious agent and the injured tissue. It is characterized in the acute form by the classical signs of
• pain (dolor), a critical component of host defense, is exacerbated by proinflammatory chemokines. When CCR1:TRPV1/HEK293 cells were pretreated with CCL3, the sensitivity of TRPV1, as indicated by the Ca²⁺ influx, was increased 3-fold. RT-PCR analysis showed that a spectrum of chemokine and cytokine receptors is expressed in rat dorsal root ganglia (DRG). Immunohistochemical staining of DRG showed that CCR1 is coexpressed with TRPV1 in >85% of small-diameter neurons. CCR1 on DRG neurons was functional, as demonstrated by CCL3-induced Ca²⁺ ion influx and PKC activation. Pretreatment with CCL3 enhanced the response of DRG neurons to capsaicin or anandamide. This sensitization was inhibited by pertussis toxin, U73122, or chelerythrine chloride, inhibitors of Gi-protein, phospholipase C, and protein kinase C, respectively. Intraplantar injection of mice with CCL3 decreased their hot-plate response latency^ref.
• heat (calor)
• redness (rubor)
• swelling (tumor)
• loss of function (functio laesa)
Histologically, it involves a complex series of events, including dilatation of arterioles, capillaries, and venules, with increased permeability and blood flow; exudation of fluids, including plasma proteins; and leukocytic migration into the inflammatory focus.
• acute inflammation : inflammation, usually of sudden onset, characterized by the classical signs (see inflammation), in which the vascular and exudative processes predominate.
• subacute inflammation : a condition intermediate between chronic and acute inflammation, exhibiting some of the characteristics of each.
• chronic inflammation : inflammation of slow progress and marked chiefly by the formation of new connective tissue; it may be a continuation of an acute form or a prolonged low-grade form, and usually causes permanent tissue damage.
• adhesive inflammation : that which promotes the adhesion of contiguous surfaces.
• atrophic, cirrhotic, fibrosing or sclerosing inflammation : a form which results in atrophy and deformity.
• hypertrophic inflammation : inflammation marked by increase in the size of the elements composing the affected tissue.
• catarrhal inflammation : a form which affects principally a mucous surface, and which is marked by a copious discharge of mucus and epithelial debris.
• focal inflammation : one that is confined to a single spot or to a few limited spots.
• diffuse inflammation : one that is both interstitial and parenchymatous or is spread over a large area.
• disseminated inflammation : one that has a number of distinct foci.
• metastatic inflammation : one that is reproduced in a distant part by the conveyance of infectious material through the blood vessels and lymph organs.
• granulomatous inflammation : an inflammation, usually chronic, characterized by the formation of granulomas
• plastic, productive, proliferous or hyperplastic inflammation : one which leads to the formation of new connective tissue fibers.
• necrotic inflammation : inflammation attended by death of the affected tissue.
• obliterative inflammation : inflammation of the lining membrane of a cavity or vessel, producing adhesions between the surfaces and consequent obliteration of the lumen.
• interstitial inflammation : one that primarily affects the stroma of an organ.
• parenchymatous inflammation : one that primarily affects the essential tissue elements of an organ.
• pseudomembranous inflammation : an acute inflammatory response to a powerful necrotizing toxin, such as the diphtheria toxin, characterized by the formation on a mucosal surface, most often in the pharynx, larynx, respiratory passages, and intestinal tract, of a false membrane composed of precipitated fibrin, necrotic epithelium, and inflammatory white cells.
• seroplastic inflammation : inflammation accompanied by both serous and plastic exudation.
• simple inflammation : that in which there is no flow of pus or other product of inflammation.
• specific inflammation : one that is due to a particular microorganism.
• toxic inflammation : one that is caused by a poison, such as a bacterial product.
• traumatic inflammation : one that is caused by an injury.
• ulcerative inflammation : that in which necrosis on or near the surface leads to loss of tissue and creation of a local defect (ulcer).
• acute phase reaction or response (APR) : a group of physiologic processes occurring soon after the onset of infection, trauma, inflammatory processes, and some malignant conditions.
• acute phase reactants (APR) : TLRs=> NFkB => IL-1, IL-6 and TNF-a =>
• acute phase proteins (APPs) :
• positive APPs (dramatic increase in their plasma concentration is the most prominent change of APR and increases ESR)
• type-1 APPs are those that require the synergistic action of IL-6 and IL-1 for maximum synthesis
• C-reactive protein (CRP) : peak up to 1000-fold the upper reference value within 8-10 hours. t = 19 hrs, so its plasma concentration decreases before ESR lowering.

CRP is a risk factor for ...
• total mortality
• acute myocardial infarction (AMI)
• sudden cardiac death
• stroke
• type II diabetes mellitus
• metabolic syndrome
• body mass index (BMI)
• cigarette smoking
• heavy drinking
• no ethanol consumption
• HRT in postmenopausal women
• age
CRP is a protective factor for ...
• weight loss
• drinking in moderation
• orosomucoid / a₁ acid glycoprotein 1 (AGP1) and orosomucoid / a₁ acid gp 2 (elevation 2-5 fold)
• serum amyloid A (SAA) component (1, 2, and 4) : peak up to 1,000-fold the upper reference value.
• type-2 APPs are those that require IL-6 only for maximal induction, while IL-1 frequently suppress their expression.
• fibrinogen ABg (2-5 fold elevation, but late-onset)
• haptoglobins (1-1, 2-1 and 2-2) (2-5 fold elevation)
• a₂-macroglobulin (in rabbits and rats, but not in humans)
• proteases inhibitors
• a₁-antichimotrypsinogen (2-5 fold elevation)
• a₁-antitrypsin / a₁-antiplasmin
• a₂-antiplasmin 1
• a₂-antiplasmin 2
• complement factors
• lectin activation pathway
• mannose-binding protein (MBP)
• alternative activation pathway
• factor-B (Bf)
• classic activation pathway
• C1 inhibitor (C1-INH)
• C2
• C4 (elevation < 2 fold)
• C4 binding protein (C4BP)
• common lytic pathway
• C3 (elevation < 2 fold)
• C5
• C8
• C9
• coagulation factors
• factor VIII
• von Willebrand factor (vWF)
• antithrombin III / heparin cofactor I
• heparin cofactor-2
• prothrombin
• plasminogen
• plasminogen activator inhibitor-1 (PAI-1)
• serum amyloid-P (SAP) component: only in mouse, no significant change in humans
• iron metabolism and reactive oxygen species
• heme oxygenase 1 (HO-1)
• hemopexin (Hpx) (elevation < 2-fold)
• apoferritin
• lactoferrin
• ceruloplasmin (Cp) / ferroxidase (elevation < 2-fold)
• SOD2
• calprotectin is a stable calcium- and zinc-binding anti-inflammatory APP found in the cytosol of neutrophilic granulocytes and monocytes : marked susceptibility to zinc deprivation is a general characteristic of pathogenic fungi^ref. This factor is composed of 8 and 14 kDa subunits, S100 calcium binding protein A8 (calgranulin A) / MRP8 and S100 calcium binding protein A9 (calgranulin B) / MRP14). Calprotectin has higher specific activity to induce apoptosis than the individual subunits, and that the mechanism is exclusion of zinc from target cells^ref. Calprotectin is stable in feces for several days after excretion, can be measured readily with commercially available assay, and correlates well with radiolabeled autologous leukocytes excretion, which is more difficult to perform. A single fecal calprotectin measurement may aid gastroenterologists in the differential diagnosis of Crohn's disease and IBS. Its use could decrease the number of invasive or radiological investigations undertaken in the latter group of patients^ref.
• fibronectin (Fn)
• kallikreins
• LPS binding protein (LBP)
• procalcitonin (ProCT / PCT) : in microbial infections and in various forms of severe systemic inflammation, circulating levels of calcitonin precursors (CTpr), including the prohormone ProCT, normally found in the serum of healthy persons, increase severalfold to several thousand-fold, and this increase often correlates with the severity of the condition and with mortality^ref. During severe systemic infections it is produced by extrathyroidal tissues. In the absence of infection, the extra-thyroidal transcription of the CALC-I gene is suppressed and confined to selective expression in neuroendocrine cells found mainly in thyroid and lung. In these neuroendocrine cells, the mature hormone is processed and stored in secretory granules. A microbial infection induces a ubiquitous increase in CALC-I gene expression and a constitutive release of CTpr from all tissues and cell types throughout the body^ref. Thus, under septic circumstances, the entire body could be viewed as an endocrine gland. Indeed, the transcriptional expression of CT-mRNA is more uniformly up-regulated in sepsis than are the mRNAs of the classical cytokines (e.g. TNF-a and IL-6). Interestingly, there is relatively low expression of CTpr in white blood cells^{ref1, ref2}. The greater CTpr mRNA induction and CTpr peptide release from parenchymal cells compared to circulating cells appears to indicate a tissue-based rather than leukocyte-based host defence mechanism. Thus, CALC gene products may be a prototype of hormokine mediators and may follow either a classical hormonal expression or, alternatively, a cytokine-like expression pathway. The production of hormokines is mediated by as yet unknown factors and may be induced either directly via microbial toxins or indirectly via a humoral or cell-mediated host response. In sepsis, the predominance of CTpr as opposed to mature CT is indicative of a constitutive pathway within cells lacking secretion granules and hence bypassing of much of the enzymatic processing. Consequently, as is the case of most cytokines, there is very little intracellular storage of CTpr in sepsis^ref. PCT is an APP with faster kinetics than CRP, its concentration in serum rising within the few hours that follow the inception of a bacterial infection. Similarly to what occurs in humans, CTpr are also increased in septic hamsters^{ref1, ref2}. In the hamster model of sepsis, ProCT does not initiate or enhance IL-1b or TNF-a expression; however, the massive and sustained elevation of this hormone seen in sepsis can be induced in normal hamsters by administration of the cytokine TNF-a. This suggests that ProCT could be a secondary mediator which may augment and amplify, rather than initiate, the septic response^ref. Both hypocalcaemia and increased serum levels of CTpr are common findings in intensive care patients, especially those with infection and sepsis. Indeed, ionised hypocalcaemia is more pronounced with increasing severity of infection, and occurs in parallel with the marked increase of CTpr^ref. In contrast, as mentioned above, serum levels of mature CT are normal or only minimally elevated in sepsis^{ref1, ref2,ref3}. Importantly, CTpr contribute greatly to the deleterious effects of systemic infection. Administration of ProCT to septic hamsters with peritonitis doubled their death rate, which reached levels exceeding 90%, and treatment with ProCT-reactive antiserum increased the survival of septic hamsters^ref. In addition, 1-hour intravenous immunoneutralisation using an antiserum reacting specifically with porcine ProCT improved the physiological and metabolic parameters of septic pigs and greatly increased their short-term survival (from 0% to 80%). Further, recent experiments have demonstrated that such immunoneutralisation is effective even when administered after the animals are moribund. Studies have demonstrated that what is beneficial is probably immunoneutralisation of the entire ProCT molecule. There are thus several observations to indicate that ProCT is not only a necessary precursor to the biosynthesis of mature CT but also, at the high concentrations occurring in the setting of sepsis, a potentially harmful mediator involved in the septic response. Several characteristics of ProCT militate in favour of this hormokine molecule as a therapeutic target in sepsis. In contrast to the transiently increased classical cytokines, for which immunoneutralisation trials in humans have been disappointing, the massive increase in circulating CTpr persists for several days^ref. Moreover, CTpr is very frequently increased in overt sepsis, its onset is early (within 3 hr), and the diagnostic accuracy of the measurement should greatly improve patient selection for any study of the therapeutic efficacy of ProCT immunoneutralisation in humans. In sepsis, several CTpr are increased, including ProCT, N-ProCT, CT:CCP-I, immature CT, and CCP-I, any of which may have agonistic, antagonistic, or neutral effects^{ref1, ref2}. Also, several members of the calcitonin gene family of peptides comprise a functional unity. The CALC-I gene, by alternative processing of the primary RNA transcript, gives rise to two different so-called mature peptides: calcitonin (CT) and calcitonin gene-related peptide I (CGRP-I). Like CT, CGRP-I is initially biosynthesised as a larger prohormone which is subsequently cleaved into smaller precursors. It is relevant that CGRP-II, amylin, and adrenomedullin, also members of the CT gene family of peptides, are encoded on the CALC-II, -IV, and -V genes respectively. In sepsis, mRNA for CGRP-I, CGRP-II and adrenomedullin also appear to be ubiquitously expressed. Based on structural homologies, different members of these CT-gene family peptides have different profiles of bioactivity, which they exert by binding to the same family of receptors. There are two subgroups of receptors for the CT-gene family: CT receptors (CRs) and CT receptor-like receptors (CRLRs). Each member of the CT-gene family of peptides binds with differing affinities to these receptors. Accessory proteins act upon these receptors, thus altering their specific responsiveness and hence the physiological profile of action of the CT-gene peptides. These accessory proteins, which are called receptor-activity-modifying proteins (RAMPs), alter the receptors’ phenotype; they act on the CRs by modification of their genes and on the CRLRs by influencing transport to the plasma membrane. The presence, concentration, and/or timing of one or more of the three RAMPs (RAMP-1, -2, and -3) determines the specific cellular phenotype of the receptor that is ultimately expressed on the cell surface^ref. The profile of RAMP expression and activity is altered by the local milieu and is subject to humoral influences. This elegant system allows for diversification of receptor function, and hence modulates the action of the CT-gene products according to ambient needs. Thus, both in health and disease, a response to different peptides of the calcitonin gene family of peptides occurs in a dynamic and varying manner. Further studies are needed to determine whether high levels of circulating ProCT or other CTpr may blunt the effects of CGRPs and/or adrenomedullin, peptides which may otherwise be beneficial in sepsis. Several clinical studies have confirmed the superior diagnostic utility of serum levels of CTpr in sepsis and their greater reliability, compared to other markers, in following the course of illness^{ref1, ref2}. Importantly, in sepsis, the extent to which any specific CTpr peptide is increased relative to the others varies; indeed, the levels of N-ProCT and CT:CCP-I may be even higher than the ProCT values^ref. PCT is a very stable molecule in vitro, and its measurement requires only 20 ml of plasma or serum (an immunoluminometric assay of 40 ml serum or plasma) and can be done within 2 hours^ref. The commercially available 2-site assay (LUMItest^® PCT, B.R.A.H.M.S. Diagnostica GmbH, Hennigsdorf/Berlin, Germany), measures both ProCT and the conjoined CT:CCP-I by means of a luminometer. This assay is useful in detecting markedly elevated CTpr levels in sepsis. However, the current assay has the disadvantage of relative insensitivity, with an accurate detection limit of ~0.3 to 0.5 ng/mL^{ref1, ref2}. A colorimetric bedside test (PCT^®-Q, B.R.A.H.M.S. Diagnostica GmbH, Hennigsdorf/Berlin, Germany) has the advantage of providing rapid determination of circulating CTpr levels (in 30 minutes); however, the assay is only semi-quantitative and is not sensitive enoughto detect mildly or moderately elevated CTpr levels^ref. Currently, the most sensitive assay capable of measuring CTpr in normal persons utilises an antibody to N-ProCT as the free peptide and within the ProCT molecule^ref. Clinically, the authors have found the determination of circulating CTpr levels to be very useful in complex, polymorbid cases with suspected bacterial infections. For example, serum CTpr measurement can be helpful in predicting the presence of serious bacterial infection in a patient with fever but without localising signs^ref. Another important use is in patients with meningeal irritation; in this context, elevated circulating CTpr levels are highly suggestive of bacterial infection^{ref1, ref2}. The CTpr assay can also be used where renal failure is present^{ref1, ref2}. In immunocompromised patients with AIDS or neutropenia, an ultrasensitive CTpr assay is probably more desirable^{ref1, ref2,ref3}. In patients with haemodynamic shock, the finding of relatively low levels of serum CTpr suggests a nonbacterial cause (e.g. acute adrenocortical failure or haemorrhage)^ref. CTpr determination can also be useful in differentiating joint infections from autoimmune inflammation (e.g. rheumatoid arthritis)^ref. Whatever the initial triggering insult may be, markedly severe systemic inflammation per se may be reflected in increased serum levels of CTpr^ref. For example, whether or not bacterial infection has been found, moderate to marked serum CTpr increases occur in pancreatitis due to biliary obstruction or in association with necrosis, in chemical pneumonitis^ref, in burns^{ref1, ref2, ref3}, in heat stroke^ref, in mechanical trauma^ref, and following surgery^ref. Additional studies are needed to determine whether the increased serum CTpr levels in such apparently non-bacterial insults are a manifestation of translocation to the blood stream of bacteria or bacterial products (e.g. endotoxin) from the gut^{ref1, ref2}. In this context administration of endotoxin to normal human volounteers increases serum CTpr values to levels seen in sepsis^ref. In addition to endotoxin, various proinflammatory mediators have also been shown to induce CTpr, e.g. TNF-a, IL-2 or IL-6. At least one parasitic infection (malaria) may substantially increase serum CTpr^ref. In fungal infections levels are occasionally^ref, but not always^ref, high. Viral infections may be associated with mildly or moderately elevated CTpr levels^{ref1, ref2} [14, 34], and here again bacterial translocation from the gastrointestinal tract may be a factor. It is of interest that, physiologically, newborns also exhibit a considerable increase in circulating CTpr which reverses spontaneously in the first week^{ref1, ref2, ref3}. This could be interpreted as a host response to initial establishment of the normal intestinal bacterial flora^ref. If the respective reference ranges are applied appropriately, CTpr can also be used to diagnose microbial infections in newborns^{ref1, ref2, ref3}. In these circumstances an ultrasensitive CTpr assay would be preferable. CTpr is an excellent marker for severe, invasive bacterial, fungal and protozoal infections (urinary tract infections and sepsis) in children^ref. Using a cut-off range of 0.5 through 2 ng/mL, the sensitivity and specificity of PCT are 100% in discriminating bacterial meningitis from viral meningitis. Some of the 7 studies published since seemed to demonstrate the usefulness of PCT in diagnosing meningitis. Finally, PCT was used effectively to shorten unnecessary antibiotic treatment for children seen in an hospital in Paris (France) during summer 2000 : on 243 patients with infections of the lower respiratory tract, such as bronchitis and pneumonia, 83% were given antibiotics when they received normal care (chest X-rays, laboratory tests and clinical symptoms) to assess whether an infection was bacterial in nature, vs. 43% when diagnoses were made on the base of plasma procalcitonin rise (a test which takes about 1 hour) : mucous culture, which is not always accurate, indicated that only about 20% of patients in both groups had bacterial infections. Even if the procalcitonin test still results in some over-prescription of antibiotics, it improves the situation dramatically^ref. In particular, serial measurements are useful in order to monitor response to therapy. PCT is present in the plasma of healthy subjects in minimal levels (< 0.5 ng/ml). Serum procalcitonin is markedly increased a few hours after the administration of endotoxin to human volunteers and in invasive bacterial infection (sepsis, septic shock, meningitis). Procalcitonin is moderately increased in local bacterial infection (pneumonia pyelonephritis) and is unchanged in viral infections or bacterial colonization. Procalcitonin is increased in serious bacterial infections in neonates, children and adults and is currently the best diagnostic marker of severe bacterial infection, being better than leukocyte, IL or CRP counts. CRP levels can be normal in severe sepsis and some viral infections^ref. CRP, PCT, WBC and ESR have only limited value in differentiating pneumococcal or other bacterial pneumonia from viral pneumonia. If there is a high value in at least one of the markers (CRP > 80 mg/L, PCT > 1.8 mg/L, WBC > 22 x 10⁹/L or ESR > 60 mm/h), viral infections are rare. There is no combination of these markers which was sufficiently sensitive and specific to be used in clinical pediatric practice^ref. 25 clinical indications, guidelines and caveats for the interpretation of serum CTpr. Many of the patient categories discussed in this table have what was initially defined a decade ago as “sepsis”, i.e. the systemic inflammatory response syndrome associated with proven or presumptive bacterial infection. However, in patients with other conditions (e.g. pancreatitis, burns, chemical pneumonitis, trauma, surgery, severe illness due to viruses, fungi, or malarial parasites, etc.), translocation of bacteria and/or bacterial products across the intestinal wall may occur, producing a syndrome that is identical to sepsis and not uncommonly just as ominous in outcome. Furthermore, there is nothing to prevent patients with a translocation-induced sepsis-like syndrome from subsequently developing a secondary, exogenous, systemic bacterial infection. Note that in all instances, as is the case of many laboratory tests, a serum CTpr level must be evaluated with proper regard to the clinical and laboratory context of the patient studied.
• the critically ill patient with sepsis and suspected bacterial infection should have a serum CTpr determination. In most such patients with marked bacterial infection, serum values will be high, and sepsis is diagnosed^{ref1, ref2, ref3}. A lower level may indicate another cause of illness, though local infection may still be present.
• in a patient with sepsis, the initial serum CTpr value may have prognostic significance (i.e. extremely high levels correlate with mortality)^ref. Similarly, a further later increase may presage a fatal outcome^ref
• very high levels of serum CTpr may occur in septic patients with severe SIRS and presumed infection, whether or not blood cultures are positive.
• in a patient with demonstrated sepsis, a progressive decrease in an initially high CTpr commonly indicates a response to therapy, an improvement in the clinical course and a favourable outcome^{ref1, ref2}. In such circumstances, however, a more sensitive CTpr assay is usually required to evaluate significant day-to-day fluctuations and reliably detect meaningful trends.
• in the elderly patient with recent onset of confusion, anorexia or incoordination, and for whom systemic bacterial infection is being considered, serum CTpr should be determined. Such patients are often normothermic or hypothermic, may not have leukocytosis, and may not manifest other signs of SIRS, such as tachycardia or tachypnoea. Furthermore, cultures in elderly patients may present positive urinary bacterial cultures and/or bacterial colonisation of the bronchial tree, without sepsis being present. If, however, sepsis is present, serum CTpr levels will be high.
• in a febrile patient, serum CTpr levels may differentiate bacterial infection from other, non-infectious causes of fever in which serum CTpr levels will usually be considerably less elevated or normal (e.g. drug fever, sarcoidosis, familial Mediterranean fever, collagen-vascular disease, lymphoma, leukaemia, sarcoidosis, solid tumors such as hypernephroma, granulomatous thyroiditis, fever of unknown origin and factitious fever)^{ref1, ref2}. The diagnostic and predictive utility of serum CTpr assay in patients with neutropenic fever is uncertain^ref
• serum CTpr levels are considerably higher in patients with septic shock as opposed to those with cardiogenic shock or with shock due to haemorrhage or acute primary adrenocortical insufficiency^{ref1, ref2}
• in the acute respiratory distress syndrome of adults (ARDS), higher CTpr levels occur if the cause is infectious than if there is a non-infectious aetiology. However, in acute respiratory distress syndrome of the premature infant, extremely high CTpr levels often occur without evidence of bacterial infection.
• serum CTpr levels are increased in most patients with pneumonitis^ref. However, if pneumonitis or pyelonephritis is minimal, SIRS may not be present. These patients may not have increased levels of CTpr when assayed commercially, but they often will have increased levels if an ultrasensitive CTpr assay is performed. This allows early identification of the problem and, if such values subsequently increase, permits the diagnosis of early sepsis. It will need to be determined whether the increased sensitivity of an ultrasensitive CTpr assay is accompanied by a decrease in specificity and positive predictive value.
• in patients with long-term intravascular catheters (e.g. central venous pressure monitoring, renal dialysis, prolonged antibiotic therapy, chemotherapy, intravenous alimentation), a sudden increase in previously normal or only mildly elevated levels of CTpr often predicts, very early, the onset of bacterial sepsis; this diagnosis of sepsis may occasionally precede the appearance of a positive blood culture or signs of sepsis. However, for this purpose an ultrasensitive CTpr assay must be used.
• significant viral infections are usually associated with only minimal to moderate increases in serum CTpr; high levels strongly suggest bacterial super-infection. In meningitis, the high levels of CTpr encountered where the aetiology is bacterial will contrast markedly with the lower levels in the vast majority of those with a viral aetiology^{ref1, ref2}
• patients with AIDS without any associated bacterial infection do not exhibit increased CTpr as assayed by the current commercially-available assay^ref. However, an ultrasensitive CTpr assay does reveal moderate elevations.
• in acute attacks of Plasmodium falciparum malaria, serum CTpr levels are markedly elevated; levels decrease with clinical improvement^ref. Most other parasitic infestations are not acute and have not been studied.
• patients with systemic fungus infections (e.g. candidiasis, aspergillosis) may have increased serum CTpr values^ref. However, some patients have slightly or moderately increased levels^{ref1, ref2}
• in severe mechanical trauma there is an early transient increase in serum CTpr. The level peaks within 1–3 days and is proportional to the severity of the tissue injury. Furthermore, a later secondary increase in CTpr is strongly suggestive of bacterial infection^ref.
• following surgical trauma, serum CTpr shows an increase in the first 1–2 days, the level of which correlates positively with the extent of surgery^ref. After some surgical procedures a high day-one postoperative serum CTpr is predictive of mortality^ref. However, after cardiopulmonary bypass there is a particularly rigorous inflammatory cascade and marked early serum CTpr elevation does not necessarily indicate infection^{ref1, ref2, ref3}. But here, as in any postoperative patient, either a persistently high serum CTpr level or a later, marked, secondary increase strongly suggests systemic bacterial infection^ref
• in patients who have undergone heart, lung, heart-lung, or liver transplantation, high serum CTpr levels indicate bacterial infection rather than acute rejection^{ref1, ref2}. Importantly, administration of monoclonal or polyclonal anti-thymocyte globulin to treat acute rejection induces a marked increase in circulating CTpr levels even in the absence of infection^ref. Also, in a study of patients after neutropenic allogenic bone marrow transplantation, serum CTpr levels were of little value in distinguishing infection from other complications^ref
• in chemical pneumonitis due to inspiration of vomitus or to inhalation burn injury or inhalation of toxic fumes, there may be a sudden, marked increase in CTpr which is often proportional to the clinical severity of the insult^{ref1, ref2}
• in pancreatitis associated with infected necrosis or caused by biliary obstruction, serum CTpr levels are higher than in the pancreatitis of alcoholism. Furthermore, in pancreatitis in general, high levels correlate with poor outcome^{ref1, ref2}
• in burns there is a subgroup of patients who exhibit extremely high serum CTpr values in the first two days; these patients often have a rapidly fatal outcome. Patients who develop high levels later in the course often die of sepsis. Patients who never show high levels usually survive^{ref1, ref2, ref3}
• heatstroke is usually accompanied by increases in serum CTpr, the level of which may correlate with the length of cooling time required for appropriate recovery^ref.
• patients with systemic lupus erythematosus or autoimmune vasculitis do not have elevated levels of CTpr as assessed by the current commercial assay^ref. Whether an ultrasensitive CTpr assay would show elevated levels has not been determined.
• mild to moderate increases in CTpr may occur in various chronic inflammatory diseases in which neuroendocrine cell hyperplasia is a commonly associated factor (e.g. chronic obstructive pulmonary disease, chronic bronchitis, pulmonary tuberculosis, regional ileitis, ulcerative colitis). In evaluating the course of such illnesses it would be preferable to use an ultrasensitive
• CTpr assay. The lack of specificity of CTpr in surgical patients, patients with burns or patients with non-infectious inflammatory disorders could reveal the potential limitations of an ultrasensitive CTpr assay.
• circulating CTpr levels show increases in newborns which reverse spontaneously in the first week. If the correct reference ranges are applied, CTpr may nevertheless be used to diagnose microbial infections.
• patients with medullary thyroid cancer always have increased serum CTpr, as do most patients with small cell cancer of the lung or carcinoid tumour and, on occasion, patients with other neuroendocrine tumours such as phaeochromocytoma^{ref1, ref2}. The usefulness of CTpr assays in evaluating the clinical course of such patients has not been studied.
• intestinal surgery and major operations more often increase PCT, whereas it is normal in the majority of patients after minor and primarily aseptic surgery. PCT can thus be used postoperatively for diagnostic means only when the range of PCT concentrations during the normal course of a certain type of surgery is considered and concentrations are followed up^ref
• sICAM-1 concentration may be used as a diagnostic tool with high sensitivity (85%) and moderate specificity (54.3%) in neonates suspected of infection.
• negative APPs / visceral proteins
• transferrin
• albumin
• transthyretin  / thyroxin-binding prealbumin (TBPA)
• C3 (elevation < 2 fold)
• ceruloplasmin (Cp) / ferroxidase (elevation < 2-fold) : IFN-g induces Cp expression and secretion in monocytes at inflamed sites, which is then subjected to translational silencing approximately 16 hours after. IFN-g  induces phosphorylation of the ribosomal protein L13a (RPL13A) / interferon-Gamma-Activated Inhibitor of Translation (GAIT) which is released from the 60S ribosomal subunit and binds to a 29 nucleotide area of the 3'-UTR of Cp mRNA^ref
• IgG
• IgM
• retinol binding protein (RBP)
• a₂-HS glycoprotein (AHSG)
• apoA-I
During APR, some acute phase reactants including serum amyloid A (SAA) component (1, 2, and 4), ceruloplasmin / ferroxidase, and apolipoprotein J / clusterin transitory integrate HDL, which in turn loses its ability to control oxidation of small dense LDL and production of oxidized phospholipids.
• oxidized LDL (oxLDL) bind to CRP and CD204 / macrophage scavenger receptor on macrophages (promoting mature dendritic cell (DC) transition from differentiating monocytes; oxLDLs engulfement leads to formation of foam cells) and lumenal surface of endothelial cells stimulating cytokine secretion via PPARd, thus being part of a nonspecific innate immune response.
• (oxLDLs)lysophosphatidylcholine (lysoPtc / LPC) acts through GPCRs PAFR, LOX-1, GPR4 and G2A, but the initiator GPCR is unknown : furthermore it is bound by the APP CRP.
• immunization with a model oxidized LDL — malondialdehyde-modified LDL (MDA-LDL) — promotes a T_h2-type response that, through the production of IL-5, increases the titre of natural IgM antibody T15/EO6 (specific for phosphorylcholine head group of oxidized phospholipids in MDA-LDL) secretion by activating B1 lymphocytes. Moreover, the presence of these natural antibodies correlates with protection from atherosclerosis as T15/EO6 natural antibody can block the uptake of oxidized LDL by macrophages, which contribute to the pathogenesis of atherosclerotic lesions through the formation of lipid-loaded foam cells. The development of atherosclerosis in LDLR-deficient mice given a high-fat diet is associated with T_h1-cell responses^ref. There are important implications for strategies that inhibit the actions of IL-5 to treat asthma and other allergic diseases.
• oxLDL is taken up much more rapidly by macrophages than native LDL and this may explain in part the generation of foam cells in the developing fatty streak lesion of atherosclerosis^ref. There is now a good deal of evidence to support the view that oxidation of LDL is a significant element in the pathogenesis of atherosclerosis, including the demonstration in several different animal models that antioxidants inhibit lesion progression^{ref1, ref2, ref3, ref4}. Most recently it has been shown that vitamin E inhibits lesion progression in the apoE-knockout model and that this correlates nicely with a decrease in the extent of isoprostane formation by nonenzymatic oxidation of arachidonic acid^ref (6). Strong support for the hypothesis is provided by the recent report that targeted disruption of the gene for 12/15-LOX profoundly inhibits atherosclerosis in the apoE-deficient mouse^ref. During oxidation of LDL there is a progressive decrease in the number of reactive lysine epsilon amino groups^ref. It has been generally assumed that these amino groups are blocked by reaction with short-chain aldehydes derived from the peroxidation of polyunsaturated fatty acids (e.g., malondialdehyde or 4-hydroxynonenal)^{ref1, ref2}. This conjugation of aldehydic fragments with lysine amino groups was believed to be responsible for converting native LDL into a form recognized by macrophage scavenger receptors. This was a logical conclusion drawn in part by analogy with the several modifications that were shown to generate modified LDL recognized by the scavenger receptors, i.e., acetylation^ref, treatment with malondialdehyde^ref, and so on. There was little or no consideration of the possibility that some of the masking of lysine amino groups might be due to conjugation with core ester aldehydes (i.e., phospholipids in which the sn-2 polyunsaturated fatty acid has been oxidatively degraded leaving a shortened fatty acid fragment, still esterified to the glycerol backbone, but terminating with an aldehydic function, or polyunsaturated cholesteryl esters in which the fatty acid has been similarly degraded leaving a new exposed aldehydic function). The possibility that such core ester aldehydes might play a significant role was raised by studies in this laboratory demonstrating that both the apolipoprotein moieties and the lipid moieties of OxLDL could bind to macrophage scavenger receptors and compete reciprocally with each other for that binding. It was further strengthened by the demonstration that a monoclonal antibody against oxidized phospholipids could react with both the isolated apolipoprotein and the reconstituted lipids from OxLDL^ref. Moreover, this antibody strongly inhibited macrophage binding of OxLDL, as well as that of the reconstituted lipid and protein moieties of OxLDL^{ref1, ref2}. In oxLDL, oxidized phospholipids bind to e-amino groups of Lys residues in apoB : the ability of the apoB from oxidized LDL to compete with intact oxidized LDL or with the apoB prepared from OxLDL varied directly with the number of phospholipid molecules bound per mole of apolipoprotein. The recognition of the oxidized LDL apoB by monoclonal antibody EO6 also varied according to the number of phospholipid molecules covalently attached^ref. Antibody EO6 has previously been shown to react with antigens present in atherosclerotic lesions^ref, suggesting that oxidized phospholipid-apoB adducts are also produced in vivo. Itabe et al. immunized mice with material from a human atheroma and cloned an antibody (DHL3) with anti-phospholipid activity very similar to that of EO6. This antibody also reacted with circulating oxidized human LDL and with human atherosclerotic lesions^ref. OxLDL has been separated into its protein moiety [apolipoprotein B isolated from OxLDL (Ox_apoB)] and its lipid moiety (Ox_lipid) using exhaustive organic solvent extraction, and the binding properties of the two fractions have been studied^{ref1, ref2}. Both of the isolated moieties bind with high affinity to the scavenger receptors of mouse peritoneal macrophages, and surprisingly, they exhibit marked reciprocal competition. Confirming this apparent "common epitope" between the two species, a monoclonal antibody, designated EO6, recognized both the protein and lipid moieties of OxLDL^ref. EO6 competed for binding to mouse peritoneal macrophages through a recently identified common epitope of oxidized phospholipid (PL), present either within the lipid phase of OxLDL or covalently associated with the lysine residues of the oxidized protein of OxLDL^{ref1, ref2}. Antibody EO6, which recognizes oxidized PL^ref, and 1-palmitoyl-2-(5-oxovaleroyl) phosphatidylcholine (POVPC), a product of LDL oxidation^ref, were both excellent competitors of OxLDL binding to CD36. The results suggested that >60% of the binding of OxLDL to CD36 is mediated by oxidized PL, present within the lipid phase or covalently associated with the OxapoB.

Anticardiolipin antibodies from patients with the antiphospholipid antibody syndrome recognize epitopes in both b₂-glycoprotein 1 and oxLDL^ref
• LDL modified by HOCl (HOCl-LDL) : hypochlorous acid (HOCl) produced by myeloperoxidase (MPO), an inflammator heme protein secreted by phagocytes oxidize tyrosine to 3-chlorotyrosine : when this occurs in the main protein in HDL, apolipoprotein A-I, this turns the 'good cholesterol' bad in the human artery wall and impairs ABCA1-dependent cholesterol transport, promoting atherogenesis by counteracting the established antiatherogenic effects of HDL and the ABCA1 pathway^ref. HOCl-LDL displays a number of pathophysiological effects on phagocytes and vascular cells, contributing to the initiation and maintenance of the inflammatory process during the early phase of atherosclerotic lesion development. HOCl-LDL induces chemokine release of monocytes and chemotactic migration of neutrophils^ref, initiates the respiratory burst of macrophages^ref, stimulates polymorphonuclear leukocytes to an enhanced production of superoxide anion radical and hydrogen peroxide, enhances neutrophil degranulation^ref, and inactivates lysosomal proteases^ref. HOCl-LDL further decreases nitric oxide-synthesis in endothelial cells^ref, causes endothelial leakage and stimulates leukocyte adherence to, and migration into, the subendothelial space^ref. HOCl-LDL enhances platelet reactivity and release reaction^{ref1, ref2}, and most importantly, HOCl converts LDL into a high uptake form for mouse peritoneal macrophages^ref, leading to the formation of cholesteryl ester (CE)-laden foam cells, which are the hallmark of fatty streaks and the earliest recognizable lesion of atherosclerosis. The presence of 3-chlorotyrosine in human atherosclerotic lesions^ref, the presence of HOCl-modified epitopes inside and outside monocytes/macrophages, endothelial cells, and smooth muscle cells in human and rabbit lesion material^{ref1, ref2, ref3}, and the presence of HOCl-modified apoB-100 extracted from advanced human atherosclerotic lesions^ref supported the view that the MPO/hydrogen peroxide/chloride system converts LDL into an atherogenic form under in vivo conditions. HOCl-LDL bind to SR-BI and CD36^ref.
• both type I and type II SR-A bind a diverse array of macromolecules, including bacterial surface lipids (endotoxin and lipoteichoic acid), b-amyloid fibrils, protein modified by advanced glycation (advanced glycation end products), and modified lipoproteins, e.g. acetylated LDL (ac-LDL) or copper-oxidized-LDL (Cu-ox-LDL), respectively. SR-AI/AII mediates 80% of the uptake of ac-LDL^ref
• APR can be truly acute, as in the case of a viral or bacterial infection, but it may also become chronic and result in inflammatory diseases like atherosclerosis. Also seen are fever, increased vascular permeability, and a variety of metabolic and pathologic changes.
• cytostasis : the closure of capillaries by white blood corpuscles in the early stages of inflammation.
Varieties :
• angioflogosis (usually acute inflammation) : prevalence of exudate
• clear, torbid or bloody look
• [Pr^-]_fluid > 3 g / dL
• [Pr^-]_fluid / [Pr^-]_serum > 0.5
• [LDH]_fluid > 200 U / L
• [LDH]_fluid / [LDH]_serum > 0.6
• [Glc] < 60 mg / dL
• WBCs > 1,000 / mL
• RBCs > 5,000 / mL
• positive Rivalta's reaction : a reaction for distinguishing fluids of transudation and exudation, utilizing acetic acid
• erythaematous angioflogosis
• exudative inflammation : one in which the prominent feature is an exudate.
• serous inflammation or angioflogosis : one which produces an exudation of serum.
• sero-fibrinous angioflogosis
• fibrinous angioflogosis or inflammation : one that is characterized by an exudate of coagulated fibrin.
• mucous or catarrhal angioflogosis
• mucopurulent angioflogosis
• purulent or suppurative angioflogosis or inflammation : one characterized by the formation of pus.
• abscess / abscessus : a localized collection of pus buried in tissues, organs, or confined spaces.
• acute abscess : one which runs a relatively short course, producing some fever and a painful local inflammation.
• hot abscess : an acute abscess with symptoms of local inflammation.
• chronic or cold abscess : an abscess of comparatively slow development with little evidence of inflammation
• bicameral abscess : one which has 2 chambers or pockets
• shirt-stud or collar-button abscess : a superficial abscess connected with a deeper one by a passage
• caseous or cheesy abscess : one containing cheeselike material
• diffuse abscess : an uncircumscribed abscess, the pus of which is diffused in the surrounding tissues
• dry abscess : one that disappears without pointing or breaking
• Welch's gas or tympanitic abscess : a localized collection of seropurulent material containing gas, caused by gas-forming bacteria such as Clostridium perfringens
• gravitation or gravity abscess : one in which the pus migrates or gravitates to a lower or deeper portion of the body
• worm abscess : one caused by or containing worms
• helminthic abscess : one caused by a worm, such as filaria.
• verminous abscess : one which contains insect larvae or other animal parasites.
• metastatic abscess : a secondary abscess, usually of embolic origin, in which organisms are carried by the circulation to a point distant from the primary lesion
• miliary abscess : one of a set of small multiple abscesses
• phlegmonous abscess : one associated with acute inflammation of the subcutaneous connective tissues.
• pyemic or septicemic abscess : one due to pyemia
• residual abscess : one recurring at the site where an incompletely resolved abscess occurred previously.
• satellite abscess : a secondary abscess arising from, and situated near, a primary abscess.
• sterile abscess : one from which microorganisms cannot be isolated
• stitch abscess : one that develops adjacent to a stitch or suture.
• sympathetic abscess : one arising some distance from the exciting cause
• wandering, hypostatic or migrating abscess : one that burrows in the tissues and finally points at a distance from the site of origin
• microabscess : a very small, localized collection of pus
• empyema : a collection of pus in a body cavity
• phlegmon / phlegmonous cellulitis : a spreading, diffuse inflammatory reaction to infection with microaerophilic streptococci, which forms a suppurative or gangrenous and undermining lesion that may extend into deep subcutaneous tissues and muscles, creating multiple small pockets of pus
• pyogenic membrane : a membrane which produces pus.
• prophylactic or pyophylactic membrane : a fibrinous membrane lining a pus cavity and tending to prevent reabsorption of injurious materials
• false membrane / neomembrane / pseudomembrane : a layer resembling an organized and living membrane, but made up of coagulated fibrin with bacteria and leukocytes, such as may be formed on mucous membranes in diphtheria or in the gut with Clostridium difficile infection
• haemorrhagic angioflogosis
• necrotic-haemorrhagic angioflogosis
• allergic angioflogosis
• histoflogosis (usually chronic inflammation) : prevalence of cellular phenomena => granuloma: a compact (organized) collection of mature mononuclear phagocytes (macrophages and/or epithelioid cells) which may or may not be accompanied by accessory features such as
• infiltration of other inflammatory leukocytes
• necrosis : tuberculization is  the formation of or conversion into tubercles.
• non-necrotizing or non-caseating granulomas
• sarcoidosis (vast majority of granulomas : hard tubercle, composed of discrete aggregations of large, pale-staining, epithelioid cells intermingled with histiocytes, lymphocytes, and Langhans' giant cells, sometimes surrounded by a narrow band of lymphocytes; when necrosis is present, it is minimal; tuberculitis : inflammation of or near a tubercle => fibrous tubercle is a tubercle that has undergone chronic inflammatory scarring)
• beryllium disease
• hypersensitivity pneumonitis
• drug reactions
• tuberculoid leprosy
• tuberculosis (minority of granulomas)
• foreign bodies in the tissues
• cutaneous leishmaniasis
• deep fungal infections
• Crohn's disease
• necrotizing or caseating granulomas / caseous tubercle (a tubercle having a central necrotic yellowish mass of cheesy material)
• tuberculosis (vast majority of granulomas)
• fungal infections
• rheumatoid nodules
• Wegener's granulomatosis
• necrobiotic post-surgical granulomas
• sarcoidosis (minimal and focal)
From the inner to the outer layer granuloma is made up of ...
• aetiological agent(s)
• living organism (see infective causes)
• unliving matter
• eleoma / elaioma : a tumor or swelling caused by the injection of oil into the tissues
• eleidoma (vegetal oils => reabsorbable)
• paraffinoma / vaselinoma : a chronic granuloma produced by prolonged continuous exposure to the irritation of paraffin (e.g. mineral oils in surgical prosthesis or anti-constipation emulsions), which cannot be reabsorbed
all pneumoconioses (except for anthracitosis).
• mature mononuclear phagocytes
• macrophages
• epithelioid cells : highly phagocytic, modified macrophages, resembling epithelial cells, having large, pale and vesicular nuclei with abundant, eosinophilic cytoplasm, which are characteristic of granulomatous inflammation
‡  coalescence of epithelioid cells or by nuclear division without cytoplasmic division of monocytes
• multinucleate giant cells
• Langhans' giant cells : have their nuclei arranged in a complete circle or in a horseshoe-shaped pattern at the periphery of the cells, characteristically seen in granulomatous inflammations, as occur in tuberculosis, leprosy (lepra cells / Virchow cells) syphilis, sarcoidosis, and deep fungal infections.
• foreign body giant cells (FBGC) : have clusters of nuclei scattered in an irregular pattern throughout the cytoplasm, formed by coalescence and fusion of macrophages, with only a rare internal nuclear division, characteristic of foreign body reaction (FBR) (a granulomatous inflammatory reaction evoked by the presence of an exogenous material (including almost all biomaterials) resistant to degradation in dermis or subcutaneous tissue). FBGC damage the surface of biomaterials. FBGC derive from blood-borne monocytes that enter the implantation site after surgery in response to the release of chemotactic signals and fuse in response to CCL-2 / MCP-1^ref. FBGC are not the primary determinants of capsule formation in the FBR.
• unusual complication of retained surgical material or instruments :
• surgical gauze pseudotumor^ref
• pseudotumor after lung volume reduction surgery^ref
• after gunshot injury^ref. Retained bullet fragments or grenade splinters in veterans of World War II are not uncommon. A large foreign-body–induced pseudotumor first presented > 60 years after the injury occurred^ref.
• activated lymphocytes
• fibroblasts
Web resources :Atlas of granulomatous diseases by Yale Rosen, M.D.
Staging :
• Neutrophils localization (see also Inflammation : the leukocyte adhesion cascade)

step 1) : reversible adhesion (tethering and rolling) (few seconds)

molecules on neutrophils	molecules on endothelial cells
	endothelial heparan sulfate has three functions in inflammation: by acting as a ligand for L-selectin during neutrophil rolling; in chemokine transcytosis; and by binding and presenting chemokines at the lumenal surface of the endotheliumref

step 2) : G_iPCRs-mediated adhesion activation  (few seconds) step 3) : adhesion / attachment & arrest (few seconds)

CLA-1  other syalyl-Lewis A and B molecules	p110CD62E / E-selectin / ELAM-1 / LECAM-2
CD11bCD18 / aMb2 integrin / C3biR / CR3 / Mac-1 (maybe in complexes with CD87/uPAR)	CD54 / ICAM-1 CD102 / ICAM-2 sulfated fucosylated proteoglycans  fucoidin

step 4) : diapedesis / transendothelial migration (TEM) (10')

CD31 / platelet endothelial cell adhesion molecule-1 (PECAM-1) / ENDOCAM was the first such protein shown to mediate neutrophil transmigration in vitro and in vivoref  CD102 / ICAM-2ref1, ref2 They are only relevant for leukocyte recruitment if the inflammation was triggered by IL-1b, but not if the tissue was stimulated by TNF-a or thioglycollate CD99 acts in vitro at a step after PECAM-1ref junctional adhesion molecule-A (JAM-A) participates in stimulus specific waysref  endothelial cell–selective adhesion molecule (ESAM) participates in a cytokine-independent way (F. Wegmann, B. Petri, A. G. Khandoga, C. Moser, A. Khandoga, S. Volkery, H. Li, I. Nasdala, O. Brandan, R. Fässler, S. Butz, F. Krombach, and D. V., manuscript submitted).

Leukocytes move through endothelial junctions between neighboring cells, and they can use an alternative transcellular route through endothelial cells. Although in vitro and in vivo evidence for the latter pathway exist, the transcellular route seems to be used with low efficiency. Whatever relevance it has in vivo, it is remarkable that more and more endothelial membrane proteins that are involved in this process turn out to be located at endothelial cell contacts.
2 peculiar neutrophil chemotactic behaviors are the neutrophil's ability to polarize in uniform concentrations of a chemoattractant (i.e. to break symmetry even in the absence of an external gradient of the stimulus) and its response to reversal of an attractant gradient by performing a U-turn instead of simply reversing their direction of motility :
• the "frontness" pathway is mediated by G_i, 3'-phosphoinositol lipids, and Rac and generates pseudopods via de novo polymerization of F-actin
• the "backness" pathway is initiated by G₁₂ and G₁₃ and is dependent on the activation of a Rho-dependent pathway that stimulates the activation of myosin II and the formation of actin–myosin complexes. Backness signals reduces the sensitivity of the rear and sides of a cell to chemoattractants
As a result of the divergent, opposed signals for front and back, the leading edge is more sensitive than the trailing edge, and the cell "follows its nose."
• chemical mediators of inflammation :
• produced in situ (previously synthetized and stored in the granules of eosinophils, PMNs, macrophages and T-lymphocytes or synthetized at the moment) :
• vasodilators : PGD₂, PGE₂, PGI₂/prostacyclin, NO^., SRS-A
• vasoconstrictors : TxA₂, TxB₂, PGF_2a, PGH₂, 5-HT
• permeabilizing : vasoactive amines (histamine, 5-HT), LTB₄, C3a, C5a, PAF, substance P, bradykinin, SRS-A, PGA₂, PGF_2a
• priming and positive chemotaxis : C5a, LTB₄, chemokines, LPS
• negative chemotaxis : PGD₂, PGE₂, PGI₂ / prostacyclin
• fever : IL-1a, IL-1b (=> IL-1R => COX-2 => PGE₂ => EP_{_} => preoptical area), IL-2, IL-6, IL-8,IFNs, TNF-a
• hyperalgesia : PGE₂, PGI₂/prostacyclin, bradykinin
• induction of platelet aggregation : NO^., R.O.S., TxA₂, PGE₂, PGH₂, PAF
• inhibition of platelet aggregation : PGD₂, PGI₂ / prostacyclin
• induction of T-cell proliferation : TxA₂, LTB₄
• inhibition of T-cell proliferation : PGD₂, PGE₂, PGI₂ / prostacyclin
• macrophage inhibition and eosinophils activation : 15-deoxy-D^12,14-PGJ₂ (15dPGJ₂) and 15-deoxy-D^12,14-PGD₂ (15dPGD₂)
• induction of pro-inflammatory cytokines production : LTB₄
• inhibition of pro-inflammatory cytokines production : PGD₂, PGE₂, PGI₂ / prostacyclin
• SMC contraction : SRS-A, PGF_2a, histamine
• produced in other places :
• coagulation cascade components
• kinin system
• complement (C') cascade components
• immune response. Lymphocyte migration occurs in focal structural dilations - microangiectasias - in the vessel, in which shear forces are reduced sufficiently to allow lymphocyte-endothelial cell interactions to occur. Shear stresses in normal vessels are 20 dyn/cm², but lymphocytes adhesion usually occurs at wall shear stresses of 1 dyn/cm². Furthermore, blood flow during inflammation is likely to increase the shear stresses rather than decrease them.
CCR5⁺ apoptotic leukocytes act as 'terminators' of chemokine signaling (CCL3 and CCL5) during the resolution of inflammation^ref
Cellular responses to hypoxia :
• fast (acute) responses (s to min) rely mainly on O₂-regulated ion channels
• during both systemic hypoxemia and local hypoxia :
• activation of glucose uptake by translocation of GLUT-1 and GLUT-4 from intracellular vesicles to the plasma membrane and activate preexisting GLUT-1 in the membrane (cardiac and skeletal muscle, fat tissue)
• activation of Na⁺-H⁺ exchanger (NHE)
• inhibition of K⁺-Cl^- cotransporter (KCC)
Hypoxia is a characteristic feature of the tissue microenvironment during bacterial infection. HIF-1a was induced by bacterial infection, even under normoxia, and regulated the production of key immune effector molecules, including granule proteases, antimicrobial peptides, nitric oxide, and TNF-a. Mice lacking HIF-1a in their myeloid cell lineage showed decreased bactericidal activity and failed to restrict systemic spread of infection from an initial tissue focus. Conversely, activation of the HIF-1a pathway through deletion of VHL tumor-suppressor protein or pharmacologic inducers supported myeloid cell production of defense factors and improved bactericidal capacity. HIF-1a control of myeloid cell activity in infected tissues could represent a novel therapeutic target for enhancing host defense^ref.
• during systemic hypoxemia only :
• hyperventilation (transmitter release by electrically excitable arterial (carotid and aortic body) and airway chemoreceptors). They may have some tonic activity at the normal P_O2 levels of arterial blood (90-100 mmHg), but they begin to be fully activated with moderate levels of hypoxia (< 50-60 mmHg). K⁺ channels are closed by low P_O2 => membrane depolarization => opening of voltage-gated calcium channel => transmitter release to the extracellular milieu and, in the innervated organs, activation of afferent sensory fibers
• decreased heart output (peripheral chemoreceptors, heart muscle)
• systemic arterial vasodilation is particularly well manifested in coronary and cerebral vessels (arterial endothelium and smooth muscle, irrigated tissues)
• decreased ATP production => opening of K_ATP channels
• direct activation of K_Ca channels
• direct inhibition of L-type voltage-gated calcium channels
• nitrite reductase activity of deoxyhemoglobin and deoxygenated erythrocytes reduces vascular nitrites into nitric oxide^ref
• hypoxic pulmonary vasoconstriction (HPV) in pulmonary arteries and veins but greatest in small resistance arteries (vascular endothelium, resistance vessel pulmonary myocytes). HPV reduces blood flow through poorly ventilated alveoli and thus contributes to matching perfusion to ventilation and preventing systemic hypoxemia when atelectasis is present.
• relaxation of ductus arteriosus (ductus muscle)
• slow (chronic) responses (hours to days) depend on the modulation of hypoxia-inducible transcription factors :
• during both systemic hypoxemia and local hypoxia :
• activation of glucose metabolism and transport (most tissues)
• neoangiogenesis and neovascularization (vascular endothelium, hypoxic or ischemic tissues)
• tissue hypertrophy and remodeling (pulmonary arterial wall, myocardium, carotid body)
• production of vasodilators (vascular endothelium and smooth muscle)
• activation of HIF-1a by low P_O2 is detectable in < 30 min, with peak response occurring in 4 to 8 hours. On return to normoxia, activity decays with a half-life under 5 min. HIF-1 DNA binding activity is modulated within physiologic P_O2 levels. It increases about 2-fold between 20 and 6% O₂ and 10-fold between 6% and 0.5% O₂. Half-maximal activation is around 1.5% O₂ (P_O2 = 11 mmHg). One of HIF's 2 transactivation domains, the C-terminal activation domain of HIF-1a (CAD), is critical to the hydroxylation of HIF and links HIF to the well-studied transcriptional coactivators p300 and CBP, as well as SRC-1, and TIF-2.
• in the presence of O₂, hypoxic switches operate in the CAD :
• hydroxylation of Pro⁵⁶⁴ by HIF-1a prolyl 4-hydroxylase (HPH) (requiring Fe²⁺, O₂, and 2-oxoglutaric acid, and activated by NO^.) : this posttranslational modification allows an E3 ubiquitin ligase complex containing the VHL tumor-suppressor protein to bind HIF-1a and polyubiquinate it in the N-terminal activation domain (NAD) overlapping the oxygen-dependent degradation domain (ODD) (interestingly, hints at the role of prolyl hydroxylation could have been gleaned earlier if researchers had looked to the field of collagen research where, for more than 2 decades, prolyl hydroxylases have been known to use O₂ and to be highly sensitive to iron chelators and cobalt). Ascorbate / vitamin C enhances the activity of HPH and might figure into an antiangiogenic treatment. Although the only known prolyl hydroxylases (prolyl hydroxylase a_I and prolyl hydroxylase a_II, which - combined to prolyl hydroxylase b - hydroxylate collagen) can function under hypoxic conditions, hydroxylation of Pro⁵⁶⁴ is far more important than other post-translational modifications of HIF-1a (e.g. serine, threonine, and tyrosine phosphorylation, asparagine hydroxylation, glutamine deamination, glutamate g-carboxylation on residues in the vicinity of Pro⁵⁶⁴). Prolyl hydroxylase domain (PHD1 / EGLN2, PHD2 / EGLN1, and PHD3 / EGLN3) are O₂-dependent, suggesting to some that these enzymes may be responsible for oxygen sensing^{ref1, ref2}.
• hydroxylation of Asn⁸⁰³ by the Fe²⁺- and O₂-dependent enzyme factor inhibiting HIF-1 (FIH-1), which inhibits binding of CAD to CBP/p300, cofactors required for initiating expression of genes involved in oxygen-response pathways. The asparaginyl hydroxylase can work reasonably well at very low O₂ levels^{ref1, ref2}. The PHDs that control HIF seem to stop functioning at less stringent hypoxia
• in the absence of O₂ no hydroxylation occurs => no proteasomal degradation occurs => HIF-1a is upregulated.


A few favor another model in which the prolyl and asparagyl hydroxylases are regulated upstream by a proximal oxygen sensor, e.g. when mitochondria release reactive oxygen species (ROS), initiating a signal transduction pathway that in turn inactivates the prolyl and asparagyl hydroxylases. The latter model helps to explain data suggesting that overexpression of FIH and the prolyl hydroxylases is sufficient to shut off HIF activity. You can have a profound effect on the transcriptional activation and the stability of HIF by manipulating the expression levels of the enzymes. If oxygen were the direct regulator of FIH and prolyl hydroxylase activities, tight controls would need to be in place to regulate the amount of these hydroxylases in the cell. By having a single proximal regulator, an oxygen sensor, that controls the activity of the hydroxylases ... ultimately HIF stability and HIF transcriptional activity could be made more independent of the precise levels of the expression of the hydroxylases themselves. Further, the model could also explain other oxygen-dependent responses that do not require HIF; for example, mitochondrial ROS mediate hypoxic pulmonary vasoconstriction, a HIF- independent process. Unfortunately ROS levels are difficult to measure, which can make it hard to draw accurate conclusions. The oxygen tension required to trigger PHD2 is in the physiologic range. There's lots of flux and the oxygen tension is very high outside of the cell; there's a gradient. The mitochondria are active consumers of oxygen, so to really get a handle on what the oxygen tension is at the very intracellular site where prolyl hydroxylase is acting, is a very tough challenge.
HIF-1 alters WBCs so that they can survive without O₂ - a trick that is essential to their defensive role, as damaged tissues are often low in the gas. WBCs from HIF-1^-/- mice stay away from low-O₂ environments, and destroy few bacteria
Genetic alterations that increase HIF-1 activity :
• decreased ubiquitylation
• VHL loss of function^ref
• p53 loss of function^ref
• increased synthesis
• PTEN loss of function^{ref1, ref2}
• ERBB2 gain of function^ref
• SRC gain of function^ref
• signalling in a component of the pathway or an upstream activator
• PI3K-AKT-mTOR^{ref1, ref2}
• MEK-ERK signalling^ref
• EGFR signalling^ref
• IGF1R signalling^ref
• PGE₂signalling^{ref1, ref2}
• decreased nucleolar sequestration : ARF loss of function^ref
• not determined : BCL2 overexpression^ref
Under certain cellular conditions, HIF-1a is also responsible for hindering the cell's mismatch repair (MMR) system, crucial for maintaining a cell's genetic integrity : HIF-1a acts by displacing Myc, the transcriptional activator of 2 nuclear proteins, MSH2 and MSH6. In the absence of HIF-1a, Myc activates the expression of the 2 compounds, which then dimerize to form the MutSa complex—one of the mammalian versions of the MMR system. In the presence of HIF-1a, Myc cannot reach the promoters, and the expression of MutSa is inhibited
• AP-1
• EGR-1
• HMG-I(Y)
• NF-IL6
• NFkB
• during systemic hypoxemia only :
• erythropoiesis (bone marrow)
Cardinal signs of localized inflammation (Aulus Cornelius Celsus, De Re Medica, 25-35 a.C.) :
• rubor = redness
• tumor = swelling
• calor = heat
• dolor = pain
• functio laesa = loss of function (added by Galenus, 129-201 a.C.)

During systemic inflammation, body temperature is either increased (fever) or decreased (anapyrexia). Either response depends on ..
• the dose of the inflammatory agent
• the ambient temperature
• under thermoneutrality, LPS always produces fever
• subthermoneutral conditions, LPS evokes ...
• fever at lower doses
• anapyrexia at higher doses
Pathogenesis : febrigenic doses of LPS => IL-1b =>
• increased production of PGE₂ by COX-2 and mPGES-1 in vein or venular-type cerebral blood vessel and leptomeningeal cells in the ventromedial preoptic region (PO) of the hypothalamus (VMPO) => decreasing the intracellular content of cAMP. Although COX-2 plays a dominant role in mediating fever responses to i.v. LPS, at least some components of the response, including avoiding hypothermia and the induction of Fos in the nucleus of the solitary tract (NTS), ventrolateral medulla (VLM), parabrachial nucleus (PB), and hypothalamic paraventricular nucleus (PVH), appear to depend on COX-1
• inhibition of NOS1  => decreased intracelullar content of cGMP in the PO
=> fever / pyrexia : elevation of body temperature above the normal. Although the role of preoptic cAMP and cGMP has not been directly assessed in the anapyrexia induced by LPS, it has been studied in that induced by hypoxia : the likeness between the thermoregulatory responses to hypoxia and to a high dose of LPS suggests that they may have similar mechanisms, ie a simultaneous increase in the levels of cAMP and cGMP in the PO as the result of an enhanced production and/or release of serotonin and nitric oxide, respectively
Aetiology :
• physiological stresses such as
• ovulation
• milk fever : a fever said to attend the establishment of lactationafter delivery
• excess thyroid hormone secretions
• fatigue fever : a febrile attack due to overexercise and the absorption of waste products.
• digestive fever : a slight rise of temperature during the process of digestion.
• functional fever : irregular defecation, dyspnea due to meteorism (hysterical pregnancy), insomnia, headache, fine tremors, frequent urination due to chronic visceral contraction, emotional instability, anxiety, normal laboratory examinations (CBC, a₁gp, ESR, CRP)

Therapy : i.v. BDZ
• central nervous system lesions
• central fever : sustained fever resulting from damage to the thermoregulatory centers of the hypothalamus, e.g. by brain tumors
• suspension of analeptics
• infection by microorganisms
• systemic infections
• Brucella melitensis (undulant fever)
• Coxiella burnetii
• otitis
• pharyngotonsillitis
• pyelonephritis (accompanied by renal colic)
• urethrocystitis (accompanied by stranguria)
• meningitis
• enteric fever : any of a group of febrile illnesses associated with enteric symptoms caused by salmonellae, especially ...
• typhoid fever (the prototype of the severe enteric salmonellal infections)
• paratyphoid fever
• stepladder stage : an early stage of enteric fever; so called from the peculiar form of the temperature curve.
• entericoid fever : any fever which resembles typhoid fever in its clinical manifestations.
• parenteric fever : a disease clinically resembling typhoid fever and paratyphoid fever, but not caused by Salmonella.
• viral hemorrhagic fevers : a group of diverse, severe epidemic viral infections, found in many parts of the world, mainly in tropical climates; causative viruses are often geographically restricted. They are usually transmitted to humans by arthropod bites or contact with virus-infected rodents; all the infections share certain clinicopathological features, including fever, hemorrhagic manifestations, thrombocytopenia, shock, and neurologic disturbances. The group comprises :
• Argentine hemorrhagic fever
• Bolivian hemorrhagic fever
• chikungunya ("bone-breaking fever")
• Crimean-Congo hemorrhagic fever
• dengue hemorrhagic fever
• Ebola hemorrhagic fever
• epidemic hemorrhagic fever
• Kyasanur Forest disease
• Lassa fever
• Marburg fever
• Omsk hemorrhagic fever
• Rift Valley fever
• yellow fever
• humidifier fever : a syndrome of malaise, fever, cough, and myalgia, caused by inhalation of air that has been passed through humidifiers, dehumidifiers, or air conditioners contaminated by fungi, amebas, or thermophilic actinomycetes
• any of a host of noninfectious processes, such as inflammation or the release of certain materials
• autoimmune diseases
• hypersensitivity reactions
• intermittent hepatic fever / Charcot's fever or syndrome : a fever occurring intermittently as the result of intermittent impaction of choledocholithiasis and inflammation of the bile ducts
• urolithiasis
• drug fever : a febrile reaction marked by prolonged temperature elevation during the course of administration of a drug, such as an antibiotic, antineoplastic, vaccine, etc.; it may be associated with vasculitis affecting small vessels, and usually disappears rapidly on discontinuance of the drug
• hydroxyurea (HU)
• duck fever : a type of hypersensitivity pneumonitis caused by sensitivity to duck proteins contained in duck feathers.
• land fever : a set of symptoms resembling seasickness sometimes experienced when, after an ocean voyage, the ship enters a relatively landlocked body of water.
• periodic fever : a hereditary condition characterized by repetitive febrile episodes and autonomic disturbances, occurring in precise or irregular cycles of days, weeks, or months. Transmitted as an autosomal dominant trait, it may begin at any time of life and may last for decades with temporary remissions, or may cease
• hereditary periodic fever syndromes
• periodic fever, aphthous stomatitis, pharyngitis, and adenopathy syndrome (PFAPA syndrome) / Marshall syndrome

Symptoms & signs : abrupt onset of fever, malaise, aphthous stomatitis, tonsillitis, pharyngitis and cervical adenopathy. The age of onset of the disease is four years, with a range of 6 months to 7 years. The syndrome is sporadic and nonhereditary
Therapy : tonsillectomy
Prognosis : long-term sequelae do not develop.
• protein fever : heightened temperature produced by the injection of protein material into the body.
• salt fever : fever associated with excess of salt in the body, due to the retention by the salt of the water normally eliminated in perspiration.
• shoddy fever : a febrile disease, with cough, dyspnea, and headache, caused by inhalation of dust in shoddy factories.
• leukemia
• Murchison-Pel-Ebstein fever or pyrexia: a cyclic fever occasionally seen in Hodgkin's disease and also associated with other diseases, characterized by irregular episodes of pyrexia of several days' duration, with intervening afebrile periods lasting for days or weeks
• thermic fever / sunstroke / insolation
• artificial fever : elevation of bodily temperature produced by artificial means, as by external heat or the injection of typhoid vaccine or malarial parasites
• therapeutic fever / pyretotherapy : treatment of a disease by raising the patient's temperature, especially by means of injecting fever-producing vaccines
• inductothermy
• aseptic fever : fever associated with aseptic wounds, presumably due to the disintegration of leukocytes or to the absorption of avascular or traumatized but uninfected tissue.
• fever of unknown origin (FUO) / pyrexia of unknown origin (PUO) / essential fever : defined in 1961 as a temperature > 38.3°C on several occasions for >= 3 weeks, with the diagnosis remaining uncertain after 1 week of hospitalization^ref, associated to systemic symptoms such as night sweats, thinning and fatigue, sometimes associated to RCCs, but for which no cause has been found. 10% remain undiagnosed. Since then, other definitions have been proposed. The initial evaluation of FUO typically involves a thorough history taking, physical examination, routine blood chemical tests, blood counts, cultures of urine and blood, and abdominal CT. Further testing is generally more invasive, and its pace depends on the patient's condition, with rapid clinical deterioration mandating an aggressive strategy. A liver biopsy yields a diagnosis in about 15% of patients with FUO^ref. Although the diagnostic yield of cultures of bone marrow in patients with FUO is usually very low, fibrin-ring granulomas in some cases suggest that a disseminated granulomatous disease is the cause of the illness.
Pathogenesis : when primed with TLR agonists or bacterial extract but not proinflammatory cytokines, hyperthermia specifically enhanced secretion of IL-12p70 by DC, without altering the secretion of IL-10, TNF-a or IL-1b. These DC induced significantly higher levels of T-cell proliferation and IFN-g production in assays of antigen presentation and MLR. Endogenous HSP70 colocalized with CD40 in DC exposed to hyperthermic conditions. Recombinant CD40-Fc fusion protein blocked the increase in IL-12p70 secretion by DC primed with bacterial extract and hyperthermia^ref
Course :
• stage of fervescence / pyretogenic or pyrogenetic stage : the stage of invasion of a febrile attack.
• continued or continuous fever : one which has gradual  onset (up to 5 days), fastigium (from day 6 to day 13) in which it varies < 1.0° to 1.5°F (< 1°C) in 24 hours, such as typhoid fever
• remittent or septic fever : a fever in which the diurnal variation is > 2° F (> 1°C), but in which the temperature never falls to a normal level
• continued-remittent fever : a fever in which in some days the diurnal variation is > 2° F (> 1°C), and in other days the diurnal variation is < 2°F (< 1°C)
• undulant fever : a particular kind of continuous-remittent fever which has increasing peaks associated with chills every 24 hrs and crisis defervescence => night sweats, arthromyalgia, headache, anorexia, alternated to afebrile periods every 1÷4 weeks such as brucellosis (some authors classify it as a type of intermittent fever)
• Haemophilus influenzae
• intermittent fever : a fever in which periods of fervescence are separted by periods of defervescence down to papyrexia, such as malaria
• cold stage / stadium frigoris : the chill or rigor period of an intermittent fever
• hot stage / stadium caloris : the period of pyrexia in an intermittent fever
• quotidian fever : fervescence each day with apyrexia at evening, such as with many infections due to decreased cortisolemia at evening
• tertian fever : fervescence each other day, such as Plasmodium falciparum (malignant), Plasmodium vivax or Plasmodium ovale (benign)
• quartan fever : fervescence every 3 days, such as Plasmodium malariae
• recurrent fever : recurrent paroxysmal fever occurring in various diseases, including borreliosis, trepanomatoses, tularemia, meningococcemia, malaria, and rat-bite fever.
• ephemeral fever : a slight fever persisting or lasting only a day or two.
• hectic fever : a fever that recurs each day, with profound sweating, chills, and facial flushing.
• defervescence : the abatement of fever.
• defervescent stage / stadium decrementi : the period of decrease in severity of a disease or fever
• defervescence by crisis : gradually
• sweating stage : the final stage of a malarial paroxysm, marked by sweating
• defervescence by lysis : rapidly
• hyperpyrexia : fever > 40°C
Associated symptoms & signs :
• asthenic fever : a fever with nervous depression, feeble pulse, and a cool, moist skin.
• sthenic fever : fever characterized by a full, strong pulse, hot and dry skin, high temperature, thirst, and active delirium.
• eruptive or exanthematous fever : any fever accompanied by an eruption on the skin during the eruptive stage / stadium fluorescentiae
• spotted or boutonneuse fever : a febrile disease typically characterized by a skin eruption, such as typhus, epidemic cerebral meningitis, and the infections caused by tick-borne rickettsiae (Rocky Mountain spotted fever, boutonneuse fever, and others)
• systemic inflammation induces various adaptive responses including tachycardia. Although inflammation-associated tachycardia has been thought to result from increased sympathetic discharge caused by inflammatory signals of the immune system, definitive proof has been lacking. The TXA₂analog I-BOP and PGF_2a each increased the beating rate of the isolated atrium of wild-type mice in vitro through interaction with TP and FP receptors, respectively. The cytokine-induced increase in beating rate was markedly inhibited in atria from mice lacking either TP or FP receptors. The tachycardia induced in wild-type mice by injection of lipopolysaccharide (LPS) was greatly attenuated in TP-deficient or FP-deficient mice and was completely absent in mice lacking both TP and FP. The b-blocker propranolol did not block the LPS-induced increase in heart rate in wild-type animals. Inflammatory tachycardia is caused by a direct action on the heart of TXA₂ and PGF_2a formed under systemic inflammatory conditions^ref.
Laboratory examinations :
• phenylbutazone test : fever disappears only if due to tumor pyrogens, not to infectious agents. no longer used due to risk to induce peptic ulcers^{ref1, ref2}.
• Naprosyn test (naproxen 375 mg p.o. every 12 hours for 3 days) was used to differentiate neoplastic from infectious causes of FUO^ref
Therapy : antipyretic drugs : COX-2 inhibitors and mPGES-1 inhibitors
Web resources : Institute for Inflammation Research
• (post-flogistic) repair
• oxygen paradox / ischemia-reperfusion injury (IRI) : adverse effects of the restoration of blood flow following an ischemic episode, including cellular swelling and necrosis, edema, hemorrhage, the no-reflow phenomenon, and tissue damage by free oxygen radicals

Pathogenesis : xanthine dehydrogenase (XDH) =reversible sulfhydryl oxidation or irreversible proteolytic modification by calpain=> xanthine oxidase (XO), which catalyzes
• hypoxanthine + O₂ => uric acid + O₂^-
• O₂^- + NO^. => ONOO^- => OH
It occurs after reperfusion in :
• acute myocardial infarction (AMI) : the progression of injury, as reflected by annexin-V–OG binding, begins minutes after reperfusion and reaches a maximum in < 30 minutes, suggesting that apoptosis occurs primarily during reperfusion rather than during ischemia.
• stroke and transient focal cerebral ischemia (FCI)
• abdominal decompression for treatment of abdominal compartment syndrome (ACS)^ref
• pulmonary infarction
• renal infarction
• tissue transplantation
• Raynaud's phenomenon
• intermittent perfusion of the placenta, secondary to reduced trophoblast invasion, causes increased secretion of TNF-a, and that this contributes to the activation of maternal endothelial cells that characterizes preeclampsia^ref
Preadministration of N-acetylcysteine (NAC) has been found to attenuate the cerebral IRI in a rat model of experimental stroke by reducing expression of proinflammatory cytokines such as TNF-a, IL-1b and iNOS, activated macrophage/microglia (ED1) and apoptotic cell death^ref. TNF appears to mediate both the early and late phases of liver IRI, but it also is an essential mediator of hepatoprotective effects brought about by ischemic preconditioning^ref. IL-12 and IL-18 cause hepatic IRI in mice by suppressing anti-inflammatory cytokine expression and facilitating neutrophil-dependent^ref. Proteasomes were found to be targets of IRI of the brain and heart. Proteasome activity increased in liver models of IRI. CCL3 / MIP-1a^ref is functionally significant in the development of lung IRI (LIRI) inducing PMN leukocyte accumulation into the interstitium => neutrophil-dependent injury. B cell-deficient (mu MT) mice are protected from renal IRI : to identify the contribution of cellular vs soluble mechanism of action, serum transfer into mu MT mice partially restored ischemic phenotype, but B cell transfers did not^ref. Recent studies indicate an important role for clonally specific natural IgM antibody from the B1 lymphocyte repertoire (and probably autoantibodies present in sera of patients with systemic autoimmune disease) that activate the classical pathway of complement^ref. Recent data have demonstrated a role for CD4⁺ cells in the pathogenesis of renal IRI. Hypoxia-mediated induction/activation of the transcription factor EGR-1 has a pivotal role in lung IRI : induction of tissue factor (TF) gene expression leads to fibrin accumulation^ref. The extent of myocardial tissue damage resulting from IRI is directly related to the extent of the inflammatory response attributed, in part, to leukocyte recruitment. The inhibitory effects of corticosteroids on endothelial-leukocyte adhesion have been shown to be mediated by GR^ref through a NO-dependent mechanism via non-transcriptional activation of eNOS / NOS3^ref : because NO also has vasodilatory^ref, anti-thrombotic^ref and anti-proliferative properties^ref, the beneficial effects of corticosteroids in cardiovascular disease may extend beyond their anti-inflammatory properties. But NO + O₂^- => ONOO^- which damages cell contractility, lipids, proteins, nucleic acids, sugars, MnSOD, aconitase and TIMP => uncontrolled activation of MMPs (despite being extracellular, MMP2 can degrade TnI and TnT, but not TnC). On reperfusion or reoxygenation it is well-documented that there is an oxygen-dependent increase in total cell calcium^{ref1, ref2, ref3} which is accompanied by an increase in total mitochondrial calcium. A number of studies have shown that reoxygention is also associated with impairment of mitochondrial function^{ref1, ref2} and ultimately cell lysis in intact hearts^ref.
Prevention : pretreatment with ...
• xanthine dehydrogenase (XDH) inhibitors
• SOD
• iron chelators
• proteasome inhibition prevented IRI of the brain, heart and kidney. A major mechanism by which proteasome inhibitors may confer tissue protection is inactivation of NFkB resulting in a block of expression of cytokines and cell adhesion molecules during the reperfusion phase^ref.
• PPAR-g agonists have an anti-inflammatory effect by preventing the activation of transcription factors such as NFkB^ref
• beneficial effects of H₂ antagonists in a rat model of renal IRI are partly mediated by decreased IL-6 and VEGF mRNA expression and significant early increase in renal VEGF abundance^ref
• cardiac protection provided by treatment with a selective Rho-kinase (Rock) inhibitor is likely via anti-apoptotic effect and attenuation of ischemia/reperfusion-induced inflammatory responses^ref
• high-dose corticosteroid therapy protects the myocardium from ischemic injury^{ref1, ref2} : however, the subsequent development of cardiac rupture, which has been attributed to the genomic inhibitory effects of GR on wound healing and cardiomyocyte remodeling^ref, has limited its use of corticosteroids in AMI^ref
• NADPH oxidase-derived O₂^- plays an important role in the pathophysiology of reduced size liver combined with ischemia and reperfusion (RSL+I/R)-induced liver injury via its ability to enhance expression of TNF-a. Therapies directed toward scavenging of O₂^-, inhibiting NADPH oxidase, and/or immuno-neutralizing TNF-a may prove useful in limiting the liver injury induced by surgical procedures that require resection and I/R such as split liver or living donor liver transplantation^ref
• MMPs inhibitors (doxycycline^{ref1, ref2, ref3, ref4, ref5}, minocycline^ref)
• quinolones ?
• K_ATP channel activators preserve capability for rapid restoration of cationic/energetic status during reperfusion^{ref1, ref2, ref3, ref4, ref5}
• endothelin-converting enzyme inhibitor (FR901533) in hepatic IRI^ref
• remnants removal : as inflammation subsides, the concentration of cytokines and chemokines diminishes, resulting in a relative deficiency of survival factors for the recruited cell populations still present in the area. Increased competition for a diminishing supply of survival factors leads to a failure to inhibit default pathway of apoptosis and, consequently, increased cell death. Rapid clearance of these apoptotic cells is essential to prevent leakage of their intracellular contents and generation of a new inflammatory focus. Professional phagocytic cells such as macrophages  have a cytokine-independent survival advantage conferred by binding and/or uptake of apoptotic cells, which leads to activation of Akt (=> inhibited default apoptotic pathway) and inhibition of ERK1 and ERK2 (=> inhibited proliferation, on the contrary of necrotic cells and danger signals).
• recovery
• relative recovery : healing or connectival reintegration (organization of the exudate or of the granulation tissue)
• wound healing : a process of cure; the restoration of integrity to injured tissue.
• healing by first intention / primary adhesion : healing in which union or restoration of continuity occurs directly without the intervention of granulations, leaving no functional menomation. It occurs only when there isn't an excessive bacterial contaminations, if tissues are vital and if are not present foreign bodies or hematomas (they facilitate bacterial growth and represent a mechanical barrier between repairing tissues)
• healing by second intention (per secundam intentionem ) / healing by granulation / secondary adhesion : union by spontaneous closure of a wound with granulations which form from the base and both sides toward the surface of the wound, with loss of substance, slower than healing by first intention.
• healing by third intention/ delayed closure : wound is deliberately left open to allow maintenance of a drainage , and then surgically closed
• healing under crust : crust allows protection from exogenous germs, is a source of nutrition for tissues and is the support for the new connective tissue. Healing under crust is dangerous if infection occurs
• healing under wet clot :
• granulation :
• epithelization / epithelialization : healing by the growth of epithelium over a denuded surface, including neoangiogenesis
• desmoplasia / fibroplasia : the formation and development of fibrous tissue, as occurs normally in the healing of wounds and abnormally in some tissues
• resolution : the subsidence of a pathologic state, as the subsidence of an inflammation, or the softening and disappearance of a swelling
• vasal regression
• collagen remodeling : reorganization or renovation of an old structure, contraction of collagen fibers makes wound borders nearer
Factors affecting cicatrization :
• nutrition (hypoproteinemia, vitamin A, C, B₂, and B₆deficiencies)
• oxygen (diabetes mellitus, obesity, corticosteroids, radiation therapy, inspection)
• temperature : despite the habit of having cold operating theatres and cooled patients (cold patients would undergo vasoconstriction, and anesthesia with cold gases would impair mucociliary elevator allowing infection), this impairs the work of the surgeons
• modulation : the normal capacity of cell adaptability to its environment
• metaplasia : the change in the type of adult cells in a tissue to another form of adult cells that are not normal for that tissue.
• cytometaplasia : alteration in the form or function of a cell
• epithelial metaplasia
• goblet metaplasia of
• simple cylindric bronchial epithelial cells in chronic bronchitis
• pseudolayered cystic epithelial cells in chronic cystitis
• Clara's cells in small airways disease
• squamous metaplasia : the transformation of pseudostratified ciliated epithelium into stratified squamous epithelium, as occurs in certain pathologic conditions or may be produced experimentally.
• simple cylindric esophageal epithelial cells in chronic esophagitis
• simple cylindric bronchial epithelial cells in chronic bronchitis
• simple cylindric endometrial cells in chronic cervicitis
• mesenchymal metaplasia
• bony metaplasia of ...
• sesamoid cartilages in trachea
• costal cartilages in elderlies
• oncocytic (oxyphil) metaplasia of ...
• thyrocytes in Hashimoto's thyroiditis^ref
• adrenal cortex secondary to metyrapone therapy
• denture hyperplasia
• melanocytic nevi
• papillary cystic type of acinic cell carcinoma of parotid
• absolute recovery : resolution / parenchymal regeneration/ restitutio ad integrum : an active process of full restoration
• basal cell hyperplasia (BCH)
Recovery in specific organs :
• in pneumonitistype II pneumocyte hyperplasia and cuboid metaplasias replace type I pneumocytes during reparative processes, so impairing diffusion of gases (alveolar bronchiolization or bronchiolarization). MMP-9 / gelatinase B is required for alveolar bronchiolization, perhaps by facilitating migration of Clara cells and other bronchiolar cells into the regions of alveolar injury
• in encephalitis, hypoxic or hypoglycemic episodes in the CNS, repair to the destruction of nearby neurons during a depends on :
• microglia
• astrocytes => gliosis / astrocytosis
Pathogenesis => apoplectic or post-hemorrhagic pseudocysts
•  adaptive immune response
• primary adaptive immune response
• secondary adaptive immune response
• immunopathological processes
• hypersensitivities / hypersensitivity reactions (HSR)(i.e. excessive or inappropriate secondary immune response). Coombs-Gell classification :
• Ab-mediated or immediated hypersensitivities
• type I hypersensitivity / allergy :

Aetiology : the incidence of type I hypersensitivities has rocketed in recent years in Western countries, and the explanation for this has been the subject of much debate. Children are born with a T_h2-biased immune system, but this bias normally diminishes during the first 2 years of life in non-allergic individuals.
• the hygiene hypothesis argues that early childhood infections provide a T_h1 stimulus for the T_h2-skewed immune system of humans. The theory is that in Western countries, the reduction of childhood infections means that the immune system lacks this T_h1 stimulus and continues to maintain a T_h2 bias, which results in an increased risk of developing type I hypersensitivities. The very low incidence of type I hypersensitivities in developing countries where helminth infection - which also result in a T_h2-biased immune response - is endemic is explained by the fact that helminth infection protect against the development of type I hypersensitivities by the production of regulatory cytokines, such as IL-10. Epidemiological studies, however, indicate that Th2 cell-stimulating helminth parasites may also counteract allergies, possibly by generating regulatory T cells which suppress both Th1 and Th2 arms of immunity and anyway the route of exposure is actually more important than the T_h polarization :
• respiratory influenzavirus A infection and IFN-g produced during the acute T_h1 response can positively regulate T_h2-dependent allergic pulmonary disease after viral clearance in animals 6 to 8 weeks old, at least in part, by altering pulmonary DC function^ref. If you would do the same with, say, a gastrointestinal virus, like HAV, you would probably see a different response : furthermore influenza virus infection may have different effects from RSV or rhinoviruses, much more common in children
• the T_h2 cell-inducing gastrointestinal nematode Heligmosomoides polygyrus influences experimentally induced airway allergy to ovalbumin and the house dust mite allergen Der p 1. Inflammatory cell infiltrates in the lung were suppressed in infected mice compared with uninfected controls. Suppression was reversed in mice treated with antibodies to CD25. Most notably, suppression was transferable with mesenteric lymph node cells (MLNC) from infected animals to uninfected sensitized mice, demonstrating that the effector phase was targeted. MLNC from infected animals contained elevated numbers of CD4⁺25⁺Foxp3⁺ T cells, higher TGF-b expression, and produced strong IL-10 responses to parasite antigen. However, MLNC from IL-10-deficient animals transferred suppression to sensitized hosts, indicating that IL-10 is not the primary modulator of the allergic response. Suppression was associated with CD4⁺ T cells from MLNC, with the CD4⁺25⁺ marker defining the most active population. These data support the contention that helminth infections elicit a regulatory T cell population able to down-regulate allergen induced lung pathology in vivo^ref

• lack of HAV infection in individuals with longer 157insMTTTVP isoform of T-cell immunoglobulin domain, mucin domain 1 (TIM-1) / HAV cellular receptor 1 (HAV-cr1) / kidney injury molecule-1 (KIM-1) (which helps the virus enter the cell more efficiently and is also expressed by CD4⁺ T cells during T_h2 differentiation; present in 66% of white and blacks and 50% of Asians) renders them more likely to develop asthma and eczema. Exposure to HAV is associated with poor hygiene, large family size and attendance at day-care centres, all factors that are also inversely associated with atopy. Antibodies that mimic the immune-boosting effects of HAV without causing the disease itself or vaccination - which promotes antibody formation - may also help to prevent or even treat allergic diseases^ref.
• perturbations in the gastrointestinal (GI) microbiota composition that occur as a result of antibacterials and diet in "westernized" countries are strongly associated with allergies^ref. Antibiotics could upset the natural balance of the microbial community in the gut, causing the population of one of the gut's natural inhabitants, the yeast Candida albicans, to explode. Both PGE₂ and TxB₂ significantly enhanced serum-induced germination by C. albicans. Since these molecules also bind to albumin, this may also explain the hyphal transforming activity in serum that associates with albumin. Interestingly, short chain fatty acids (butyric acid), the product of lactic acid bacteria (LAB), inhibited germination. In addition, LAB culture supernatants as well as live LAB also inhibited C. albicans morphogenesis^ref. The yeast makes prostaglandin-like immune response modulators that can modulate immune responses and make the immune system overly sensitive. A broad range of pathogenic eukaryotic microbes (fungi^{ref1, ref2}, protozoa, and helminths) produce eicosanoids and other oxylipins by novel synthesis pathways. Why do these organisms produce oxylipins? Accumulating data suggest that phase change and differentiation in these organisms are controlled by oxylipins, including prostaglandins and lipoxygenase products. The precise role of pathogen-derived eicosanoids in pathogenesis remains to be determined, but the potential link between pathogen eicosanoids and the development of T_h2 responses in the host is intriguing. Thus, eicosanoids and oxylipins (host or microbe) may be mediators of a direct host-pathogen "cross-talk" that promotes chronic infection and hypersensitivity disease, common features of infection by eukaryotic pathogens^ref. In earlier experiments, the team had shown that these animals' lungs, unlike those of mice that had received neither the antibiotics nor the fungi, produce a moderate allergic reaction to mold. A new study shows that the treated mice also develop allergies to another substance, egg white protein. C57BL/6 mice are treated for 5 days with cefoperazone in the drinking water, followed by a single oral gavage of C. albicans. This results in alterations of GI bacterial populations and increased yeast numbers in the GI microbiota for at least 2 to 3 weeks and can drive the development of a CD4⁺ T-cell-mediated allergic airway response to subsequent mold spore (Aspergillus fumigatus) exposure in immunocompetent mice without previous systemic antigen priming. The allergic response in the lungs is characterized by increased levels of eosinophils, mast cells, IL-5, IL-13, IFN-g, IgE, and mucus-secreting cells. In the absence of antibiotics, mice exposed to Aspergillus spores do not develop an allergic response in the airways. This study provides the first experimental evidence to support a role for antibiotics and fungal microbiota in promoting the development of allergic airway disease. In addition, these studies also highlight the concept that events in distal mucosal sites such as the GI tract can play an important role in regulating immune responses in the lungs^{ref1, ref2}. To investigate the role of host genetics, BALB/c mice were tested. As with C57BL/6 mice, microbiota disruption promoted the development of an allergic response in the lungs of BALB/c mice upon subsequent challenge with mold spores. In addition, this allergic response required IL-13 (the response was absent in IL-13^–/– mice). To investigate the role of antigen, we subjected mice with disrupted microbiota to intranasal challenge with ovalbumin (OVA). In the absence of systemic priming, only mice with altered microbiota developed airway allergic responses to OVA. The studies presented here demonstrate that the effects of microbiota disruption are largely independent of host genetics and the nature of the antigen and that IL-13 is required for the airway allergic response that follows microbiota disruption^ref.
• susceptibility loci : MHC class II, FceRII, IL-4, calcium-binding atopy-related autoantigen 1, PHD finger protein 11 (PHF11), IL-13+2044G=>A is expected to result in the nonconservative replacement of arginine 130 (R130) with glutamine (Q)^ref
• maternal total IgE levels correlate with cord blood and infant IgE levels and the development of infant eczema (specificity = 83%; sensitivity = 34%)
• traffic-related air pollution is statistically connected with enhanced allergies in the population : a combination of NOx and ozone can nitrate tyrosine, an amino acid present in all proteins, which may then go on to trigger an allergic response. Roadside dust and airborne particulate matter (PM₁₀ and PM_2.5), including pollen, contain up to 5% of proteins. Paved road dust present on the surface of streets in Southern California consists of a complex mixture of soil dust, deposited motor vehicle exhaust particles, tire dust, brake lining wear dust, plant fragments, and other biological materials. The research presented here shows that allergens from at least 20 different source materials are found in the paved road dust. These include pollens and pollen fragments, animal dander, and molds. When paved road dust is resuspended into the atmosphere by passing vehicle traffic, allergen concentrations in the air are increased above the levels that would prevail without the vehicle traffic. Using immunological assays that measure the proteins extracted from environmental samples that bind to IgE antibodies present in the blood serum of allergenic patients, it is possible to measure the allergen concentrations present in paved road dust and in airborne particle samples. Total protein contributions to monthly average airborne TSP and PM₁₀ concentrations are found to be in the range from 1 to 5.8 mg/m³, potentially accounting for a significant fraction of the airborne particulate organic material that has not been identified to date by GC/MS techniques. Results show that up to 5-12% of the allergenicity of atmospheric total suspended particulate matter samples at Long Beach and Rubidoux, CA, is attributable to paved road dust emissions. In an industrial area of urban central Los Angeles where there is less proximity to vegetation and domestic activities, the paved road dust contribution to airborne allergen concentrations is lower, accounting for approximately 0.5% of the total allergenic activity of the atmospheric particle samples. In conclusion, paved road dust when entrained into the atmosphere by passing traffic is a source of allergen exposure for the general population and could be more important in areas with more abundant vegetation or with closer proximity of populations to major highways than is the case for the Southern California air monitoring sites studied here^ref. Research from as far back as the 1930s points to nitro derivatives, particularly those of proteins, as strong immune stimulators. High concentrations of NOx and ozone under “summer smog” conditions—heavily polluted, hot, humid air—could nitrate the tyrosine residues in proteins. These modified proteins might then induce inflammation and allergy after inhalation. A reference protein and a birch pollen extract containing the prominent allergen Bet v 1 (which has 7 Tyr residues) were exposed to parts-per-billion (ppb) concentrations of NO₂ and ozone mixtures : both proteins were substantially nitrated after 30 hours at 100 ppb, a level found under low-smog conditions, and more affected after 30 hours at 200 ppb, which is reached during severe summer smog alerts. Tyrosine was also nitrated when the proteins were exposed to ambient air collected from Munich’s heavily trafficked intersections. Enzyme immunoassays have been used to determine equivalent degrees of nitration (EDN) for protein samples exposed to urban outdoor air and synthetic gas mixtures. The observed rates of nitration were governed by the abundance of nitrogen oxides and ozone, and concentration levels typical for summer smog conditions led to substantial nitration within a few hours to days (EDN up to 20%). Moreover, nitrated proteins were detected in urban road dust, window dust, and fine air particulate matter (EDN up to 0.1%), but for allergenic proteins from birch pollen left at a busy Munich road junction for a few days, that figure rises to 10%. And for allergenic proteins exposed to smog in the laboratory, nitration rose to 20%^ref. Previous research has shown that nitrated proteins bind more strongly to IgE antibodies. Preliminary results suggest that the nitrated proteins are significantly more active at boosting allergies by giving normal and nitrated proteins to mice. Proteins that are easiest to nitrate might trigger the strongest allergic reaction. It's not clear how nitrate groups increase the allergic response, but previous studies have suggested that the body uses tyrosine nitration as a marker to attract antibodies to inflamed tissue. The finding could help to develop drugs that stop nitrated proteins disrupting our immune systems. Scientists are still unsure what makes a protein allergenic, and nitration is unlikely to be the only factor. Others have suggested that traffic pollution could make urban trees emit more allergens.
During the primary immune response in predisposed individuals :
• epithelial cells produce TSLP : TSLP expression was increased in the lungs of mice with antigen-induced asthma, whereas TSLP receptor-deficient mice had considerably attenuated disease. Lung-specific expression of a Tslp transgene induced airway inflammation and hyperreactivity characterized by T_h2 cytokines and increased IgE. The lungs of Tslp-transgenic mice showed massive infiltration of leukocytes, goblet cell hyperplasia and subepithelial fibrosis. TSLP was capable of activating BMDCs to upregulate costimulatory molecules and produce the T_h2 cell-attracting chemokine CCL17. These findings suggest that TSLP is an important factor necessary and sufficient for the initiation of allergic airway inflammation^ref
• DC activation
• DCs capture antigens in mucosal surfaces and travel to regional lymph nodes
• polarization of T_h0 cells toward T_h2 cytokine secretion profile by directly producing IL-4 or by inducing IL-4 production by NK cells
• T_h2 cells induce class swith recombination (CSR) in plasma cells up to IgEs / reagins against the allergen(s) (usually it is an enzyme with 10 < M_r < 20 kDa, acting as an immunogen or as an hapten that use host molecules as immunogenic carriers)
• IgEs bind to FceRI on mast cells and basophils (up to 5^.10⁵ IgE / _cell : sensitization).



At the second challenge ...

• allergen binding to cell-bound IgEs
• anaphylotoxins
• C3a
• C3a-desArg
• C5a
... induce mast cells and basophils to undergo exocytosis of granules (degranulation : early reaction (within seconds); amplification mechanism => requires mg of Ag) containing :
• histamine : it causes its biological effects in 1-2' (mantaining activity up to 10') acting on ...
• H₁ :
• contraction of smooth muscle cells of bronchi => bronchospasmus, small bowel (abdominal colics) and uterus (Schultz-Dale reaction : an in vitro test for local anaphylactic sensitization in the guinea pig: a small amount of the antigen added to a tissue bath in which is immersed the excised uterine horn (smooth muscle) of an anaphylactically sensitized guinea pig causes contraction of the sensitized uterine muscle. Schultz employed intestinal muscle; Dale used uterine muscle from virgin guinea pigs)
• dilatation of small veins =>
• oedema
• erythema (allergic angioflogosis : increased blood flow and blood vessels permeability)
• hypotension
• axonic reflex (induction of substance P exocytosis by antidromic conduction in sensitive fibers)
• negative inotropic and dromotropic effect on ventricular and left atrial cardiomyocytes at doses < 10^-8 M
• H₂ :
• hypersecretion of H⁺Cl^- by parietal cells of stomach
• positive inotropic and chronotropic effects on ventricular and right atrial cardiomyocytes at doses > 10^-8 M
• H₃ :
• inhibition of neural norepinephrine release from orthosympathetic terminals of myocardium
• heparin activates antithrombin III acting as an anticoagulant
• tryptases
Further they produce and release :
• LTB₄ : chemotaxin for granulocytes and macrophages
• SRS-A : smooth muscle cells contraction
• PGD₂
• TNF-a
• PAF
These cause infiltrations of activated T_h2 lymphocytes, activated eosinophils, basophils and some neutrophils (late phase reaction) 5 to 12 hours after exposure to antigen, after the wheal and flare reactions of immediate hypersensitivity have diminished; it is characterized by pruritis and minor cellular infiltration. Prostaglandins are not formed. Inflammation subsides after reaching a maximum around 24 hours after exposure.
Activated mast cells, basophils and eosinophils up regulate CD40L / CD154 expression and produce IL-4, allowing a positive feeedback for a T_h2-polarized immune response.
PGD₂ and PGE₂ are produced during allergic reactions. Although PGD₂ is an important mediator of allergic responses, aspirin-like drugs that inhibit prostaglandin synthesis are generally ineffective in allergic disorders, suggesting that another prostaglandin-mediated pathway prevents the development of allergic reactions : such a pathway may be mediated by PGE₂ acting at the PGE₂ receptor EP₃. Mice lacking EP₃ developed allergic inflammation that was much more pronounced than that in wild-type mice or mice deficient in other EP subtypes. Conversely, an EP₃-selective agonist suppressed the inflammation. This suppression was effective when the agonist was administered 3 h after antigen challenge and was associated with inhibition of allergy-related gene expression. Thus, the PGE₂-EP₃ pathway is an important negative modulator of allergic reactions^ref.
The reaction may be :
• local (atopy), if the allergen remains localized at entry door, which may be :
• epidermis => dermis => wheal and erythema or wheal and flare reaction : the characteristic local cutaneous reaction consisting of an elevated, blanched wheal surrounded by a spreading “flare” of erythema that occurs within a few minutes at the site of a minor nonpenetrating skin injury and also in response to administration of allergen to an atopic individual; it is caused by release of histamine from mast cells : atopic dermatitis, angioedema and/or acute urticaria, rarely persisting for > 48 hrs
• respiratory tract =>
• increased nasal secretions (allergic rhinitis)
• bronchostenosis (bronchial asthma) => dyspnea
• conjunctiva => allergic conjunctivitis
• gastro-intestinal tract =>
• irritable bowel syndrome (IBS) : hyperperistalsis and increased secretions => diarrhoea, abdominal pain
• nausea and vomiting
• systemic (anaphylaxis), if the allergen enters the bloodstream (directly by intravenous injection or indirectly, mainly by parenteral routes, rarely by oral route) : symptoms are those of more than a kind of atopy, plus systemic arterial hypotension => anaphylactic shock (AS) => syncope => death. The target organs of guinea pigs and humans are the bronchi of the lungs. In dogs the target organ is the liver, which may accumulate up to 60% of the animal's blood. The target organs of the rabbit are the heart and lungs. Anaphylactic shock is estimated to affect up to 15% of the US population. Previous research in mice, which have a similar immune system to humans, had hinted that extreme amounts of nitric oxide (NO) throughout the body might be responsible. By injecting nitric-oxide blockers into some of the mice before attempting to give them anaphylactic shock, the researchers were able to confirm that nitric oxide produced by eNOS was indeed the culprit^ref. Mice genetically engineered to lack eNOS were immune to potentially fatal allergens, whereas mice missing iNOS fell victim to them. eNOS was always presumed innocent. Doctors could potentially use nitric-oxide blockers to help treat the condition.Mice injected eNOS inhibitors before trying to induce a shock experienced just a brief drop in blood pressure followed by a quick recovery. Unfortunately, these types of drugs are very slow-acting against anaphylaxis as they need time to accumulate in the body, so would presumably not do much good as an emergency response. But a patient sensitive to penicillin, anaesthetics, or latex could be pre-loaded with these drugs before an operation
• food-dependent exercise-induced anaphylaxis (FDEIA) : initially described in 1979^ref, FDEIA is an important and underrecognized cause of seemingly unprovoked anaphylaxis. Ingestion of particular food antigens (typically wheat), followed by exercise, triggers anaphylaxis within hours in specific FDEIA. In the nonspecific form, however, plain physical exertion on a full stomach suffices^ref. A putative mechanism is exercise-related mesenteric ischemia, leading to increased absorption of food antigens^ref. Acetylsalicylic acid is known to aggravate symptoms^ref. Pruritus, hives, or angioedema is present in more than 90% of cases and is often accompanied by upper or lower airway symptoms^{ref1, ref2}
Type I hypersensitivity can be transferred via IgE contained in transfused serum (Prausnitz-Küstner (P-K) reaction) or transplanted organs (expecially liver).
• Hektoen phenomenon : when antigens are introduced into the animal body in allergic states, there may exist an increased range of new antibody production which may include production of antibodies concerned in previous infections and immunizations.
• parallergy : a condition in which an allergic state, produced by specific sensitization, predisposes the body to react to other allergens with clinical manifestations that differ from the original reaction
• pseudo-allergic reactions (if at systemic level : anaphylactoid reaction) : same late pathogenesis and hence same symptoms of type I hypersensitivity can be caused by same mediators released via IgE-independent mechanisms.
• complement-mediated hypersensitivities have no amplification mechanism => require mg of Ag
• type II hypersensitivity : molecular mimicry cause Ab to cross-react with self Ags on host cells, resulting in cell killing by :
• immune or opsonic phagocytosis
• induction of the classical activation pathway for complement cascade followed by the lytic pathway.
• ADCC
• type III hypersensitivity / immune complex disease

Deposition of IgG- or IgM-containing immunocomplexes on :
• basal laminae of renal tubules (=> glomerulonephritis)
• joints
• choroid plexuses
• small blood vessels
... causes :
• complement activation => generation of
• C3b, which may
• attach to innocent bystander cells which may lyse due to formation of the MAC
• cause platelet aggregation =>
• release vasoactive amines contributing to edema
• microthrombi => ischemia
• anaphylotoxins => mast cell activation => edema
• C5a => attracts neutrophils which try to phagocyte iccosomes but they undergo autolysis releasing proinflammatory enzymes that local damage
• ischemia
Varieties :
• with Ab excess (Arthus phenomenon or reaction) : the development of an inflammatory lesion, with induration, erythema, edema, hemorrhage, and necrosis, a few hours after intradermal / subcutaneous injection of antigen into a previously sensitized animal producing precipitating antibody (repeated Ag injections for a long period induce high Ab titre). The lesion results from the precipitation of antigen-antibody complexes, which causes complement activation and the release of complement fragments that are chemotactic for neutrophils; large numbers of neutrophils infiltrate the site and cause tissue destruction by release of lysosomal enzymes. Symptomatology peaks within 3÷8 hrs. Sometimes it is used as an experimental method in immunological investigations :
• passive cutaneous Arthus reaction : a variation of the Arthus reaction in which a nonimmune host is inoculated with large quantities of precipitating antibody intravenously, followed by local cutaneous inoculation of antigen.
• reverse passive Arthus reaction : the reaction produced when precipitating antibody is inoculated into a skin site in an experimental animal followed in 30 minutes to 2 hours by inoculation of the homologous antigen, either intravenously or intracutaneously at the same site. Thus the usual anatomical locations of precipitating antibody and antigen in an Arthus reaction are reversed.
• with Ag excess after massive inhalation or parenteral injection of Ag. 8 to 12 days following treatment with the heterologous Ag(s), the individual experiences generalized swelling of the lymph nodes, with itching, urticaria or erythmatous eruptions : following the episode, the complexes are removed in about 2 days and the reaction subsides.
Thermal phenomena :
• cryoglobulins are Igs that undergo precipitation in the cold (standard procedure : > 48 hr at + 4°C) due to high content of hydrophobic amino acids, and can be resolubilized by heating at 37°C.

Epidemiology : prevelence = 2-3% after age 60 years
Brouet immunochemical classification :
• type I cryoglobulins (5-15%) : monoclonal IgM >> IgG > IgA > light chain from secreting B cell lymphomas (malignant paraproteinemia : immunocytoma/Waldenström macroglobulinemia, multiple myeloma). Monoclonal cryoglobulinemia is frequently asymptomatic. Symptoms are rare. When present, clinical manifestations derive from hemorheologic disturbances and include acrocyanosis, Raynaud phenomena and gangrene.
• mixed cryoglobulins : rheumatoid factors (RF) occurring in ...
• essential mixed cryoglobulinemia (EMC)
• systemic lupus erythematosus (SLE) (lupus glomerulonephritis)
• rheumatoid arthritis (RA)
• infectious diseases
• type II cryoglobulins (50-60%) : monoclonal rheumatoid factors (RF) (IgMk >> IgG) which form cryoprecipitating circulating immune complexes with polyclonal intact IgG and the F(ab')₂ fragment, as well as with the Fc fragment of autologous IgG. Symptoms only in 10%
• type II/III / oligoclonal or microheterogeneous type : oligoclonal IgM which form cryoprecipitating circulating immune complexes with polyclonal IgG. Characterized using more sensitive methods (immunoblotting, 2D-PAGE), defined by > 2 heterogeneous bands of H chains (oligoclonal IgMs or a mixture of polyclonal and monoclonal IgMs). The oligoclonal type seems to be an intermediate stage in the course of type III evolving to type II MC. In fact, during chronic active HCV infection MC may change its immunochemical type: type II MC is more stable over time than type III and oligoclonal^ref
• type III cryoglobulins (30-40%) : polyclonal rheumatoid factors (RF) which form cryoprecipitating circulating immune complexes with polyclonal IgG. Symptoms only in 10-15%
Symptoms & signs :
• palpable purpura in lower limbs (90-100%)
• Raynaud's phenomenon, subungueal hemorrhages, pustular hemorrhagic folliculitis, erythema multiforme, livedo reticularis, urticaria and ulcers (10-35%)
• intermittent arthralgias (50-95%)
• renal involvement (10-55%)
• peripheral sensitive or sensitivie-motor neuropathy
• hepatomegaly (70%)
• splenomegaly (50%)
• lymphadenomegaly (5-15%)
• Sjogren syndrome (3-40%)
• pulmonary involvement is rare
Laboratory examinations :
• blood samples are stored at 37°C until complete clotting occurs and are then centrifuged. The serum is kept at 4°C for 3-4 days. If a cryoprecipitate is visible, the sample is centrifuged at 4°C for 15 minutes to measure a cryocrit. Cryo-globulins are purified by washing at 4°C and characterized using immunofixation electrophoresis
• low C4 (90%)
• transaminitis (30%)
• proteinuria and microhematuria (50%)
• pyroglobulins (pyroglobulinemia)^ref : first described in 1954^ref, heating of the serum at 56°C for 30 min induced an irreversible gel (pyrogel)  formation that fails to redissolve on heating to 100°C; for IgM, observed with pentamers as well as monomers, but not with separated H or L chains. Amino acid analysis showed quantitative abnormalities of aspartic acid, glycine, cystine and leucine within the H chains. Solubility of the pyrogel in sodium dodecyl sulfate, the pyroglobulin's tendency to aggregate and the cystine deficit of H chains implicate conformational changes leading to hydrophobic bonding at 56degreesC in the formation of pyrogel^ref
• rubella virus^ref
• Sjogren syndrome (IgA)^ref
• paraproteinemia^ref
• ovarian lymphoma (IgMl)^ref
• systemic primary amyloidosis^ref
• k light-chain disease associated with cryoglobulinemia, pyroglobulinemia, and hyperviscosity syndrome^ref
• Waldenstrom's macroglobulinemia^{ref1, ref2}
• multiple myeloma^{ref1, ref2, ref3}
• MM and plasma cell leukemia with pyroglobulinemia and cryoglobulinemia^ref
• IgAl^{ref1, ref2} and k^ref
• IgGk^ref
• IgEk pyroglobulinemia^ref
• IgDl MM^ref and plasma cell leukemia associated with pyroglobulinemia and l pyroglobulinuria^{ref1, ref2}
• type IV or delayed-type hypersensitivity (DTH)
• during primary immune response B-1 B cells produce IgM
• complement activation by subunit or pentameric IgM generates C5a
• C5a binds C5aR on ...
• ... mast cells, causing release of the vasoactive serotonin and TNF-a
• ... platelets, causing release of the vasoactive amine serotonin
Both molecules induce expression of addressins on the luminal surface of local endothelium
• T cell recruitment into the tissues
• specific Ag/MHC complexes expressed on local APC drive CD4⁺ T_h1 polarization
• production of proinflammatory cytokines, such as IFN-g, to mediate classical late DTH. Granuloma appears gradually 10 hours after injection, with maximal intensity and size occurring between 24 and 72 hours (48 hours average) : this time is required to recruit CD4⁺ T_h1, CD8⁺ T_c1, and macrophages.
DTH responses are downregulated by T_h2 cells. Mice devoid of T cells and B cells demonstrated substantial contact hypersensitivity responses to 2,4-dinitrofluorobenzene and oxazolone. Those responses were adaptive in nature, as they persisted for at least 4 weeks and were elicited only by haptens to which mice were previously sensitized. No contact hypersensitivity was induced in mice lacking all lymphocytes, including natural killer cells. Contact hypersensitivity responses were acquired by such mice after adoptive transfer of natural killer cells from sensitized donors. Transferable hapten-specific memory resided in a Ly49C-I⁺ NK subpopulation localized specifically in donor livers^ref.
• Jones-Mote reaction / cutaneous basophil hypersensitivity : a weak type IV hypersensitivity reaction to protein antigens associated with basophil infiltration, occurring on challenge a few days after priming with a protein antigen in aqueous solution or in Freund's incomplete adjuvant
• autoimmune reactions => diseases

Presuntive evidences of autoimmune pathogenesis :
• major
• evidences of AMI and/or CMI against self antigens
• detection of infiltrated antibodies and/or lymphocytes in injured tissues
• demonstration that antibodies and/or lymphocytes can cause tissue damage
• transplacental transmission
• passive transfer on animals
• in vitro effects on cell functions
• minor
• compatible animal models
• ex juvantibus criterion : improvement of symptoms with immunosuppressive treatments
• associations with other autoimmune reactions
• no other evident aetiology
They affect 3-5% of the population : 75% are females as estradiol (E₂) affects immature B cells through E₂R up-regulating CD22, SHP1 / PTPN6, Bcl-2 and CD106 / VCAM-1 / INCAM-110 : this leads to rescue of autoreactive B cells that would have been deleted. Some autoimmune illnesses occur more frequently after menopause, others suddenly improve during pregnancy, with flare-ups occurring after delivery, while still others will get worse during pregnancy.
Aetiology :
• exogenous aetiology
• immune response against cross-reacting heterophilic Ags (molecular mimicry). E.g. :
• Forssman-Skoog Ag is Gb5 glycosphingolipid whose sugar group is as follows : a-D-GalNAc-(1->3)-b-D-GalNAc-(1->3)-a-D-Gal-(1->4)-b-D-Gal-(1->4)-D-Glc). It was first identified in sheep RBCs, and then in bacterial cell walls. It is absent in human, rabbit, rat, porcine and bovine tissues.
• Streptococcus pyogenes Ags and a human heart tissue antigen
• Escherichia coli O86 and human blood group Ag B.
• Streptococcus pneumoniae serotype XIV and human blood group Ag A.
• Proteus vulgaris OX-19 Ag and Ag(s) on Rickettsia conorii,Rickettsia prowazekii, Rickettsia typhi
• Proteus vulgaris OX-2 Ag and Ag(s) on Rickettsia conorii
• Proteus mirabilis OX-K Ag and Ag(s) on Orientia tsutsugamushi
An approximately 5-fold difference in the TcR affinity for the activating ligand results in a 50% reduction in the incidence of autoimmunity. A reduction in TcR-ligand affinity to approximately 20 times lower than normal does not induce autoimmunity despite the unexpected induction of CTLs and insulitis. Furthermore, in the absence of a key negative regulatory molecule, Cbl-b, 100% of mice develop autoimmunity upon infection with viruses encoding the lower-affinity ligand. Therefore, autoimmune disease is sensitive both to the affinity of the activating ligand and to normal mechanisms that negatively regulate the immune response^ref.
• aspecific polyclonal lymphocyte activation
• B lymphocytes
• HHV-4 / EBV
• mitogens
• T lymphocytes
• superantigens
• mitogens
• bacterial adjuvants
• endogenous aetiology : the concordance of autoimmune diseases among monozygous twins is virtually always < 50%, often in the 25-40% range. This incomplete penetrance may be due to :
• extrinsic aetiology
• distinct lymphocyte receptor repertoires in otherwise identical individuals
• initiation of disease is a quantal, stochastic event that depends on the behavior of only a single lymphocyte among the mature peripheral ones whose TcR affinity for a particular autoantigen approaches that required for strong signaling. Susceptibility alleles can affect the mean and range of net activation energy (signaling competence, the amount of costimulatory ligand available, the number and activity of inhibitory T cells, and/or the ambient levels of suppressive cytokine) closer on average to a level suitable for cellular activation. The exponential nature of cell division ensures that only a few cycles are needed to generate a large pool of effector cells, whose intracellular signaling competence seems to have increased sensitivity. This "rogue cell" model has substantial implications with respect to the possible use of prophylactic immunosuppressive strategies in gentically susceptible individuals.
• alterations in antigen presentation
• autoantigen dissequestration from an immune-privileged site
• autoantigen alteration during
• inflammation
• drug exposure
• cell death
• during necrosis, e.g.
• acute myocardial infarction (AMI)
• X-rays
• metal-catalyzed oxidative modifications
• changes induced by :
• infectious disease agents
• xenobiotic chemotherapeuticals
• during apoptosis : cleavages by caspases and granzyme B
The immune response may be cross-reactive with unaltered self proteins.
• epitope spreading : T cell reactivity is shifted from a narrow set of inducer determinant to
• other responder determinants (cryptic epitopes) on the same Ag (intramolecular spreading)
• other antigens (intermolecular spreading)
It may contribute to the variation of the autoantibody-response pattern observed in different individuals with a given autoimmune disease.
• altered functionality of APCs
• cytokine production
• costimulatory molecules
• increased activity of T_h lymphocytes
• T cell lymphomas
• cytokine production
• costimulatory molecules
• increased activity of B lymphocytes
• B cell lymphomas
• apoptosis deficiencies
• unusual TCR-binding properties can permit autoreactive T cells to escape deletion : rather than being centered on the peptide-MHC, the TCR trimer from a patient with multiple sclerosis that causes autoimmunity in transgenic mice contacted only the N-terminal peptide segment and made asymmetrical interactions with the MHC helices. The interaction was dominated by the hypervariable CDR3 loops, indicating that unconventional topologies are possible because of the unique CDR3 sequences created during rearrangement. This topology reduces the interaction surface with peptide and alters the geometry for CD4 association^ref
• disequilibrium in cytokine production
• impaired immunoregulatory mechanisms
• proteolytic cleavage (shedding) of cell-surface TNF receptor p55 (TNFRp55) modulates TNF function, and in humans, impaired receptor shedding is linked to a group of autoinflammatory syndromes, but also increased resistance to infection with Listeria monocytogenes in knock-in mice heterozygous or homozygous for a non-sheddable TNFRp55^ref
• anti-idiotype network
• immune response against a peptide that is antisense or complementary to the autoantigen, which then induces anti-idiotypic antibodies (autoantibodies) that cross-react with the autoantigen. (e.g. proteinase-3 (PR-3))
• T_Reg lymphocytes : the Autoimmune regulator (Aire) gene, which is defective in the disorder autoimmune polyglandular syndrome type 1, has been shown to promote the thymic expression of self-antigens. Autoimmune eye disease in aire-deficient mice develops as a result of loss of thymic expression of a single eye antigen, interphotoreceptor retinoid-binding protein (IRBP). In addition, lack of IRBP expression solely in the thymus, even in the presence of aire expression, is sufficient to trigger spontaneous eye-specific autoimmunity. These results suggest that failure of thymic expression of selective single self-antigens can be sufficient to cause organ-specific autoimmune disease, even in otherwise self-tolerant individuals^ref.
• B lymphocytes that had undergone BcR revision have been isolated from ectopic germinal centers in inflamed tissues in patients with rheumatoid arthritis (RA)
• common mutations : disruption of RUNX1 binding sites in different genes have been associated with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and psoriasis
These explanations cannot account for the diversity of pathogens associated with specific autoimmunity, nor can they explain the association of one pathogen with many autoimmune diseases. General characteristics of infections, rather than specific Ag, could partake in disease induction : nonspecific, nontissue damaging (hence irrespective of additional endogenous Ag release), inflammatory events byproduct of an infection (e.g. adjuvants such as CT, anti-CD40 mAb) can induce APC maturation, causing cross-presentation of endogenous Ag to shift from cross-tolerization to cross-priming, and  hence to tissue-specific destructive autoimmune reaction.
According to some theories an autoimmune disease is the outcome of failure to control an autoimmune reaction whose original purpose was beneficial : the beneficial and the destructive response share the same antigenic specificity. E.g. an autoimmune response targeting CNS can prevent release of compounds toxic even for noninjured neurons.
Let us think about latent infections, cancer or other chronic diseases. Can it be possible that some damages may take place in form of Autoimmunities to starve the "disease causing agents" from their required nutrients (including oxygen) to resist or restrict their growth & spread or to encourage anorexia to withhold intestinal absorptions for this purpose? Can pancreatic islet cell damages in diabetes mellitus, be meant to save us from hyperinsulinemia's persistant effects? Other autoimmune diseases can also be thought for some natural body's mechanism hidden behind these "to save us as a whole at the cost of some".
Genes with a distinct expression pattern in autoimmunity (conserved among the different autoimmune diseases) are not necessarily immune response genes, but are genes that encode proteins involved in apoptosis, cell cycle progression, cell differentiation, and cell migration.
Autoantibodies can be pathogenetic or represent just an epiphenomenon or an activity marker ; they can be pathognomonic or be associated to a whole group of autoimmune diseases. Anyway they are directed against the most conserved regions of the most conserved molecules. A long CDR3 and a positively charged amino acids in CDR3 in the Ig heavy chain are 2 features that have been associated with self-reactive antibodies.
Autoantibodies are assayed on ...
• tissue sections or cultured cell lines : this doesn't allow Ag definition, but sensitivity is usually excellent (except for under-representation of Ro antigen in cellular extracts)
• cell extracts
• defined sets of autoantigens (with authentic 3D structure in order to prevent false negative results for conformation epitopes)
• ... isolated from natural sources like human tissues
• ... produced by recombinant DNA technology
Autoantigens can be classified as follows :
• autoantigens reachable by antibodies => CMI, type II and type III hypersensitivity reactions
• secreted autoantigens
• enzymes
• acetylcholinesterase (AChE)
• structural proteins
• gastric intrinsic factor (IF) (GIF)
• collagen type II (CII), a₁ chain
• collagen type IV, a₃ chain (non-collagenous NC1-domain)
• collagen type XVII, a₁ chain / BP180 / BPAG2
• human cartilage glycoprotein 39 (HCgp39) / YKL-40
• chondroitin sulfate carbohydrate moiety in decorin (a.k.a. BJ antigen)
• hormones
• insulin
• T₃, T₄ or both (hapten)
• antibodies (=> rheumatoid factor (RF)) : IgM (the most commonly detected isotype, as serologic tests measure only IgM), IgG, IgA, and/or IgE (rarely detected) containing a region outside the CDRs which can bind the C_H2 and C_H3 domains (Gm antigenic determinant) in the Fc region of human and rabbit IgGs (serum RF binds Fcg₁, Fcg₂ and Fcg₄ > Fcg₃; synovial fluid RF binds Fcg₃ > Fcg₁, Fcg₂ and Fcg₄). They are detected by :
• agglutinations
• passive hemagglutination (RF fixed on RBCs with CrCl₃) : Waaler-Rose reaction
• immunonephelometry
• ELISA
They are found in the following situations :
• autoimmune diseases
• essential mixed cryoglobulinemia (> 90%)
• classical or definite rheumatoid arthritis (RA) (50-85% of RA patients; 30% of all RF⁺)
• Sjögren's syndrome (SS) (50-85%)
• systemic lupus erythematosus (SLE) (25-50%)
• juvenile chronic arthritis (JCA)(10-25%)
• ankylosing spondylitis (AS) (5-10%)
• psoriatic arthritis (PsA) (5-10%)
• idiopathic pulmonary fibrosis (IPF) (10-25%)
• sarcoidosis
• infections
• HBV
• post-infectious mononucleosis (IF) (< 5%, self-limiting)
• leprosy (25-50%)
• tuberculosis (TB) (10-25%)
• subacute bacterial endocarditis (25-50%)
• rheumatic fever (RF) (5-10%)
• Treponema pallidum subsp. pallidum
• Plasmodium spp.
• Leishmania spp.
• Schistosoma spp.
• hepatopathies(25-50%)
• gout
• osteoarthritis (5-10%)
• healthy subjects (5%)
• preformed IC containing vitronectin (VN) and fibronectin (FN) present in sera of patients with CSS and WG bind to endothelial cells (anti-endothelial cell antibodies (AECA))
• autoantigens exposed on external layer of plasma membrane
• CD41 / a_IIb integrin / p136^gpIIb / CD31 / PECAM-1 / b₁ integrin / p130^gpIIa (95-100%)
• CD42b / glycocalicin / gpIb_a42c / gpIb_b / CD2a / p17^gpIX
• CD49 / a₂ integrin / p153^gpIa / CD31 / PECAM-1 / b₁ integrin / p130^gpIIa
• CD49f / a₆ integrin
• CD104 / b₄ integrin
• H⁺ / K⁺ ATPaseb subunit
• myelin-associated glycoprotein (MAG)
• myelin basic protein (MBP) (homologous to HBV protein and Human adenovirus type 2 protein)
• myelin oligodendrocyte protein (MOG)
• myelin protein P₀ (MPZ)
• proteolipid protein 1 (PLP1)
• P-type Ca²⁺ channel
• BP230 / BPAG1
• claudin 11
• desmoglein 1
• desmoglein 3
• SSN / ABCA7
• CYP2D6 / LKM-1
• anti-endomysial antibodies (EMA)
• transglutaminase 3 / tissue transglutaminase (tTG)
• anti-phospholipid antibodies (IgG or IgM) are directed against ...
• immunogenic protein carriers :
• a dominant target epitope defined by residues 40-43 in domain I of apoH / b₂ glycoprotein I (a member of the SCR protein family with 5 SCRs : the 5^th SCR contain an extra 20-aa C-terminal loop ending in an extra terminal disulfide bond and a Lys-rich region that functions as the major anionic phospholipid binding site)
• prothrombin
• hapten phospholipids :
• cardiolipin (CL), associated with HLA-DR3
• phosphatidylinositol (PtI)
• phosphatidylglycerol (PtG)
• phosphatidylserine (PtS), exposed during trophoblast syncytium formation
• phosphatidylcholine (PrC)
• phosphatidic acid (PA)
The antiphospholipid antibodies (aPL), but not control Ig or Ig from aPL^- patients, bound to apoptotic cells, but not to viable cells. Nuclear antigens were not recognized. Opsonization of apoptotic cells by aPL substantially enhanced recognition and binding by scavenger macrophages, with massive TNF-a secretion, possibly enhancing the immunogenicity of the autoantigens they contain^ref.
3 kinds of operative definitions exist :
• lupus anticoagulant (LAC) (increase of aPTT,INR, and dRVVT not correctable with addition of  normal plasma)
• anti-cardiolipin antibodies (aCL) / anti-M1 antibodies (see below)
• biological false-positive (BFP) test results for syphilis
Despite their name, they don't predspose to hemophilia but rather to to clotting in vivo, predominantly by interfering with the antithrombotic role of phospholipids, inhibiting of endothelial cell activation (decreased expression of adhesion molecules E-selectin, ICAM-1, and VCAM) and of monocyte adhesion, and up-regulating platelet aggregation. They are found in 2-4% of otherwise healthy individuals.The SWISS PROT protein database revealed high homology between the hexapeptides that bind to ILA-1, ILA-3, and H-3 mAbs and the membrane particles of different bacteria and viruses :
• LKTPRV sequence shows homology to 8 different bacteria (eg, Pseudomonas aeruginosa) and 5 types of viruses (ie, polyoma virus, HHV-5 / CMV, adenovirus)
• TL-RVYK sequence shows homology to 8 different bacteria, including Haemophilus influenzae, Neisseria gonorrhoeae, and Shigella dysenteriae, and to viruses such as HHV-4 / EBV and HIV-1.
• receptors
• ACTH-R
• M₃ AChR (second extracellular loop)
• gastrin receptor
• nicotinic acetylcholine receptor (N AChR) < 0.4 nmol/L
• PTPRN / IA2 / ICA 512
• PTPRN2 / IA2b / phogrin / IAR
• glycosylated TSH-R : TSH-receptor antibodies (TRAb)
• thyroid stimulating antibodies (TSAb) / thyroid-stimulating immunoglobulins (TSI) / long-acting thyroid stimulator (LATS) occur in Graves' disease
• TSH-stimulation blocking antibodies (TSBAb) (dosed with TSH-binding inhibitor immunoglobulin (TBII) assay) occur in anti-TSH-R antagonist antibody syndrome
• FceRI a chain
• autoantigens not reachable by antibodies : CMI only. Autoantibodies against such autoantigens have pathogenetic meaning only after cell death, when they can bind to their target and trigger type III hypersensitivity-like reaction. Anyway some kinds of autoantibodies can enter cell via RME or RME-independent pathways and cross the plasma membrane and reach them.
• autoantigens in the inner layer of plasma membrane
• PtS
• PtI
They are translocated on the external layer of plasma membrane (i.e. become reachable by antibodies) in cells undergoing apoptosis.
• cytoplasmic autoantigens
• amphiphysin
• glutamic acid decarboxylase 1 (GAD1) => GAD67
• glutamic acid decarboxylase 2 (GAD2) => GAD65 < 1 U/mL
• PA28g / Ki autoantigen
• restin
• cyclic nucleotide phosphodiesterase 1 (CNP-1)
• UDP glycosyltransferase 1 family members (LKM-3)
• neutrophil cytoplasm autoantigens (=> anti-neutrophil cytoplasmic antibodies (ANCA)) : after ethanol fixation ...
• diffusely granular cytoplasmic neutrophil fluorescence pattern with internuclear accentuation (C-ANCA / c-ANCA)
• proteinase 3 / AGP7 / p29 / myeloblastin (IL-1 and TNF-a induce translocation on cell membrane of neutrophils and endothelial cells). Several patients with vasculitiis and PR3-specific antibodies also have antibodies specific for complementary PR3 (cPR3) - a peptide translated from the antisense DNA strand of the PR3 gene - in the sera, absent in hose without PR3-reactive antibodies or healthy individuals. Mice injected with the middle region of human recombinant cPR-3(105-201) developed both cPR3- and PR3-specific antibodies, indicating that peptides translated from the anti-sense strand of an autoantigen, or microbial-derived peptides that mimic complementary peptide, could initiate autoimmunity. Both human and mouse cPR3 and PR3-specific antibodies did not cross-react with the same antigen but bound to each other, forming an idiotypic pair antibodies^ref.
• perinuclear fluorescence pattern with nuclear extension (P-ANCA / p-ANCA) : even these autoantigens are widely dispersed in cytoplasm but migrate in the perinuclear region during ethanol fixation procedure.
• MPO (10%)
• human leukocytary elastase (HLE) / medullasin
• a-enolase / enolase 1 / non-neural enolase
• lysozyme
• C-terminal part of BPI / CAP-57
• azurocidin
• lactoferrin
• cathepsin G
• HMG1
• HMG2
• human lysosome-associated membrane protein-2 (hLAMP-2)
  
A substantial proportion of the granulocyte-specific ANA (GS-ANA) belongs to the p-ANCA family.
• atypical diffuse cytoplasmic pattern (A-ANCA / a-ANCA) are ANCA detected by IIF and not directed against the predominant ANCA antigens (i.e. PR3 and MPO), but against a variety of still ill-defined neutrophil antigens. They most frequently yield a perinuclear pattern (P-ANCA) and are found in
• inflammatory bowel diseases (IBDs)
• primary sclerosing cholangitis (PSC)
• rheumatoid arthritis (RA)
• cystic fibrosis (CF)
• leprosy
• malaria
• APSGN
• HIV-1
• Golgi apparatus autoantigens
• golgin a1 (GOLGA1)
• macrogolgin (GOLGB1)
• mitochondrial autoantigens detected from beef heart mitochondria (M2), rat liver (M4), or pig kidney (M8) (=> anti-mitochondrion antibodies (AMA))
• inner mitochondrial membrane
• anti-M1 / anti-cardiolipin antibodies (aCL) (see above) in syphilis (Treponema pallidum subsp.pallidum)
• anti-M2 / E2 component of pyruvate dehydrogenase complex (PDC) in primary biliary cirrhosis (PBC)
• anti-M7 / sarcosine dehydrogenase in certain forms of acute and chronic cardiomyopathy (infectious myocarditis ?)
• outer mitochondrial membrane
• anti-M3 (major : a firmly bound component; minor : a mercurial-insensitive Ag present in the chloroform-released ATPase preparation) in phenopyrazone-induced pseudolupus syndrome
• anti-M4 / sulfite oxidase in primary biliary cirrhosis (PBC) (always associated with anti-M2)
• anti-M5 (a digitonin-sensitive component) in UCTD :  a small population cross-reacts with cardiolipin
• anti-M6 / liver MAO-B  in iproniazid-induced autoimmune hepatitis (AIH)
• anti-M8 in primary biliary cirrhosis (PBC) (always associated with anti-M2)
• anti-M9 / glycogen phosphorylase in primary biliary cirrhosis (PBC) (do not fix complement)
Considering the similarities between bacterial and mitochondrial membranes, it is suggested that the formation of AMA of different specificities may be induced by cross-reacting antigens.
• E3 component of pyruvate dehydrogenase complex (PDC)
• dimethylglycine dehydrogenase
• mitochondrial adenine nucleotide translocase (ANT) / SLC25A6 in idiopathic dilated cardiomyopathy (IDCM). The most significant epitopes belong to the M2 and M3 hydrophilic loops.
• ribosome autoantigens
• ribosomal protein L7
• ribosomal protein P0 (expressed also on cell surface !) => psychiatric symptoms (?), more active disease, photosensitivity and malar and discoid rash in SLE
• ribosomal protein P1
• ribosomal protein P2
• ER autoantigens
• calreticulin / mobilferrin
• CYP1A2
• CYP11A1 / cholesterol 20,22-desmolase / side-chain-cleaving enzyme (P450_scc)
• CYP11B / steroid 11b-hydroxylase
• CYP17A1 / steroid 17-hydroxylase
• CYP21A2 / steroid 21-hydroxylase
• smooth muscle autoantigens (SMA) (=> anti-smooth muscle antibodies (ASMA))
• leiomodin
• myosin
• F-actin (autoimmune hepatitis type I)
• caldesmon
• cytoskeletal autoantigens (=> anti-cytoskeleton antibodies (ACTA))
• nuclear autoantigens (=>anti-nucleus antibodies (ANA)) : detemined by indirect immunofluorescence (IIF) as fluorescent ANA (FANA) on
• rat hepatocytes
• human cell lines
• HEp-2 (laryngeal carcinoma : polyploid, ectopic gene expression)
• WIL-2 (splenic lymphocytes of B-cell origin)
Fluorescence pattern relates to subnuclear localization (e.g. :
• nuclear type
• centromere
• fine speckled
• coarse speckled
• diffusely grainy
• homogeneous (diffuse) = DNA + histones
• peripheral or rim-pattern = DNA
• nucleolar type
• speckled = snRNP, SSA or SSB, ACA
• clumpy
• homogeneous
• DNA : it is an hapten but becomes immunogen when conjugated to immunogenic carriers such as most nuclear proteins. In fact anti-DNA antibodies cross-react with protein nuclear antigens. The rare hypomethylated DNA motifs found in mammalian DNA, which might be enriched in apoptotic chromatin, are an essential component of chromatin-immune complexes (ICs) from dying cells. The binding of haptenated CpG dsDNA to hapten-specific B cells induces a suboptimal signal from a minimally crosslinked BcR. The BcR also functions to shuttle the CpG DNA antigen to an intracellular endocytic compartment containing TLR9. TLR9 signalling through a MyD88-dependent pathway, together with the signal from the BcR, leads to DNA-specific B-cell activation and proliferation^ref. This might also be a general mechanism for the recognition of opsonized foreign antigens such as bacterial DNA. The AM14 autoimmune mouse strain produces B cells that recognize autologous IgG_2a specific for DNA. In particular, the IgG_2a antibodies that are recognized can complex with chromatin released from dying cells to form multivalent DNA-containing immune complexes (chromatin-ICs) : in addition to the BcR recognition of IgG_2a, the DNA portion of chromatin-ICs is involve in the activation of AM14 B cells through TLR9
• dsDNA autoantigens : determined as peripheral nuclear pattern on whole cells or by ...
• hemoflagellate Crithidia luciliae (eventually pretreated with hydrochloric acid to remove histone-like proteins from kinetoplast) indirect immunofluorescence (IIF) (CLIF) test (CLIFT)
• solid-phase enzyme-linked immunosorbent assay (ELISA) (high sensitivity)
• Farr test (liquid-phase RIA using ³H-labeled dsDNA from the Enterobacteria phage PM2 as antigen) (for high avidity antibodies; high specificity) : antibody is reacted with radiolabeled antigen and precipitated with ammonium sulfate; bound antigen or hapten is precipitated while free antigen remains in solution. This test is based on the capacity of antibody to combine with antigen rather than on such secondary properties as precipitation and therefore measures all immunoglobulin classes and subclasses
• immune complexes polyethylen glycole (PEG) precipitation assay (for low avidity antibodies)
• ssDNA autoantigens : even during chronic infections and hepatopathies
• histone autoantigens
• centromere autoantigens (=> anti-centromere antibodies (ACA))
• centromere protein A (CENP-A) : G/A-P-R/S-R-R motif that occurs 3 times in the N-terminal amino acids. Mimotopes of this motif are present in a vast number of autoantigens and in EBNA-1.
• centromere protein B (CENP-B)
• centromere protein C1 (CENP-C1)
• centromere protein E (CENP-E)
• SSSCA1
• NHEJ machinery proteins autoantigens
• Ku70 / Ki / thyroid autoantigen
• Ku80 / XRCC5 / ATP-dependent DNA helicase II
• DNA-PK_CS / XRCC7
• DNA ligase IV
• XRCC4
• striatin
• follistatin-like 1
• SSNA1
• TNRC6
• U2AF1
• Sp100
• extractable nuclear antigens (ENA) from calf thymus nuclei isotonic phosphate-buffered saline extract (CTE), far more sensitive than rabbit thymus extract (RTE) : the 60-80% ammonium sulfate fraction (ASF) of thymus extract contains SS-B, the 30-60% ASF Sm and U₁ snRNP, and the chromatographically purified 30-60% ASF only Sm antigen. Autoantibodies are dosed with :
• Ouchterlony double diffusion
• immunoelectrophoresis
• countercurrent immunoelectrophoresis / counterimmunoelectrophoresis (CIE)
• immunoblotting / immunotransfer techniques (IBT)
• dot blot
• line immunoassay (LIA) is a development of ELISA in which the antigens to be tested are adsorbed onto a nylon test strip to detect single specificities
• SS-A autoantigens : a number of environmental factors (exposure to UV radiation, viral infections) may cause translocation of the Ro complex to nucleocytoplasmic and membrane sites where it is not normally found. AutoAbs are cross-reactive with calreticulin
• SS-A1 / 52-kDa Ro is expressed in lymphocytes
• 54-kDa Ro is expressed in erythrocytes
• SS-A2 / 60-kDa Ro is associated with the 1-5 hYRNAs and is expressed in lymphocytes and erythrocytes
• SS-B / La / 48-kDa autoantigen (linear epitope)
• DNA topoisomerase I / Scl-70 => idiopathic pulmonary fibrosis in scleroderma
• PCNA
• tRNA synthetases (this is a working localization given that 98% of protein has cytoplasmic localization; nuclear localization has unknown biological role)
• alanyl-tRNA synthetase / PL-12
• glycyl-tRNA synthetase / EJ
• histidyl-tRNA synthetase / Jo-1 => antisynthetase syndrome in polymyositis/dermatoyositis (ILD, subluxing arthropathy sine miositis and periarticular calcinosis)
• lisyl-tRNA synthetase
• isoleucyl-tRNA synthetase / OJ
• threonyl-tRNA synthetase / PL-7
• nucleolar autoantigens
• nucleolar Cys-rich protein
• polymyositis / scleroderma antigen 1
• polymyositis / scleroderma antigen 2
• SC65
• RNA polymerase I
• RNA polymerase II
• RNA polymerase III
• nuRNA autoantigens
• spliceosome-associated antigens
• anti-A1 autoantibodies are directed to 2 proteins
• hnRNP A1
• hnRNP A1b
... encoded from a same gene which also codes for hnRNP B2.
• anti-A2 / RA33 autoantibodies target the RA33 complex (some seem to shuttle between the nucleus and the cytoplasm)
• hnRNP B2 (encoded from the same gene which also encodes hnRNPA1 and hnRNP A1b)
• hnRNP A2
• hnRNP B1
The latter 2 proteins are coded from a same gene.
Epitope-mapping studies have revealed that the major antibody binding sites are located in the RNA-binding regions. Furthermore, there is some indication of disease-specific epitope recognition.
• snRNP autoantigens :
• U₁ snRNP subunits :
• RNA-binding domain of the 70-kDa subunit, cross-reactive with retroviral antigens => MCTD
• snRNP A
• snRNP B and snRNP B1 / snRNP B'
• snRNP C
• U₂ snRNP subunit B''
• fibrillarinis associated with the U₃ (=> U₃ snRNP), U₈, and U₁₃ snRNAs
• Sm autoantigens (for Smith) : cross-reactive with VSV proteins.
• SmD (Arg are simmetrically methylated in D1 and D3)
• Sm D1
• Sm D2
• Sm D3
• Sm E
• Sm F
• Sm G
• Sm N
Murine B cells containing the Y-linked autoimmune accelerator (Yaa) locus are intrinsically biased toward nucleolar antigens because of increased expression of TLR7, a single-stranded RNA-binding innate immune receptor. The TLR7 gene is duplicated in Yaa mice because of a 4-Megabase expansion of the pseudoautosomal region. These results reveal high divergence in mouse Y chromosomes and represent a good example of gene copy number qualitatively altering a polygenic disease manifestation^ref.
• LE factor : an antinuclear antibody present in the blood serum in systemic lupus erythematosus (SLE), having a sedimentation rate of 7S and reacting with leukocyte nuclei in the LE cell test
• unknown
• a cleavage product of 120-kDa a-fodrin
• annexin A11 56-kDa Ag
• calpastatin
• cerebellar degeneration-related autoantigen 3
• COCH5B2
• formiminotransferase cyclodeaminase / LC1
• islet cell autoantigen (ICA)
• MI-2 / Mi 2 / RBBP4
• RCD-8
• SE20-4
• S-arrestin / S antigen
• soluble liver antigen (SLA)
• thyroglobulin (TG) (TgAb < 5 U/mL)
• thyroid peroxidase (TPO) (conformational epitope on C2 segment) (TPOAb < 10 U/mL)
• titin
• uveal autoantigen with coiled-coil domains and ankyrin repeats
• anti-citrullinated peptide antibodies
• anti-filaggrin autoantibodies recognize different epitopes on various isoforms of (pro)filaggrin, expressed in synovial lining, lung, eyes and heart
• antifilaggrin antibodies (AFA)
• "antikeratin" antibodies (AKA)
• antiperinuclear factor (APF)
• cyclic citrullinated peptide (CCP)
• Sa Ag (detected in immunoblots using human spleen and placenta extracts as antigens)
• p50/46
• p68 purified from a mitochondrial extract (amino terminal sequence is DEPKXEVP)
In inflammatory arthridities such as rheumatoid arthritis, cognate lymphocytes have long been considered instigators of autoimmunity, but accumulating evidence indicates that innate immune cells such as neutrophils and mast cells are responsible for a vast majority of acute and ongoing inflammation requiring the forkhead transcription factor Foxo3a: Foxo3a-deficient mice are resistant to 2 models of neutrophilic inflammation, immune complex-mediated inflammatory arthritis and thioglycollate-induced peritonitis. This reflects a need for Foxo3a to maintain neutrophil vitality during inflammation by suppressing FasL; because Foxo3a can bind and suppress the Fasl promoter, Foxo3a-deficient neutrophils upregulate Fas ligand and undergo apoptosis in response to TNF-a and IL-1, and FasL blockade renders Foxo3a-deficient mice susceptible to both arthritis and peritonitis. Thus, Foxo3a ensures neutrophil survival during inflammation, identifying Foxo3a as therapeutic target in inflammation^ref.
• major transplant-related complications (MTCs) => transplant-related mortality (TRM) : post-reduced-intensity cord-blood transplantation (RI-CBT) immune reactions were classified according to time course^ref:

• pre-engraftment immune reactions (PIR)
• veno-occlusive disease of liver (VOLD)
• idiopathic pneumonia syndrome
• systemic capillary leak syndrome (SCLS) / engraftment syndrome (ES)
• host-versus-graft reaction / transplant rejection

In a humanized SCID mouse model, the infiltration of human T cells into skin allografts is enhanced by VEGF-A-induced endothelial expression of MCP-1 and IL-8. In synergy with IFN-g, VEGF also enhances endothelial expression of the T-cell chemoattractant IP-10. In line with this, an anti-VEGF mAb inhibits T-cell infiltration and acute rejection of heart transplants in mice^ref.
• hyperacute or antibody-mediated rejection (AMR) (minutes to a few hours) due to preformed donor specific antibodies (DSA) (heterophilic IgMs and IgGs)

Aetiology : pregnancy or blood transfusion
Laboratory examinations : flow-cytometry cross-match (FC-XM) tests looking for percent of reactive antibodies (PRA) are performed to avoid this complication. However, present techniques do not allow determination of HLA specificity of donor-reactive antibodies in the acute necro-donor situation. The development of HLA class I and class II single antigen (SA) beads technique (Luminex) has considerably improved HLA antibody identification^ref. This technique enables accurate identification of antibody specificities, undetectable by standard techniques such HLA-DP^{ref1, ref2, ref3}, HLA-DQA or allele-specific antibodies. It also provides an ideal approach for the identification of potential HLA antibody epitopes^{ref1, ref2, ref3, ref4, ref5}.  Nevertheless it is known that patients with DSA have a lower graft survival than patients without DSA, even if antibodies are produced long before the failure and before the increase in serum creatinine. De novo DSA are known to be detrimental to graft^{ref1, ref2, ref3}.
Prevention : Historically, transplantation for such patients is possible only when the donor, whose tissues do not express any HLA recipient has antibodies against to be found. Thus, the waiting time for those patients frequently extends to > 3 years^{ref1, ref2, ref3, ref4, ref5, ref6}
• desensitization (DS) with pooled human hyperimmune IVIG and/or plasmapheresis have been shown to produce elimination of some or all of DSA^{ref1, ref2, ref3, ref4, ref5, ref6, ref7}. Many studies reported that sensitized transplant candidates required different DS regimens. For example, a transplant group from Chicago reported that the combination of IVIG and PP allowed successful conversion from positive to negative flow-cytometry (FC) XM in 75% of cases^ref. Other clinical studies showed a high degree of variability of IVIG dose and number of PP cycles required to reduce DSA level and make particular donor–recipient combination FC XM compatible^{ref1, ref2, ref3, ref4, ref5}. The rate of anti-HLA antibody disappearance has been reported to be different also^ref. Furthermore, efficacy of DS has been reported to be different regarding DSA and third party HLA antibodies^{ref1, ref2, ref3, ref4, ref5}. Significant relationship between SNP of genes encoding IL-4Ra, IFN-g and IL-12 (p70) and susceptibility to DS was demonstrated. Correlation between elevated serum level of IL-12 (p70) and A/A or C/A genotype at - 1188 position was found in resistant to DS TCs (p = 0.015)^ref.
• accelerated rejection
• second-set phenomenon or rejection : the accelerated and intensified rejection by the recipient of a second graft of tissue from the same donor as a consequence of the primary immune response (i.e., antibody production and cell-mediated immunity) induced by the first graft
• acute rejection : secondary CMI response to alloantigens.
• acute vascular rejection (days to weeks) : white-graft reaction (an immune reaction to a tissue graft, e.g., a skin graft, as a result of which the grafted tissue does not become vascularized due to rapid rejection)
Memory T lymphocytes that have been primed during primary immune response to previous graft from the same donor or environmental Ags that exhibit chance cross-reactivity to alloantigens :
• allogeneic blood transfusions
• organ allotransplantation
• pregnancy
• infectious agents
• vaccines
Previously primed or memory T cells in "immune-experienced" patients may even be resistant to immunomodulation or tolerance induction, an issue that is extremely relevant to present clinical trials in transplantation in which tolerance is being attempted in the human transplant recipients. Virus-induced CD8⁺ central memory T lymphocytes that are cross-reactive with donor alloantigens have a lower threshold for activation and are a potent barrier to the induction of tolerance in transplant recipients : this is one potential explanation why strategies that effectively promote long-term allograft acceptance by blockade of T-cell costimulation in naive (specific pathogen-free (SPF)) rodent models have been unsuccessful in animals with memory cells (non-human primate models and human patients). Studies demonstrating the importance of alloreactive T-cell deletion during tolerance induction have promoted use of peritransplant T-cell-depleting (TCD) therapies in clinical trials. But potentially complicating wide-scale, nonspecific T-cell depletion is the finding that conditions of lymphopenia induces extensive homeostatic T-cell proliferation, which may induce acquisition of functional memory T cells. In this setting, costimulatory blockade neither significantly suppresses homeostatic proliferation nor prevents allograft rejection. In addition, T cells that have completed homeostatic proliferation show dominant resistance to tolerance when adoptively transferred into wild-type recipients, consistent with known properties of memory cells in vivo^ref.
Alloantigen recognition by CD8⁺ CTLs :
• major histocompatibility antigens : MHC molecules
• direct pathway : host T cells are stimulated by intact allogeneic MHC:peptide complexes on the surface of donor cells. The majority of the T cells responding via this direct pathway of allorecognition derive from the naive T cell repertoire and are primed only in secondary lymphoid organs when the alloantigens are coexpressed in the context of  appropriate costimulatory molecules. Direct allorecognition is a high frequency event; some evidence suggests that 1-10% of the T cells in a given individual are capable of directly recognizing allo-MHC:peptide complexes. This is the predominant pathway, as substantiated by a number of in vivo findings :
• the importance of MHC Ag matching in prolonging allograft survival
• the prolongation of allograft survival via depletion of donor APCs
• the restoration of passenger cell-depleted renal graft immunogenicity by addition of donor strain dendritic cells
• indirect pathway : allogeneic MHC molecules are processed and cross-presented by host APCs to responding host T cells. This indirect alloresponse also seems to derive primarily from naive T cells and comprises only a minority (< 10%) of the total alloimmune T cell repertoire.
In discussing a donor-recipient pairing the degree of matching will often be summarized as a fraction of the number 6, refering to the 6 critical antigens (HLA-A, HLA-B, and HLA-DRB1) only. HLA-A and HLA-B play a major role in antibody-mediated rejection of kidney grats while class II seem to play a role in cell-mediated graft rejection. HLA-DPa and HLA-DPb do not play a significant role in solid organ or bone marrow transplantation. HLA-DQa is not detectable serologically and is generally not considered for transplantation purposes.
DNA strands extending across 2 million base pairs of the MHC were isolated to determine the physical linkage of HLA-A, -B, and -DRB1 alleles in 246 HCT recipients and their HLA-A, -B, -C, -DRB1, -DQB1 allele-MUDs. MHC haplotype mismatching was associated with a statistically significantly increased risk of severe aGVHD (OR 4.51; 95% confidence interval [CI], 2.34–8.70, p < 0.0001) and with lower risk of disease recurrence (hazard ratio 0.45; 95% CI, 0.22–0.92, p = 0.03). The MHC harbors genes that encode unidentified transplantation antigens. The 3-locus HLA-A, -B, -DRB1 haplotype serves as a proxy for GVHD risk among HLA-identical transplant recipients. The phasing method provides an approach for mapping novel MHC-linked HSCT determinants and a means to decrease GVHD-related morbidity after HCT from unrelated donors^ref
• minor histocompatibility antigens (mHags) : all peptides coded by non-self alleles that can be presented on allogeneic MHC molecules of donor cells (to host CD8+ CTLs) or donor APCs (to host CD4+ T lymphocytes).
• HA-1
The minor antigens are presented by a small fraction of HLA molecules and initiate weaker responses than the major antigens do. Minor antigens encoded by genes on the Y chromosome account for the higher incidence of GVHD^ref and lower rate of relapse of underlying disease^ref among male recipients of marrow transplants from female donors than among male recipients of transplants from male donors.
Some transplant centers consider paternal HLA antigens as unacceptable mismatches for mothers awaiting transplantation : it is feared that a pregnancy may cause priming of the maternal immune response directed toward paternal HLA antigens. Should a woman receive an organ from a donor who shares those paternal HLA antigens, the risk of graft rejection might be increased. It is known that some women, as a consequence of pregnancy, develop antibodies specific for paternal HLA antigens. Naive CTL responses are detected against paternal antigens that have never been inherited and those that have been inherited but have not induced antibody formation. In contrast, inherited paternal antigens that have induced HLA-specific antibodies in the mother gave rise to elevated CTL precursor frequencies, as compared with the response to third-party antigens. Also, the CTLs are found to be more resistant to inhibition by anti-CD8, suggesting that these cells have been primed in vivo. These results suggest that not all paternal HLA antigens have to be considered as unacceptable mismatches. Only those individuals who share a paternal HLA antigen against which a mother has formed HLA-specific alloantibodies should be excluded from organ donation^ref
Alloantibodies not only mediate hyperacute rejection, but may also participate in the acute rejection of organ grafts.
• chronic rejection : for kidney transplant recipients, the major risk factor for development of chronic rejection appears to be a previous acute rejection episode. Other risk factors include low-dose maintenance immunosuppression and previous infection. For extrarenal transplant recipients, CMV and HLA mismatch have been implicated. Noncompliance probably plays a role for all patients. Laboratory studies have suggested that the development of chronic rejection (or lack thereof) may be due to an interplay of immunoregulatory factors. Patients with anti-HLA antibodies have an increased incidence of late graft loss whereas those with antiidiotypic antibodies or with donor-specific hyporesponsiveness (in MLC) have improved outcome and less chronic rejection. These findings have led to testable hypotheses as to the pathogenesis of chronic rejection:
• chronic rejection is inadequately treated acute rejection
• chronic rejection can be prevented by maintaining adequate long-term immunosuppression
• preventing or adequately treating infection will prevent chronic rejection
• the balance of immunoregulatory factors determines chronic rejection
• chronic rejection is the result of noncompliance
Each may play a role in some patients.
• first-set phenomenon or rejection : the immunological reaction of the body against a tissue or organ in a host not previously sensitized against the graft antigens
• delayed graft failure (DGF)
Donor allorecognition of the recipient after hematopoietic transplantation can result in :
• graft facilitation effect
• graft-versus-host disease (GvHD / GVHD) or reaction / homologous disease
Aetiology :
• allogeneic hematopoietic stem cell transplantation (HSCT). Both acute and chronic GVHD are more common after allogeneic peripheral-blood stem-cell transplantation (PBSCT) than allogeneic bone marrow transplantation, and this may be associated with lower rates of malignant relapse. The magnitude of the transfused T-cell load may explain the differences in chronic GVHD risk. These diseases are described as occurring with a lower incidence and lesser severity when human umbilical cord blood (HUCB) cells are used for transplantation, probably related to the immaturity of neonatal HUCB T lymphocytes. HHV-5 / CMV infection triggers autoantibody formation against CD13 in transplanted patients which, in turn, has been shown to be associated with the development of chronic GvHD
• maternal-fetal blood transfusion
• therapeutic blood transfusion in which the recipient has a cellular immunodeficiency disease.
Pathogenesis : high-dose preparative regimens damage tissues, particularly in the gut, allowing lipopolysaccharide from bacteria in the bowel to leak into adjacent tissues and the bloodstream. Distinct classes of conserved microbial molecules and necrotic-cell elements (including high-mobility group B1 protein (HMGB1)) activate TLRs on various cells, which leads to release of inflammatory cytokines (including TNF-a and IL-1b, IL-6, and IL-12) => allogeneic donor's T lymphocytes attack the tissues of the recipient due to recognition of recipient's Ags on recipient's MHC molecules expressed by APCs (not necessarily by recipient's tissues). All 3 critical loci (HLA-A, HLA-B, and HLA-DRB1) are important in GvHD in bone marrow transplants. In many cases a single alloreactive CD4⁺ T cell clone might mediate severe GvHD (in a case by day 69 post-SCT almost 100% of all the circulating CD4⁺ T-cells in the peripheral blood of the patient was due to the expansion of this clone^ref). Therefore, this method of using clonotypic quantitative real-time PCR (qPCR) for identifying T-cell motifs, can be applied to detect nascent GvHD alloreactive T cells and might allow potential immunological intervention to prevent GvHD. Cytokines are critical to GVHD, and their genetic variants influence its development. Indeed, the inactivation of the chemokines or the adhesion molecules that attract donor T cells to Peyer's patches eliminates most deaths from GVHD in mice^ref.
• the incidence and severity of GVHD correlates with the relative numbers of CD4⁺ Foxp3⁺ regulatory T cells contained in the marrow. Patients receiving stem cell grafts with relatively high numbers of CD4⁺Foxp3⁺ regulatory T cells had a significantly lower incidence of GVHD compared with patients receiving grafts that had low numbers of regulatory T cells. Of additional interest is the finding that the regulatory population recovers rapidly and is not affected by either treatment with the immunosuppressive drug cyclosporine A or the infusion of donor lymphocytes. The transfer of regulatory T cells along with the marrow graft appears to prevent the subsequent development of GVHD, limiting one of the major complications of allogeneic bone marrow transplantation. The studies of Rezvani et al set the foundation for the development of strategies to engineer the HSC graft expanding the CD4⁺ Foxp3⁺ regulatory population ex vivo/in vitro prior to transplantation. One caveat still remains, however: will the transfer of high numbers of regulatory T cells compromise the antitumor efficacy of HSCT^ref. By suppressing the release of inflammatory cytokines and the activation of T cells, IL-10 promotes tolerance. Homozygosity for a common variant of the IL-10 promoter appears to increase the production of IL-10 and reduce the incidence of GVHD^ref. The genetic polymorphisms of other cytokines also appear to influence GVHD^ref. In addition, small variations in donor or recipient genes that encode NOD2/CARD15 are associated with severe GVHD^ref
• histoincompatibility between donor and recipient is necessary but not sufficient to elicit GVHD. Some donors may be “stronger alloresponders” than others, and consequently more likely to elicit GVHD. The gene-expression profiles of CD4⁺ and CD8⁺ T cells from 50 allogeneic HSCT donors with microarrays. pre-HSCT gene-expression profiling segregates donors whose recipient suffered from GVHD or not. Using quantitative PCR, established statistical tests, and analysis of multiple independent training-test datasets, for chronic GVHD the “dangerous donor” trait (occurrence of GVHD in the recipient) is under polygenic control and is shaped by the activity of genes that regulate TGF/b signaling and cell proliferation^ref. These findings strongly suggest that the donor gene-expression profile has a dominant influence on the occurrence of GVHD in the recipient. The ability to discriminate strong and weak alloresponders using gene-expression profiling could pave the way to personalized transplantation medicine.
• if allogeneneic hematopoietic stem cell transplantation (HSCT) occurs in a recipient with cancer, transplanted donor's T lymphocytes can also attack the tumor cells (graft-versus-tumor (GVT)) :
• leukaemia or lymphoma cells (graft-versus leukemia (GvL / GVL) effect). At present, 50% of patients who could benefit from BMT do not receive it because of the high incidence of GvHD in mismatched donors. The recognition that the incidence of relapse after autografting for recurrent or refractory lymphoma is usually substantial and the emerging use of reduced-intensity (RI) allogeneic conditioning regimens have resulted in the need to evaluate critically which lymphomas are amenable to GVL effects. A GVL effect can be convincingly demonstrated only by the durable resolution of biopsy-proven residual progressive disease after allografting in response to immunomodulation such as withdrawal of immunosuppression or donor leukocyte infusion (DLI). Indirect evidence may include eradication of active disease by minimally cytotoxic conditioning regimens and allogeneic cell infusion and statistical association between GVHD with a lower risk of relapse and T-cell depletion with a higher risk of relapse. Although a substantial reduction in relapse with a plateau in disease-free survival (DFS) after allogeneic versus autologous transplantation in comparable patient groups would also be consistent with a GVL effect, the possible contribution of reinfused autologous tumor cells to relapse^ref [1] means that such observations are not definitive proof. A number of comprehensive reviews of single-institution and registry studies of allografting in lymphoma with both myeloablative and RI conditioning regimens have been published^{ref1, ref2} [2 and 3].

Minor histocompatibility antigens on leukemic cells can provoke a graft-versus-leukemia response^{ref1, ref2, ref3}. Donor T cells reactive to recipient minor histocompatibility antigens inhibit the growth of leukemic colonies and, in one study, prevented the development of AML derived from human cells in immunologically susceptible mice^ref.
Difficulties in assessing the literature : there are a number of difficulties in interpreting the literature on GVL effects.
• registry analyses :
• many have incorporated a number of lymphoma subtypes with distinct histopathologic, molecular, and clinical characteristics into a single group for the purposes of analysis of GVL effects, making it difficult to evaluate responses in specific entities. For example, diffuse small cleaved (often mantle cell [MCL]) and follicular large-cell lymphomas have been regarded by the European Bone Marrow Transplant (EBMT) registry as intermediate-grade lymphomas under the Working Formulation and analyzed together^ref [4], whereas another transplant analysis from this group classified patients as having only low- or high-grade lymphoma^ref [5]. In addition, many of these registry reviews include patients undergoing different conditioning regimens and GVHD prophylaxis.
• lack of centralized histologic review, so that the reported diagnoses have not been independently verified.
• probable imbalances in baseline pretransplantation characteristics in studies comparing the outcome of autografts versus allografts^ref [4], together with potential variability in patient selection and standards of supportive care over long periods of analysis.
• not surprisingly, the results of these studies are often inconsistent.
• retrospective single-institution studies comparing autografts with allografts are subject to various potential confounding factors, such as referral bias, physician preference, and improvements in outcome over time, such that definitive conclusions are rarely possible.
• there are substantial differences in the intensity of RI conditioning regimens. Fludarabine/melphalan 140 to 180 mg/m², for example, may well be myeloablative, because chimerism early after transplantation is usually exclusively donor and because the risk of GVHD is comparable to that with traditional myeloablative regimens^ref [6]. In contrast, fludarabine/low-dose cyclophosphamide is clearly not ablative, generally results in mixed chimerism, and has a relatively low incidence of GVHD^ref[7]. Moreover, most RI conditioning studies from single institutions or registries have short-term follow-up, so the durability of responses is not established.
• the importance of documentation by biopsy of viable residual disease after transplantation in assessing the response to DLI or withdrawal of immunosuppression is particularly relevant because functional imaging with positron emission tomography or gallium scanning may not always reliably distinguish inflammatory from neoplastic tissue or indolent from aggressive lymphoma, which may coexist.
• although responses to DLI have been well documented in some lymphomas, particularly follicular and MCLs, this does not necessarily mean that all such lymphomas are immunologically responsive. Some cases have documented a GVL effect in response to DLI that seems to alter the natural history of previously progressive lymphoma but that is not durable, suggesting that malignant clones may differ in their susceptibility to immunotherapy.
GVL effects in histologic subtypes and proposed allograft policies : data for each subtype will be discussed as follows: (1) DLI, (2) single-institution and registry reviews of RI transplants, and (3) institution and registry reviews of myeloablative transplants, including, where available, an evaluation of the effect of GVHD on relapse.
• follicular lymphoma (FL) : a graft-versus-follicular lymphoma (FL) effect was suggested by case reports of resolution of active disease by DLI^{ref1, ref2} [8 and 9] and was confirmed more recently in a British survey in which 8 of 13 patients with overt FL after allografting achieved complete remission (CR) after DLI^ref [10]. A high rate of remission after RI conditioning has been reported^ref [7], although most patients had nonbulky chemosensitive disease at transplantation and although follow-up was relatively short in a disease with a propensity for late relapse. An EBMT registry analysis of RI allografts in low-grade lymphoma (Working Formulation) using a variety of conditioning regimens in heavily pretreated patients, most with chemosensitive disease and with sibling donors, demonstrated a 1-year probability of disease progression of 21% and 2-year progression-free survival (PFS) of 54%; pretransplantation chemosensitivity was the only significant factor predicting for progression^ref [5]. No relapses were seen beyond 1 year, although few patients were followed up beyond 2 years. There are minimal data on the outcome after unrelated donor transplantations for FL and other lymphomas^{ref1, ref2, ref3, ref4} [4, 5, 11 and 12]. Single-institution studies with long-term follow-up have reported a low incidence of relapse after myeloablative allografts for refractory or recurrent indolent non-Hodgkin lymphoma (NHL), predominantly follicular^{ref1, ref2} [13 and 14], which seems less than after autografts^ref [15]; this is consistent with an EBMT registry analysis^ref [4]. Recently the International Bone Marrow Transplant Registry (IBMTR) and the Autologous Bone Marrow Transplant Registry, in a retrospective study, compared the outcome of myeloablative allogeneic versus purged autologous versus unpurged autologous transplants^ref [16]. Allografts had a higher treatment-related mortality and a lower risk of recurrence. Few recurrences occurred after 2 years, although the maximum follow-up of 5 years is still relatively short. Intriguingly, the relapse rate was higher in unpurged versus purged autografts, and there was no association between acute or chronic GVHD and recurrence. The EBMT registry analysis also found no effect of acute GVHD on relapse, although in the British review, the responses of FL to DLI correlated with both acute and chronic GVHD^ref [10]. Overall, these data suggest that the major benefit of allografting in FL may relate to effective high-dose chemoradiotherapy followed by infusion of uncontaminated stem cells rather than a GVL effect. The absence of a significant difference in relapse risk after syngeneic versus allografts for FL and the reduction in recurrence rate after syngeneic versus autologous unpurged transplantation is consistent with this^ref [17], although a syngeneic GVL effect cannot be excluded. To complicate the issue, however, a prospective randomized autograft study found no benefit in PFS or overall survival between purged and unpurged marrow^ref [18]. Long-term results of minimally cytotoxic conditioning regimens in patients with active disease are needed to clearly evaluate the clinical effect of a graft-versus-FL effect. Additional questions include the durability of responses after RI allografts, the effect of the histologic grade in FL on the incidence of relapse, the outcome after unrelated donor allografts, and the identification of tumor antigens that are immunologically relevant for allogeneic responses. Because of a relatively high risk of both early and late relapse after autografting, in general an allograft is the transplant option recommended if a compatible sibling is available. We consider patients up to age 60 to 65 years, depending on their general fitness and level of donor compatibility. A well-matched unrelated donor transplant is considered in selected younger patients, generally younger than 45 years and with a good performance status. The indications for transplantation are at least 1 of (1) progressive symptomatic disease within a year of CHOP-like (cyclophosphamide, hydroxydaunomycin, vincristine, and prednisone) or purine analog-based therapy either as induction or for relapse or (2) multiply relapsed disease. Our preference is to attain a minimal residual disease state (usually with fludarabine with or without cyclophosphamide with or without rituximab, depending on prior therapy), and we use a very-low-intensity conditioning regimen such as fludarabine/low-dose cyclophosphamide or fludarabine/low-dose total body irradiation^ref [12], with the addition of rituximab if not used previously^ref [7]. This is based on our acceptance that a graft-versus-FL effect exists to a degree. The intention is to achieve mixed chimerism early after transplantation to reduce the risk of severe acute GVHD and to gradually convert to donor chimerism either from the natural course of the transplantation or with DLI. More intensive conditioning with increased organ toxicity and a higher risk of GVHD due to early establishment of full donor chimerism (fludarabine/melphalan or cyclophosphamide/total body irradiation) is reserved for patients with bulky, aggressive, or chemorefractory disease.
• mantle cell lymphoma (MCL) : the role of allografting in MCL has been reviewed by Sweetenham^ref [19]. A graft-versus-MCL effect has been unequivocally documented in case reports of resolution of progressive disease after withdrawal of immunosuppression^{ref1, ref2} [20 and 21] or DLI [22]. This is broadly consistent with single-institution studies reporting a low relapse rate after RI^ref [23] and myeloablative^ref [21] conditioning for advanced or recurrent chemosensitive disease, although the follow-up in both of these studies was relatively short. This contrasts with a poor outcome in an EBMT registry survey in 22 older patients undergoing RI allografts^ref [5]: this was due to a high early transplant-related mortality and a substantial rate of relapse. The ability of a GVL effect to eradicate chemoresistant MCL or to improve the poor outcome observed in patients autografted in CR1 with high b₂-microglobulin^ref [24] is not proven. Moreover, although a GVL effect may occur in the diffuse form of the disease^ref [21], its activity against the more aggressive blastoid variant is not known. The effect of GVHD on relapse in MCL has not, to our knowledge, been evaluated. Although there is some conflicting evidence, we believe that an allograft is appropriate therapy for patients with relapsed MCL, because the autograft results with readily available conditioning regimens are poor^ref [25]. The intensity of conditioning used depends on disease status and on patient age and performance status. Ideally, the allograft should be offered in first relapse rather than subsequent relapses and limited to patients with chemosensitive disease. The optimal approach to patients in CR1 is controversial. Options include an autograft^ref [26] or observation, the latter based on promising results with short-term follow-up in patients receiving aggressive induction therapy, including rituximab, without an autograft^ref [27]. This has to be balanced against the strong evidence for an allogeneic graft-versus-MCL effect. Currently our preferred option is a very-low-intensity allograft in CR1 in patients younger than 60 years with a sibling donor, particularly in those with increased ?2-microglobulin at diagnosis. A randomized study addressing observation versus autograft versus RI allograft in CR1 is needed.
• B-cell chronic lymphocytic leukemia (B-CLL) : a durable response to DLI has been reported in chronic lymphocytic leukemia (CLL)^ref [28], although British data suggest that the response may be less frequent than in FL^ref [10]. Promising early results of RI conditioning allografts have been reported by the EBMT registry^ref [29] and Schetelig et al.^ref [30]. The former study reported a 2-year probability of relapse of 31%, with no relapses beyond 12 months in patients receiving T cell-replete grafts, contrasting with ongoing late relapses in those receiving T cell-depleted grafts. The development of chronic GVHD was very significantly associated with a lower risk of relapse. In the latter study, a graft-versus-CLL effect was suggested by the late occurrence of remissions and the effectiveness of DLI in early relapse; DLI was ineffective in patients with a high tumor burden. Also consistent with, but not proof of, a graft-versus-CLL effect are registry data suggesting a lower relapse rate after allografts than autografts, with a plateau in DFS after allografting^ref [31]. Our policy is similar to that with FL. Grafting is offered to suitable patients with disease relapsing early after, or refractory to, purine analog-based therapy. The intensity of conditioning depends on the chemosensitivity and bulk of disease before transplantation.
• Hodgkin lymphoma : the largest series documenting the response to DLI for Hodgkin lymphoma (HL) relapsing after an allograft has been reported recently^ref[32]. Of 7 patients with progressive (n = 6) or residual (n = 1) disease who received DLI in the absence of chemotherapy, 3 achieved a CR, 2 of whom remained in remission with follow-up > 12 months. No response was seen in 2 patients, and a partial remission was seen in the other 2. Responses correlated with the development of GVHD. Of note, most patients had nodular sclerosing histology, had undergone a prior autograft, had disease that had run a relatively indolent course over a number of years, and did not have a rapidly progressive recent relapse. Updated data from this group, reported in abstract form, are consistent with a response rate in approximately half of this selected patient cohort^ref [33]. Responses with and without GVHD after DLI have been reported by other authors, but concomitant chemotherapy, absence of histologic detail, and only short-term follow-up make these data difficult to interpret^{ref1, ref2} [34 and 35]. Longer follow-up of various recently published single-institution and registry series of RI conditioning allografts^{ref1, ref2, ref3} [36, 37 and 38] may provide useful information. The results of the largest of these studies, published in abstract form^ref [37], argue against the existence of a profound graft-versus-HL effect, because the outcome was poor in chemoresistant disease and there was no plateau in PFS in the second year after transplantation. A smaller single-institution study demonstrated little effectiveness of this approach in patients with rapidly progressive disease relapsing early after autografting^ref [39]. Another study used fludarabine/low-dose cyclophosphamide followed by DLI or peripheral blood stem cells in 8 patients with HL relapsing after autografting^ref [36]; 5 did not respond, 2 died of acute GVHD, and only 1 patient was alive in CR, notably, without evidence of donor engraftment. Results of myeloablative allografts for HL are disappointing. The IBMTR reported a 3-year probability of relapse of 65% and a DFS rate of 15% in 100 patients with advanced HL undergoing sibling allografts^ref [40]. There are conflicting data about the effect of GVHD on relapse and whether allografts have a lower rate of relapse than autografts. A trend for a lower probability of relapse after allografting compared with autografting in patients with chemosensitive disease has been reported [41]. An early EBMT registry analysis found a significantly lower risk of relapse with acute GVHD grade II or higher [42]. A more recent EBMT analysis, however, reported no influence of acute GVHD on the rate of relapse and in fact reported a higher risk of relapse after allografts versus autografts for HL, although the former group almost certainly contained a higher proportion of patients with advanced chemoresistant disease^ref [6]. A collaborative international effort is under way among various investigators examining the outcome of patients undergoing RI allografts for HL relapsing after autografts. The effect of factors such as histology (classic versus lymphocyte predominant; the latter clinically behaves as a low-grade B-cell lymphoma and is likely to be immunologically responsive), different conditioning regimens, and acute and chronic GVHD on relapse and survival will be examined in addition to DLI/withdrawal of immunosuppression in biopsy-proven active disease. Prospective studies under consideration include an autograft followed by an RI allograft in poor-prognosis patients, the feasibility of which has been established^ref [43]. An allograft for classic histology is considered only if all of the following conditions apply:
• biopsy-proven relapsed disease after autografting in which the duration of remission after autografting is >6 months.
• the relapse is not rapidly progressive, is not chemosensitive, is not amenable to local radiotherapy (eg, pulmonary relapse), and is preferably not associated with "B" symptoms.
• a second autograft is not feasible (eg, insufficient stem cells)^ref [44].
The choice of conditioning regimen and whether to consider an unrelated donor depends on factors such as patient age, time since previous high-dose therapy, organ function, and degree of donor match. Ideally, these patients should be enrolled on a prospective study (see below).
• diffuse large B-cell lymphoma (DLBCL) : there are only 2 reports of a durable response to DLI after allografting: 1 in a patient with relapsed mediastinal B-cell NHL^ref [2] and the other with Richter transformation of CLL^ref [45]. A remission lasting 140 days after cessation of tacrolimus for relapsed T cell-rich B-cell NHL has been reported; 3 other patients with relapsed DLCL did not respond to withdrawal of cyclosporine and DLI^ref [46]. Failure of DLI despite GVHD has been documented in anaplastic large-cell NHL^ref [47] and in lymphomatoid granulomatosis and B-cell DLCL^ref [48]. 2 patients with high-grade NHL relapsing after a nonmyeloablative allograft did not respond to DLI^ref[35]. Single-institution and registry data in this area are difficult to interpret for the reasons outlined previously. Of note, many RI conditioning studies have been published as abstracts only (without peer review). Most studies have varied with respect to histology, status at transplantation, and T-cell depletion. The EBMT registry reported the results of RI conditioning regimens (mainly fludarabine based and varying from low-dose cyclophosphamide to high-dose melphalan) in 62 patients with high-grade lymphoma, including transformed low-grade disease^ref [7]. The results were disappointing despite most patients having chemosensitive disease before transplantation: the probability of disease progression at 2 years was 79%, with a PFS of 13%. The myeloablative allograft data are inconclusive. There were no survivors in a series of 14 patients who received allografts with a myeloablative regimen for advanced intermediate-grade NHL (predominantly DLCL) reported by the M.D. Anderson group in the mid 1990s; all died of progression or toxicity^ref [49]. A French review found no effect of acute or chronic GVHD (although the data were not provided) in a series of allografts for aggressive NHL, predominantly DLCL^ref [50]. In this series, the 5-year survival was 23% in 48 patients not in CR at transplantation—results not obviously different from those of autografting in a similar patient group. A more recent survey from the EBMT registry reported a lower relapse rate after myeloablative allografting compared with autografting for intermediate-grade NHL by using the Working Formulation [6]. As discussed previously, this category includes follicular large-cell, diffuse large-cell, and diffuse small cleaved (most likely mantle cell) histologies, so the specific GVL effects in these individual histologies cannot be elucidated. Acute GVHD was associated with a reduced rate of relapse, but insufficient data were available to analyze the effect of chronic GVHD. In contrast, the recent review by Bierman et al.^ref [17] showed no difference in relapse rate for intermediate- or high-grade NHL between syngeneic, allogeneic T cell-replete or-depleted, or autologous transplants, although the mix of histologies, imbalances in pretransplantation characteristics, and small numbers (particularly in the syngeneic and T cell-depleted groups) make interpretation of these results difficult. There are few specific data on the outcome of allografting for peripheral T-cell lymphomas. The Milan group have reported in abstract form the outcome of RI conditioning in 8 patients with relapsed nodal peripheral T-cell lymphomas, most with chemorefractory disease and in half of whom a previous autograft had failed^ref [51]. With a short median follow-up of 18 months, all were alive and in remission. Questions that need to be addressed in properly designed studies include the effect of immunophenotype (B versus T cell), histology (de novo B-cell DLCL versus follicular large cell versus transformed low grade), and location (nodal versus site specific, eg, mediastinal or bony) on the outcome after transplantation. We are not convinced that a clinically relevant graft-versus-de novo B-cell DLCL effect commonly exists. Hence, an allograft is not generally offered to patients with primary refractory disease, chemorefractory relapse, or postautograft relapse. Possible exceptions include those with transformed low-grade NHL (based hypothetically on a higher chance of a GVL effect in the presence of an underlying low-grade component) relapsing more than 6 months after autografting without rapid disease progression or mediastinal lymphoma [2]. An allograft is also considered in patients with chemosensitive first relapse in whom sufficient autologous stem cells cannot be collected or in whom the collection is overtly contaminated with lymphoma. Rituximab is offered after allografting to patients with CD20⁺ B-cell tumors who have not previously received this drug, on the basis of promising preliminary experience with this approach [52].
• lymphoblastic lymphoma : a single report of late-onset cutaneous relapse of T-cell lymphoblastic lymphoma (LBL) that responded durably to withdrawal of cyclosporine and a flare of cutaneous GVHD has been published^ref [46]. A recent publication from the IBMTR and the Autologous Bone Marrow Transplant Registry compared autologous versus myeloablative non-T cell-depleted allografts for LBL^ref [53]. The relapse rate beyond 6 months after transplantation was significantly lower in the allograft patients and was independent of disease status at the time of transplantation. A graft-versus-LBL effect cannot be concluded unequivocally from these data, however, because infusion of contaminated autologous cells may have contributed to a higher risk of relapse. GVHD had no significant effect on the risk of relapse, but the study was not powered adequately to detect a small effect. EBMT studies have also demonstrated a lower risk after allografting for LBL and suggested a reduced risk with acute^ref [6] and chronic^ref [54] GVHD. A myeloablative allograft is offered to suitable patients with high-risk LBL in CR1 or those with chemosensitive relapse. Immunosuppression is tapered early in the absence of GVHD^ref [55].
• Burkitt's lymphoma : there is a paucity of data in this area and no convincing evidence that allografting can cure adult patients with Burkitt lymphoma relapsing after, or refractory to, intensive induction regimens. A response to withdrawal of immunosuppression has been reported, but this was not durable^ref [56]. The M.D. Anderson group reported the results of myeloablative allografts in 10 patients with diffuse small noncleaved lymphoma and noted a very high rate of rapid disease progression after transplantation^ref [49]. No difference in relapse rate between autologous and allogeneic transplants for Burkitt lymphoma and no effect of acute GVHD on the rate of relapse have been reported by the EBMT^ref [6]. Allografts are not offered to patients with Burkitt lymphoma, because most patients are either cured by aggressive induction regimens^ref [57] or relapse early with rapidly progressive chemoresistant disease.
• cutaneous T-cell NHL : a graft-versus-mycosis fungoides effect has been reported by a number of investigators. In one case, histologically and molecularly documented persistent disease at day +60 after a nonmyeloablative allograft regressed after withdrawal of cyclosporine and development of cutaneous GVHD. Remission was maintained for ?24 months of follow-up^ref [58]. In a second case, mycosis fungoides recurring 9 months after a myeloablative allograft subsequently resolved after cessation of cyclosporine and development of lichenoid chronic GVHD. However, low-grade cutaneous disease subsequently recurred over the next 4 years and responded temporarily to DLI ^ref[59]. The latter case points out the necessity of long-term follow-up in evaluating GVL effects. We have observed a patient with large-cell transformation of mycosis fungoides in whom the low-grade cutaneous disease regressed with the onset of chronic GVHD but in whom the large-cell component progressed. Resolution after withdrawal of immunosuppression of a cutaneous CD30? large T-cell lymphoma persisting after a nonmyeloablative allograft has been reported^ref [60]. A GVL effect was suggested by infiltration of the skin tumors by donor lymphocytes. Suitable patients with relapsed mycosis fungoides are considered for an allograft. Large-cell transformation is a contraindication.
GVL criteria for various types of lymphoma : there are insufficient data to comment meaningfully for histologies such as peripheral T-cell NHL, hepatosplenic lymphoma, and nodular lymphocyte-predominant HL.

NE indicates not evaluable, ie, insufficient data or duration of follow-up; Allo, allogeneic transplantation; Auto, autologous transplantation; IS, immunosuppression.
A number of collaborative groups have phase II trials under way evaluating the role of RI conditioning regimens in diseases such as HL, in which the existence of a GVL effect is controversial. Enrollment of patients in these prospective studies is strongly encouraged because without a collaborative systemic approach in a large number of patients, it is unlikely that the questions raised in this article will be answered. Ultimately, however, randomized trials will be required for definitive conclusions.
GVL immunobiology : by examining the mechanisms by which malignant B cells may be induced to function as effective antigen-presenting cells (APC) to allogeneic T cells, we may better understand (1) the observed variations in sensitivity of lymphoma subtypes to GVL effects and (2) how to successfully manipulate interactions between T cells and lymphoma to maximize eradication of residual disease while minimizing GVHD. The induction of a GVL effect after allogeneic HSCT is dependent on tumor-antigen recognition, subsequent CTL generation, and sensitivity of the lymphoma to cytotoxicity effector mechanisms. Activation of resting donor T cells follows recognition of alloantigens or tumor-specific antigens expressed on the surface of either professional APCs, such as DC, or lymphoma cells themselves. Host APCs are central to donor T-cell activation, as demonstrated in animal models in which GVHD and GVL effects diminish and are eventually lost as initial mixed APC chimerism evolves to full donor chimerism^{ref1, ref2, ref3} [61, 62 and 63]. Once activated, CTLs mediate the effector phase of GVHD/GVL via cytotoxicity produced either by cell/cell contact mechanisms (Fas, perforin, and TNF-a) or by the production of soluble mediators, including IFN-g and soluble TNF-a^ref [64].

• B-cell lymphoma : the question arises whether B lymphoma cells represent competent APCs for direct activation of alloresponsive T cells or whether B lymphoma cells are subsequent targets for CTLs generated initially by the allostimulatory effects of host DCs. Normal B cells constitutively express both major histocompatibility complex (MHC) class I and II, along with co-stimulatory molecules, including CD80 and CD86, which are essential for generation of CTLs from naive T cells. It is important to note that the activation status of B cells is central to their ability to act as APCs. Resting normal B cells have been variously shown to induce direct tolerance of antigen-specific CD8⁺ T cells^ref [65], induce T-cell anergy via TGF-b production^ref [66], downregulate IL-12 production by DCs^{ref1, ref2} [67 and 68], and influence T_h1 T_h2 differentiation via the production of regulatory cytokines, including IL-10^ref [69]. Similarly, resting B cells exert a regulatory function in in vivo models of T-cell immunity, including tumor rejection^{ref1, ref2} [70 and 71]. Conversely, B cells activated via ligation of CD40 are potent inducers of T-cell activation, which in turn can deliver antitumor immune responses against both non-B-cell^ref [72] and B-cell^{ref1, ref2, ref3} [73, 74 and 75] malignancies in vivo. The expression of co-stimulatory surface-bound molecules is significantly enhanced after B-cell activation by a range of stimuli, including CD40L and LPS^ref [76]. B cells activated by LPS and IL-4, however, fail to induce effector T-cell responses^ref[75], and this may lead to T-cell tolerance^ref [66], thus underlining the importance of the different outcomes of B-cell activation depending on the stimulating signal used. These studies indicate that normal B cells can potentially be induced to act as effective APCs for the generation of T-cell immune responses.
• follicular lymphoma (FL) and mantle cell lymphoma (MCL) : parallel to findings in normal B cells, some malignant lymphoma cells may also demonstrate an APC phenotype. B cells isolated from FL and SLL/CLL share phenotypic similarities to normal resting B cells in both their expression of MHC and co-stimulatory molecules and in their responses to CD40L. These lymphomas consistently show high levels of CD80, CD86, and CD40 expression^{ref1, ref2} [77 and 78]. Similarly, the B cells from MCL express CD40 and show intense upregulation of CD80 and CD86 after treatment with CD40L^ref [79]. The sensitivity of SLL to T-cell cytotoxicity has been demonstrated by studies of autologous vaccination with either DCs^{ref1, ref2} [80 and 81] or killed autologous lymphoma cells^ref [82]. In these studies of autologous immunotherapy, CTL generation against B-cell idiotype was successfully achieved from within the patient’s naive-T-cell population. In the post-allogeneic SCT setting, there is potential not only for a greater range of CTLs to be generated from the donor T-cell repertoire, but also for residual lymphoma B cells to act as functional APCs. Activation of resting lymphoma B cells may occur in response to CD40L provided by donor CD4⁺ T cells alloreactive to host major or minor histocompatibility antigens. When activated by CD40L, the lymphoma B cells, in turn, can drive the subsequent activation of allogeneic CD8⁺ T cells to CTLs. Once generated, both activated CD8⁺ T cells^ref [83] and subsets of CD4⁺ T cells^ref [84] are capable of inducing the cytotoxicity of the stimulatory APCs and eradicating residual disease. This hypothesis is supported by the observation that these lymphoma subtypes are those that show convincing responses to an allogeneic GVL effect.
• diffuse large B-cell lymphoma (DLBCL) and B-cell LBL : in contrast to small-cell lymphoma, DLBCL and B-cell LBL express significantly less CD86^ref [77] and demonstrate MHC class I downregulation in both animal models^ref [85] and clinical samples^ref [86] of B-cell DLCL. These biological features of large-cell lymphomas lead to failure of recognition by CTLs and likely escape from immune attack. There may, however, not be uniformity in the apparent inability of DLBCL to induce T-cell activation. Lymphochip technology has demonstrated that DLBCL can be separated on the basis of gene expression into either a normal germinal center phenotype or an activated peripheral blood B-cell phenotype^{ref1, ref2} [87 and 88]. Although the latter group shows a poorer prognosis when treated with conventional chemotherapy, these genotypic differences may indicate a greater ability to initiate a cytotoxic response by allogeneic T cells. This is countered by the finding that the antiapoptotic genes PDCE4B and PKC are overexpressed in these subgroups. Clinical studies are required to clarify the effect of genotypic differences on susceptibility to GVL effects.
• CTL trafficking : to take advantage of the potential APC function of B cells, T cells must first gain access to the sites of lymphoma involvement. Although naive T cells can freely access the secondary lymphoid tissues, activated CTLs are excluded from doing so by the downregulation of the chemokine receptor CCR7^ref [89]. As a consequence, T cells activated by exposure to alloantigens expressed on APCs outside of the lymph nodes, such as DCs resident within epithelial tissues, are actively excluded from "seeing" residual lymphoma cells within a lymph node. Thus, for a GVL effect to occur, both the activation and effector phases of the GVL effect must occur within the lymphomatous node, as occurs in the lymphoid hypoplasia associated with GVHD^ref [90]. T-cell trafficking to sites of lymphomatous involvement may also vary between lymphoma subtypes. For example, CLL B cells actively attract CD4⁺ T-cell help via the expression of chemokine ligand 22^ref [91]. The implication of these variations in T-cell trafficking is that after allogeneic BMT, residual CLL may actively recruit allogeneic T cells and be activated into APC function, as outlined previously. The requirement for both activation and effector responses to occur within the lymph node may limit the application of adoptive transfer of ex vivo-activated CTLs and may necessitate in vivo CTL generation either by allogeneic transplantation or vaccination strategies. To this end, manipulation of allogeneic T-cell trafficking by using the sphingosine-1-phosphate receptor agonist FTY720, which traps allogeneic T cells within the secondary lymphoid tissues, has been used to promote a GVL effect while preventing the development of GVHD in the peripheral tissues in a haploidentical mouse model^ref [92]. This agent has not, however, been successful in treating established GVHD in a nonidentical canine model^ref [93]. The differences in outcome observed between these models likely reflect the fundamental differences between preventing the onset of GVHD and attempting to suppress activated circulating T cells.
• T-cell lymphoma : the ability of T lymphoma cells to function as APCs in the activation of antilymphoma CTLs is less well described than for B cells. Normal T cells express MHC class I and, when activated, high levels of MHC class II in addition to low levels of co-stimulatory molecules. In vitro studies demonstrate that T cells may also be targeted by CTL clones^ref [94]. Low-grade CTCLs express high levels of MHC class II, which initiate allogeneic cytotoxicity sufficient to eradicate the lymphoma^ref [95]. The challenges faced in the application of allogeneic HSCT to the treatment of lymphomas center on limiting GVHD and identifying those subtypes of lymphoma that are sensitive to CTL-mediated cytotoxicity. It is likely that the B-cell malignancies that are genotypically or phenotypically similar to normal B cells able to differentiate into an effective APC phenotype, sensitive to apoptosis induction and permissive to T-cell trafficking, will be more sensitive to GVL effects. Whether genotype analyses with microarray technology can help in identifying immunoresponsive lymphoma subgroups is unknown. To date, lymphoma-specific antigenic targets for the GVL effect remain limited; hence, it is not yet possible to achieve adoptive immunotherapy with highly selected, ex vivo-expanded GVL-specific CTLs. As a result, means of controlling allogeneic T-cell function are the current focus of research. It has been suggested that delaying T-cell infusions until after resolution of the cytokine storm may maintain the antitumor efficacy of engrafted T cells while limiting nonspecific alloreactivity. This is an attractive hypothesis and has been effectively demonstrated in animal models^{ref1, ref2} [96 and 97], but it is hindered by the dual observations that maximal GVL effect is dependent on a state of mixed APC chimerism and that prediction of GVHD risk at any given dose of DLI is imprecise^ref [98]. Given the difficulties of minimizing GVHD by manipulating T-cell doses or timing, there has been increased interest in the use of regulatory cell populations to restrict the action of allogeneic CTLs. Both regulatory T cells^ref [99] and regulatory DCs^ref [100] are capable of limiting GVHD while preserving the GVL effect. These phenomena are yet to be tested in the clinical setting. Given the central role of host APCs in the induction of GVL after SCT, vaccination with either autologous DCs or irradiated, ex vivo-activated lymphoma cells may lead to enhanced re-stimulation of engrafted allogeneic T-cell responses against minimal residual disease and result in lower rates of recurrence. Mouse models of pretransplantation vaccination of donors^{ref1, ref2, ref3} [101, 102 and 103] and posttransplantation vaccination^{ref1, ref2} [104 and 105] of recipients have been shown to result in enhanced GVT effects without exacerbation of GVHD. These strategies have not yet been demonstrated in a clinical setting, but they offer great potential in the delivery of enhanced immunotherapy as an adjunct to allogeneic HSCT.
GVL effects can be induced in tolerant mixed chimeras prepared with nonmyeloablative conditioning. GVL effects can be amplified by post-grafting DLI. Unfortunately, DLI is frequently associated with GVHD. Intentionally mismatched DLIs, which induce potent GVL effects, can be followed by elimination of alloreactive donor T cells by cyclophosphamide to prevent lethal GvHD. Mice inoculated with BCL₁ leukemia cells and mismatched DLIs were treated 2 weeks later with low-dose or high-dose cyclophosphamide. All mice receiving cyclophosphamide 2 weeks after DLI survived GvHD, and no residual disease was detected by PCR; all control mice receiving DLI alone died of GVHD. Analysis of host (female) and donor (male) DNA showed that cyclophosphamide treatment eradicated most alloreactive donor cells, yet mixed chimerism was converted to full donor chimerism following transient self-limited GVHD. Short-term yet effective and safe adoptive immunotherapy of leukemia may be accomplished early post-transplantation using alloreactive donor lymphocytes, with prevention of GVHD by elimination of GVL effector cells^ref.
Various immunoregulatory cells that inhibit GVHD and induce GVL effect are found after allogeneic HSCT. These cells comprise :
• CD4⁺CD25⁺ regulatory T-cells
• regulatory dendritic cells (rDCs) display high levels of MHC molecules and extremely low levels of costimulatory molecules. A single injection of rDCs following allogeneic BMT controls the ability of the transplanted T cells to induce acute GvHD and GvL effect in the recipients bearing leukemia. This results in protection from the lethality caused by acute GvHD and tumor burden. In conclusion, the use of rDCs might be therapeutically useful for the treatment of acute GvHD and leukemia relapse in allogeneic BMT^ref.
• gd T lymphocytes
• NKT lymphocytes
• NK cells and T lymphocytes with inhibitory NK receptors.
Although the first 4 types of cells effectively inhibit GVHD in animal models, with rDCs showing an inhibitory effect on GVHD in humans as well, the GVL effect was observed only in rDCs. Additional analyses are required to determine whether these cells can inhibit GVHD and exert the GVL effect in humans. In contrast, NK cells and T-cells with inhibitory NK receptors have been shown in humans to possess a suppressive activity against GVHD while preserving the GVL effect. These results indicate that immunoregulatory cells may be used to modulate GVHD and the GVL effect in clinical settings^ref.
Surprisingly, antitumor responses can occur in patients who reject donor grafts following nonmyeloablative HSCT. In murine mixed chimeras prepared with nonmyeloablative conditioning, recipient leukocyte infusions (RLI) induced loss of donor chimerism, IFN-gproduction, and antitumor responses against host-type tumors. Donor marrow rejection is induced by RLI-derived alloactivated T cells, which activate non-RLI-derived, recipient IFN-g-producing cells. RLI-derived CD8 T cells induce the production of IFN-g by both RLI and non-RLI-derived recipient cells. The antitumor responses of RLI involve mainly RLI-derived IFN-g-producing CD8 T cells and recipient-derived CD4 T cells and do not involve donor T cells. The pathways of donor marrow and tumor rejection lead to the development of tumor-specific cell-mediated cytotoxic responses that are not due to bystander killing by alloreactive T cells^ref. In mixed chimeras prepared with nonmyeloablative conditioning, RLI induced loss of donor chimerism and anti-tumor responses against the A20 BALB/c B cell lymphoma. RLI-mediated tumor rejection involved IFN-g-producing RLI-derived CD8⁺ cells and non-RLI, recipient-derived CD4 T cells, leading to the generation of anti-tumor cytotoxic cells. However, the mechanisms of such paradoxical anti-tumor responses remained to be clarified. In the present study, we further explored the cellular mechanisms of the anti-tumor effects of RLI in fully MHC-mismatched and haploidentical strain combinations. In both cases, RLI breaks the tolerance of chimeric T cells toward donor antigens, in association with the in vivo expansion of recipient splenic T, B and CD4^-CD8^- cells and the production of IFN-g. RLI leads to the development of 2 types of tumor-specific responses. The first is mediated by indirect presentation of donor antigens and occurs independently of tumor injection. The second is observed only in recipients of RLI and tumor and may involve responses to self antigens. Anti-tumor cytotoxicity was mediated by CD8⁺ or CD4^-CD8^- effector cells. Thus, anti-tumor cytotoxic responses are generated following complex interactions between recipient APCs presenting donor and recipient antigens and host-type CD4⁺, CD8⁺ and CD4^-CD8^- cells^ref.
• syngeneic or autologous GVHD : cyclosporine A is an immunosuppressive agent widely used to treat autoimmune diseases, allograft rejection, and graft-vs-host disease (GVHD)^ref. However, under certain experimental conditions, CSA can itself induce organ-specific autoimmunity and autologous GVHD (autoGVHD). For example, thyroiditis, gastritis, insulitis, oophoritis, and/or orchitis occur in certain strains of neonatal mice treated with CSA before thymectomy (Tx) and in athymic mice after engraftment of thymic lobes from CSA-treated syngeneic donors^{ref1, ref2}. autoGVHD also occurs in adult rats treated with CSA either before Tx or after irradiation and syngeneic bone marrow transplantation^{ref1, ref2}, similar to acute GvHD after allogeneic HSCT. CSA-induced autoGVHD is mediated by autoeffector T cells that recognize self-MHC class II Ags^ref. Generation of these autoeffector T cells has been linked to defects in both positive and negative selection of thymocytes, the former with interference in calcineurin activation^ref and the latter with alteration in the expression of MHC class II (and to a lesser extent MHC class I) molecules by thymic epithelial and dendritic cells^ref. These and related defects result in the arrested development^{ref1, ref2} and massive release of cortical thymocytes (predominant phenotype in rats, TCR-ß^low,CD4⁺CD8⁺,Thy-1^high,TdT⁺) to peripheral lymphoid tissues during the first week of CSA treatment^{ref1, ref2, ref3}. Eradication of the peripheral immunoregulatory compartment by the preparative regimen provides a permissive environment for the activation of the syngeneic/autologous GVHD effector T cells. Although the repertoire of autoreactive effector T lymphocytes is highly conserved, these T cells promiscuously recognize MHC class II determinants. Interestingly, CD8⁺ T cells that develop during autologous GVHD are also able to lyse myeloma, lymphoma and breast cancer cell lines^{ref1, ref2}. Moreover, the antitumor effect can be enhanced by administration of IFN-g that is principally thought to be due to the up-regulation of the MHC class II target antigen (Ag) on the tumor cells^{ref1, ref2}. T-cell clones associated with autologous GVHD have been isolated in rodents^{ref1, ref2} and are restricted to the TCRb-chain variable region (BV)8.5 allele. Analysis of the effector mechanisms involved in both autologous and syngeneic GVHD unexpectedly showed that there is promiscuous recognition of MHC class II determinants including self by CD8⁺ T cells^{ref1, ref2, ref3, ref4}. The novel specificity and the lack of typical MHC class II restriction element (CD4) on the autoreactive T cells suggest that either a highly conserved structural element or an endogenous superantigen governs recognition of the target antigen^ref. Surprisingly, an element commonly associated with MHC class II molecules exists that may satisfy both possibilities^{ref1, ref2, ref3, ref4, ref5}. CLIP, a peptide from the invariant chain, is highly conserved and has a supermotif for binding to MHC class II molecules^{ref1, ref2, ref3, ref4}. These cells recognize MHC class II determinants in association with a peptide from the invariant chain (CLIP)^{ref1, ref2}. CLIP is a peptide associated with MHC class II  chain and occupies the MHC class II groove^{ref1, ref2}. Certain TCR BV elements, including BV15, interact directly with the superantigen SEB^{ref1, ref2}. It is thought that HLA-DR-bound CLIP stimulates autologous GVHD T cells through the SEB binding site in a superantigen-like fashion, resulting in the induction of T-cell cytotoxicity against MHC class II⁺ cells^{ref1, ref2}. Post-transplantation autocytotoxic activity was inhibited by the pretreatment of target cells with anti-CLIP and anti-HLA-DR antibodies. The main involved organs are skin and bowels^ref : in humans, the BV15 allele appears to be the dominant TCR in cells infiltrating the skin during autologous GVHD^ref. To examine the human effector T-cell response, the T-cell repertoire of peripheral blood lymphocytes was assessed by CDR3 size distribution analysis and T-cell clonotype analysis in 26 patients treated with CsA after autologous HSCT. Autologous GVHD developed in 3 of 4 patients with HLA-DRB1*0701, and clonal expansions of b-chain variable region (BV)16⁺ T cells were shared. Clonal expansions within BV15⁺ and BV22⁺ T cells were also detected in 4 of 6 patients with HLA-DRB1*1501 and in 3 of 4 patients with HLA-DRB1*0401, respectively. Sequencing of BV16 cDNA for which the CDR3 size pattern exhibited apparent clone predominance revealed an identical CDR3 peptide sequence in 2 different patients, one with HLA-DRB1*0701 and the other with HLA-DRB1*1502. These findings indicate that the discrete antigen-driven expansion of T cells is involved in autologous GVHD^ref. Although the induction of syngeneic/autologous GVHD is an untoward effect of CsA immunosuppression, mobilization of these autoimmune mechanisms provides a promising immunotherapeutic approach for certain neoplastic diseases^ref. Inasmuch as thymocytes obtained during the first 12 days of CSA treatment, but not thereafter, can adoptively transfer GVHD to irradiated syngeneic recipients^ref, it has been suggested that autoeffector T cells are exported to the peripheral lymphoid tissues soon after their generation in the thymus^ref. Conversely, reconstitution of the thymic medulla after cessation of CSA treatment seems necessary for the prevention of autoGVHD^ref. The autoeffector cells appear in and are released from the thymus during the first week of CSA treatment, whereas the immunoregulatory thymocytes appear during the second week but are not released until several days after cessation of CSA treatment. Cell separation and mixing experiments demonstrated that the autoeffector T cells persist in LN after cessation of CSA treatment, but their activity is not detectable in the presence of recently exported CD4⁺ T cells^ref. It has been suggested that many self MHC-reactive thymocytes escape negative selection in CSA-treated animals due to reduced MHC class II expression in the thymus medulla^{ref1, ref2}. CSA can also affect positive selection by interfering with calcineurin activation^{ref1, ref2}. However, due to its variable absorption from the gut^ref, it is possible that positive selection may not be blocked completely during the first few days of CSA treatment, when the bulk of autoeffector T cells are exported. This is important, as intrathymic positive selection unopposed by negative selection has been shown to favor the production of autoreactive T cells^ref. The failure of clinical autologous GVHD to develop may be attributed to peripheral regulatory cells that are transferred with the stem cell inoculum^{ref1, ref2}. These findings are consistent with the demonstration that rigorous T-cell depletion of the stem cell inoculum is associated with a greater frequency of autologous GVHD^ref. The determination of gene expression by RT-PCR revealed that IL-10 mRNA levels by PBMCs in patients with autologous GVHD were 29-fold higher than in healthy individuals. IFN-g (4-fold), IL-2 (3-fold), and MIP-1a (44-fold) mRNA levels were also increased in GVHD-induced patients compared with healthy individuals. The ability of PBMCs to lyse autologous PHA-blasts and T47D tumor cells exhibited an identical temporal relationship with expression of IL-10 and IFN-g during autologous GVHD. Moreover, the susceptibility to autologous GVHD as assessed in 75 patients was significantly associated with the IL-10^-1082 G/G polymorphic alleles, allelic variants in the promoter region that govern IL-10 production. These findings indicate that IL-10 may play an unexpected but critical role in autologous GVHD and could be utilized to enhance a graft-versus-tumor effect after transplantation^ref. IL-10 can promote the growth of activated CD8⁺ T cells^{ref1, ref2} leading to the exacerbation of GVHD and the maintenance antitumor function in vivo^{ref1, ref2}. In contrast, IL-10 has a direct inhibitory effect on CD4⁺ T cells in vitro suppressing clonal expansion^{ref1, ref2, ref3, ref4, ref5}. Moreover, IL-10 inhibits APC-dependent T-cell activation by preventing expression of MHC class II, costimulatory molecules, and chemokine ligands on APCs^{ref1, ref2}.

Several clinical trials suggest that the induction of autologous GVHD with CsA (1 mg/kg/day i.v. for 28 days) can reduce the rate of relapse in patients with :
• acute myeloid leukemia^{ref1, ref2, ref3}
• chemo-resistant NHL (10), acute lymphoblastic leukemia (2), chemo-resistant HL (2), acute myeloid leukemia (1), and multiple myeloma (1) (also IFN-g 0.025 mg/m² every other day was started when the total white cell count was >200 cells/ml for 2 consecutive days and continued for a total of 10 doses after ABMT) NHL^ref
• NHL (11), HL (5), AML (4), ALL (1), and MM (1) (also IFN-a_2a)^ref
• various hematological malignancies of childhood^ref and adults^ref
• NHL^ref in remission (also IFN-g)^ref
• relapsed or refractory intermediate grade NHL (also IFN-a)^ref
• NHL of HL relapsed from a prior autologous HSCT (also IFN-a)^ref
• multiple myeloma^ref : anyway MM cells do not express antigen MHC II, which is necessary for GVT effect^ref
• breast cancer^{ref1, ref2} (also IFN-g^ref)
• induction of immune tolerance by intrathymic injection of alloantigens has been used to prevent allograft rejection^{ref1, ref2, ref3, ref4}
Timing :
• acute GvHD (aGvHD / AGVHD) occurs during the first 100 days after transplantation in 40-80% of graft recipients, favored by ... :
• donor host : HLA disparity, minor HA disparity, sex mismatch, age
• graft source : PBSC > BM > cord blood
• HSC dose
• intensity of conditioning
• GvHD prophylaxis
• cytokine gene polymorphisms (IL-6, IL-10, TNF-a)
Localizations & staging^ref :
• skin : maculopapular rash beginning from hand palms, foot soles, face or trunk. Skin responses should be evaluated with skin biopsies
• stage 0 : no rash
• stage I : maculopapular rash on < 25% of body surface
• stage II : maculopapular rash on 25-50% of body surface
• stage III : generalized erythroderma
• stage IV : generalized erythroderma with bullous formation and desquamation
• gastrointestinal tract : diarrhea, abdominal pain, nausea and vomiting
• stage 0 : < 500 ml diarrhea/day
• stage I : 500-1000 ml diarrhea/day or nausea or vomiting or anorexia
• stage II : 1000-1500 ml diarrhea/day
• stage III : > 1500 g diarrhea/day
• stage IV : severe abdominal pain with/without ileus
• liver : destructive lesion of biliary ducts (less intense inflammation than in acute rejection) with variable hepatocellular involvement => hyperbilirubinemia
• stage 0 : < 2 mg/dl (< 34 mM)
• stage I : > 2 mg/dl (34-50 mM)
• stage II : > 3 mg/dl (51-102 mM)
• stage III : > 6 mg/dl (103-255 mM)
• stage IV : > 15 mg/dl (> 255 mM)
• [aGvHD of the lung with atypical presentation^ref]
• endothelium :
• transplant-associated microangiopathy (TAM)
• veno-occlusive disease (VOD) of the liver
• diffuse alveolar hemorrhage
• engraftment syndrome
• capillary leak syndrome
They are more likely to occur after allogeneic HSCT, in unrelated transplantation, or with non-T-cell-depleted grafts. In addition, acute GvHD is a risk factor for all these endothelial syndromes^ref.
Grading^ref :
• Glucksberg-Seattle Transplant Team criteria (GSC) :
• grade 0 :
• grade I : skin stage I to II, no gut or liver involvement, no decrease in clinical performance
• grade II : skin stage I to III or bowel/liver stage I
• grade III : skin stage IV or bowel/liver stage II/III
• grade IV : bowel/liver stage IV
• Severity Index by International Bone Marrow Transplant Registry (IBMTR)

index	skin involvement		liver involvement		gut involvement
	stage max	rash extent	stage max	total bilirubin	stage max	diarrhea volume ml/day
A	1	< 25%			0	< 500
B	2	25-50% or	12	34-102 or	1-2	500-1000
C	3	> 50% or	3	103-255 or	3	> 1500
D	4	bullae or	4	> 255 or	4	severe pain and ileus

It appears more predictive of transplant failure (TF), i.e. relapse or transplant-related mortality (TRM).
• acute GVHD activity index (aGVHDAI)^ref
• total serum bilirubin concentration
• oral intake
• need for treatment with prednisone
• performance score
Risk factors :
• age
• sex matching
• donor parity
• cyclosporin and methotrexate dose intensities
• donor responsiveness to G-CSF is associated with aGVHD following PB HSCT^ref
• donor PBPC grafts mobilized with GM-CSF alone result in prompt hematopoietic engraftment despite lower CD34⁺ cell doses and may reduce the risk of grades II-IV acute GVHD following HLA-matched PB HSCT, without increase in relapse risk or overall survival^ref
• CD56⁺ cells in G-CSF-mobilized PBPC allografts from HLA-identical sibling donors may play an important role in preventing the development of GVHD^ref
Pathogenesis : triggered by alloantigens recognized by T cells that mature in the thymus of the donor. Endogenous donor-derived IL-15 has been implicated in the pathogenesis of aGVHD, yet the mechanism by which it impacts this lethal process remains unclear. Using the well-described and clinically relevant C57BL/6 => B6D2F1 murine model of aGVHD, it was demonstrated that in trans presentation of IL-15 by donor bone marrow-derived cells is required for the rapid onset of aGVHD. Recipients of IL 15^-/- C57BL/6 bone marrow cells show diminished T_h1 polarization of T cells yet there is no decrease in donor T cell reconstitution. A molecular basis for these findings is provided with the observation that expression of T-bet, the master control gene for T_h1 cell functions, is necessary for IL-15-mediated aGVHD lethality. Finally, in the absence of donor-derived IL-15, the GVT effect is maintained. These findings thus establish a mechanism by which endogenous donor-derived IL-15 impacts the pathobiology of aGVHD and GVT activity^ref. Since following NST in comparison with myeloablative conditioning, higher proportions of host immunohematopoietic cells may persist while donor-derived alloreactive lymphocytes are being infused, thus possibly serving as host antigen presentation for continuous stimulation of donor T cells, GVHD may be similarly amplified by conditioning followed by intentional administration of host cells. This hypothesis was tested in a preclinical animal model. Increased incidence of GVHD, higher mortality and increased levels of chimerism were observed in recipients reconstituted with host cells, particularly with non-irradiated spleen cells. GvL effect was not impaired by post transplant cell administration. These results suggest that GVHD may be amplified by recipient cell infusion using either irradiated or viable stimulatory host cells, thus possibly explaining in part higher than anticipated incidence of GVHD and rapid displacement of host cells and conversion to 100% donor type cells following NST. Administration of irradiated host antigen-presenting cells post transplantation may thus represent a potential approach for amplification of the alloreactive capacity of donor lymphocytes following HSCT^ref. In 8 patients with aGVHD of grades II to IV, the CD27⁺/CD27^- ratios of CD4⁺ (but not CD8⁺) T-lymphocytes were significantly higher after allo-BMT, even at day 21, than the ratios in patients with aGVHD of grade 0 or I and remained high after day 21. In contrast, the ratios were low after day 21 following allo-BMT in 14 patients with aGVHD of grade 0 or I^ref.
Laboratory examination :
• hepatic GvHD :
• cutaneous GvHD :
• intestinal GvHD :
• EGDS and colonoscopy : endoscopic findings in GVHD, linked to stage of disease, range from normal mucosa to erythema, edema, erosions,

ulcerations and mucosal sloughing^{ref1, ref2, ref3}. In a retrospective study of patients undergoing upper gastrointestinal (UGI) endoscopy and biopsy between days 20 and 100 following HSCT, no significant differences in symptoms or endoscopic appearance were observed between patients with or without UGI GVHD^ref. Given the nonspecific symptoms and endoscopic findings of acute GVHD and the significant side effects of immunosuppressive medications used to treat GVHD, a histologicdiagnosis should be sought before treatment is initiated^{ref1, ref2}.. The biopsy site with the highest yield was the distal colon (82%). A combination of upper endoscopy with sigmoidoscopy and colonoscopy with ileal biopsies were equivalent (94%). In patients with diarrhea at risk for GVHD, biopsies of the distal colon had the highest diagnostic yield suggesting the importance of sigmoidoscopy and biopsy. Colonoscopy and ileoscopy or flexible sigmoidoscopy plus upper endoscopy had the highest diagnostic yields^{ref1, ref2}
• FDG-PET is a sensitive and specific non-invasive imaging technique to assess intestinal GvHD, map its localization and to predict and monitor treatment responsiveness^ref

 
Prevention :
• host Langerhans cells that persist in the skin following bone-marrow transplantation are responsible for the induction of GvHD
• when T cells are included in the transplant, host Langerhans cells are replaced by donor-derived cells. This occurs by 2 donor-T-cell-mediated processes:
• CD178 / FasL-mediated depletion of host Langerhans cells
• induction of expression of inflammatory chemokines in the skin, which recruit donor Langerhans-cell precursors
However, mice that received whole bone marrow still develop GvHD, because the depletion of host Langerhans cells occurs after donor T cells cause disease.
• when T cells are depleted from the bone-marrow transplant, host Langerhans cells persisted in the skin of lethally irradiated mice, even though the blood and spleen are repopulated with donor-derived dendritic cells. A strategy to eliminate host Langerhans cells before donor T-lymphocyte infusions (DLIs) is the following : first, irradiated mice are reconstituted with T-cell-depleted (TCD) bone marrow, allowing complete chimerism to be established in the blood but not the skin. These mice are then exposed to UV light, which causes host Langerhans cells to migrate out of the skin and allows repopulation with donor-derived cells. After several weeks, the mice are then retransplanted with DLIs. No GVHD develops in these mice, indicating that Langerhans-cell replacement in the skin is required to prevent GVHD following transplantation of T cells. Future experiments will address whether exposing patients to ultraviolet light before transplantation might help prevent GVHD in human recipients^{ref1, ref2}.
Mouse models of minor histocompatibility Ag-mismatched HSCTwere used to study donor DC reconstitution after conditioning, variables influencing the persistence of residual host DCs in different compartments, their phenotype, and their role in governing DLI-mediated alloresponses. Reconstitution of all splenic DC subsets occurred rapidly after HSCTand before T cell reconstitution. However, in contrast to MHC-mismatched chimeras, residual host-derived DCs persisted in the cutaneous lymph nodes (CLNs) of MHC-matched chimeras despite the presence or addition of donor T cells to the graft. The phenotype of these residual host-derived DCs in CLNs was consistent with Langerhans' cells (LCs). Their skin origin was confirmed : there is near-complete preservation of host-derived LCs in the skin. Host-derived LCs retained their ability to continuously traffic to the CLNs, expressed homogeneously increased levels of costimulatory molecules, and could capture and carry epicutaneously applied Ags. To determine the role of residual host LCs in governing DLI-mediated alloresponses, DLI was administered alone or after topical application of the TLR7 ligand imiquimod, which is known to enhance the LC emigration from the skin. DLI administration resulted in a decrease in host-derived DCs in the CLNs and increased recruitment of donor-derived DCs to the skin, whereas imiquimod augmented their alloreactivity. These results suggest uniqueness of the MHC-matched setting in relation to the persistence of host-derived DCs in the skin and points to a previously unrecognized role of host-derived LCs in the induction of DLI-mediated graft-vs-host alloresponses^ref.
• IL-10 plays a negative role in the development of aGVHD and graft rejection : a specific type of the IL-10 gene, found in 25% of North Americans, protects patients against severe aGvHD and dying from the transplant. The 3-year death rate from the transplant was 13% in those with the favorable gene type and as high as 26% in those without the variation. 9% of the patients with the gene variation developed the disease in the 3 months after the transplant, compared with 21% of patients without it. The gene variant didn't protect against cGvHD. Host B cells produce IL-10 following TBI and attenuate aGVHD after allogeneic bone marrow transplantation^ref
• donor APC are required for aGvHD but not for full-strength GVL : using a mouse model of bone-marrow transplantation with or without CD8⁺ T cells between MHC-matched but minor histocompatibility antigen-mismatched donor and recipient, as is the case for most human transplants, mice that were transplanted with b₂-microglobulin-deficient (b₂m^–/–) bone marrow + wild-type CD8⁺ T cells suffered less severe GVHD than did mice receiving wildtype bone marrow and T cells. Because donor APCs derived from b₂m^–/– bone marrow do not express MHC class I and therefore cannot cross-present host antigens, this indicates that donor APC function is required to maintain maximal GvHD and that donor APC depletion would reduce disease. In a mouse model of CML, irradiated mice that received bone marrow plus CD8⁺ T cells generally survived,whereas T-cell depletion of the transplant resulted in death  from CML. The use of wild-type or b₂m^–/– bone marrow did not affect the protective effect of the T cells, showing that donor APCs are not required for GVL killing. Also, because APCs can quickly regenerate from donor stem cells, the immunosuppression that is associated with APC depletion would not be as long lasting as that caused by other strategies, such as T-cell depletion, that are currently used to treat GVHD^ref.
• residual host APCs are required to initiate aGVHD mediated by CD8 T cells
• since following NST in comparison with myeloablative conditioning, higher proportions of host immunohematopoietic cells may persist while donor-derived alloreactive lymphocytes are being infused, thus possibly serving as host antigen presentation for continuous stimulation of donor T cells, GVHD may be similarly amplified by conditioning followed by intentional administration of host cells. In a preclinical animal model, increased incidence of GVHD, higher mortality and increased levels of chimerism were observed in recipients reconstituted with host cells, particularly with non-irradiated spleen cells. GVL effect was not impaired by post transplant cell administration. These results suggest that GVHD may be amplified by recipient cell infusion using either irradiated or viable stimulatory host cells, thus possibly explaining in part higher than anticipated incidence of GVHD and rapid displacement of host cells and conversion to 100% donor type cells following NST. Administration of irradiated host antigen-presenting cells post transplantation may thus represent a potential approach for amplification of the alloreactive capacity of donor lymphocytes following stem cell transplantation^ref
• genetic modification of T lymphocytes with a retroviral vector (RV) expressing the herpes simplex virus-thymidine kinase (TK) suicide gene confers selective sensitivity to the prodrug ganciclovir (GCV). In patients, the infusion of TK⁺ lymphocytes and the subsequent administration of GCV resulted in a time-wise modulation of antihost reactivity for a GvL effect, while controlling GvHD. Because activation required for genetic modification with RV may reduce antihost reactivity, we investigated the requirements for maximizing the potency of human TK⁺ lymphocytes. Whereas T-cell receptor triggering alone led to effector memory (EM) TK⁺ lymphocytes, the addition of CD28 costimulation through cell-sized beads resulted in the generation of central memory (CM) TK⁺ lymphocytes. In a quantitative model for GvHD using nonobese diabetic/severely combined immunodeficient mice, CM TK⁺ lymphocytes were more potent than EM TK⁺ lymphocytes. GCV administration efficiently controlled GvHD induced by CM TK⁺ lymphocytes. These results warrant the clinical investigation of CM suicide gene-modified human T lymphocytes for safe and effective allo-HCT^ref.
• immunization of recipient and donor mice with LPS prior to transplantation, in an attempt to reduce mortality caused by gram-negative bacterial infections posttransplantation. In one experiment, recipient mice were immunized with LPS prior to BMT, whereas in another experiment, donor mice were immunized prior to BMT. The mice were evaluated for development of GVHD and for survival. Injection of low-dose LPS to mice prior to induction of GVHD with allogeneic spleen cells saved > 40% of the recipients, whereas all mice in the untreated control group died. The survival of recipients of spleen cells from immunized donors rose to 54% and clinical signs of GVHD were attenuated as compared to control mice inoculated with spleen cells obtained from unimmunized donors^ref
• 2 types of regulatory T cell in mice, NKT cells and CD4⁺CD25⁺ T cells, can prevent acute GVHD^{ref1, ref2, ref3, ref4}. These regulatory T cells inhibit the proliferation of and cytokine secretion by CD4⁺ and CD8⁺ donor T cells that injure the intestines, liver, and skin in acute GVHD^{ref1, ref2}. Nevertheless, direct tumor-killing activity mediated by donor CD8⁺ T cells remains unaffected^ref. Thus, regulatory T cells can separate GVHD from the antitumor activity of the graft. Regulatory NKT cells of either donor or host origin have the unique capacity to prevent aGVHD by secreting IL-4^{ref1, ref2, ref3}. NKT cells recognize a nonpolymorphic, MHC class I–like antigen-presenting molecule, CD1d, which is present on both the donor and host antigen-presenting cells^ref. NKT cells constitute only 1 to 3% of all T cells in the spleen in normal mice. After repeated treatment with low-dose irradiation targeted to the spleen, thymus, and lymph nodes, however, the proportion of these cells progressively increases until ultimately they constitute the majority of T cells in the spleen and bone marrow^{ref1, ref2}. This change probably results from the resistance of NKT cells, as compared with conventional T cells, to radiation-induced apoptosis due to increased expression of antiapoptotic genes by the NKT cells^ref. In preclinical studies, murine recipients of allogeneic bone marrow that underwent conditioning with anti–T-cell antibodies and repeated low-dose irradiation targeted to the lymphoid tissue (total lymphoid irradiation) were fully protected from GVHD, whereas mice that underwent conditioning with anti–T-cell antibodies and a single dose of total-body irradiation were not protected^{ref1, ref2}. Studies of the pattern of cytokine secretion by donor T cells in protected hosts showed a polarization toward a pattern of T_h2 cells, with increased secretion of IL-4^ref. Although we could not distinguish the separate contributions of total lymphoid irradiation and antithymocyte globulin to protection against GVHD, preclinical studies have shown that the combination of total-body irradiation and antithymocyte globulin fails to provide protection against GVHD^{ref1, ref2}. It is likely that the changes in the function of donor T cells in the patients who underwent total lymphoid irradiation reduced the risk of aGVHD, since donor T cells with a profile of increased Th2 cytokine secretion have a decreased capacity to induce GVHD in rodents^{ref1, ref2, ref3}
• ex vivo methods of treating donor lymphocytes to inhibit GVHD while preserving GVL activity in murine models of allogeneic HSCT. Donor/recipient pairs included MHC fully mismatched, MHC haplomismatched, and MiHA mismatched strain combinations. T cell-depleted BM (TCD-BM) was transplanted in combination with untreated, fludarabine-treated, 7.5-Gy g-irradiated, or psoralen/UVA (PUVA)-treated splenocytes. GVL activity was studied by adding a lethal number of recipient-type lymphoma cells. Posttransplant survival was determined, and GVHD and GVL activity were assessed by clinical and pathological scoring. Hematopoietic chimerism and donor T-cell expansion were analyzed by flow cytometry of peripheral blood samples at 30 and 60 days posttransplant. Allogeneic splenocytes treated with fludarabine, 7.5 Gy g-irradiation, or PUVA had significantly diminished GVHD activity, and all treated donor T cells facilitated engraftment by low-dose TCD-BM. Allogeneic splenocytes treated with fludarabine and, to a lesser extent, PUVA retained GVL activity and contributed more to donor T-cell chimerism compared to g-irradiated donor splenocytes. Among ex vivo methods of treating donor T cells to limit their proliferative capacity, fludarabine exposure had the greatest differential ability to inhibit the GVHD activity of allogeneic lymphocytes, while preserving their GVL activity and ability to engraft recipients. Thus, ex vivo treatment with fludarabine was superior to g-irradiation or PUVA in separating GVL from GVHD activity in murine models of allogeneic HSCT^ref.
• drugs : combination regimens have been shown to be more effective than single agents in preventing GvHD. In the first study that randomized patients to receive cyclosporine or cyclosporine + methotrexate, the combination showed a significant reduction (33% vs 54%, P = 0.014) in the cumulative incidence of clinically significant GvHD^ref.
• cyclosporine A (CsA), along with methotrexate, is the standard of care in HLA-matched sibling transplants. The target levels are between 200 ng/ml and 400 ng/ml, measured by various assay techniques^ref
• tacrolimus : levels may be measured and the target range is 10–20 ng/ml^ref. Because of increased toxicity in the range 15–20 ng/ml, some centers maintain levels 5-15 ng/ml
• methotrexate (MTX)5–10 mg/m² on days +1, +3, +6, and +11 is used in combination with agents such as cyclosporine and tacrolimus. Although the doses of methotrexate used are low, adverse effects such as mucositis, delayed engraftment and liver toxicity can be associated with its use^ref.
• MMF was approved for use in preventing rejection in renal transplants. Recently, it has been studied in combination with cyclosporine as prophylaxis for GvHD related to stem-cell transplantation^{ref1, ref2}. MMF may have a role as a steroid-sparing immunosuppressant in the treatment of acute and chronic GvHD^ref. The usual doses used are 1000 mg orally every 12 hours. In situations where a patient is unable to take oral medication, there is an intravenous formulation available. The conversion to the intravenous dose is 1:1, as the bioavailability of the oral tablet is nearly 100%. Adverse effects related to MMF use are bone marrow suppression and gastrointestinal intolerance.
• sirolimus / rapamycin is an agent approved to prevent graft rejection in solid organ transplants. This agent is not truly a calcineurin inhibitor, in that cyclosporine and tacrolimus inhibit T-cell production of cytokines whereas sirolimus inhibits T-cell response to these cytokines^ref. The side-effect profile of sirolimus is different from cyclosporine and tacrolimus, in that it does not cause renal dysfunction or hypertension but does cause thrombocytopenia, hypertriglyceridemia and hypercholesterolemia^ref. Experience in the setting of stem-cell transplant is limited. Sirolimus may find a niche as an alternative for individuals who are intolerant of traditional calcineurin inhibitors.
• alemtuzumab : initial studies explored the use of this agent to prevent acute GvHD through in vitro purging of T lymphocytes. More recent studies have examined the role of alemtuzumab as part of the transplant conditioning regimen^ref. Infectious complications are the most serious adverse effects associated with alemtuzumab use.
• prednisone has been studied as part of prophylaxis regimens^ref. One study prospectively evaluated a combination of cyclosporine, methotrexate and prednisone against cyclosporine and methotrexate^ref. The results of this trial demonstrated that the addition of prednisone is associated with a lower incidence of early post-transplant complications. However, there was no reduction in the overall incidence of acute or chronic GvHD, event-free survival or overall survival.
Treatment of aGvHD :
Despite measures to prevent GvHD, 30–60% of patients will still develop some degree of GvHD^ref [23], which must be treated.
• steroids remain the standard initial treatment for acute GvHD. Corticosteroids inhibit cytokine production by T cells and macrophages. This halts T-cell activation and macrophage-mediated tissue injury^ref [2]. In the setting of acute GvHD, intravenous methylprednisolone is usually used in doses of 2mg/kg^ref [24]. Adverse effects associated with high-dose steroid use include hypertension, dyslipidemia, glucose intolerance, acne and euphoria
• polyclonal antibodies such as ATG have been used to treat steroid-refractory acute GvHD^ref [25]. The primary target is surface antigens on T lymphocytes. The result is elimination of antigen-reactive T lymphocytes in the peripheral blood and alteration of T-cell function. Antilymphocyte immune globulin has some activity against B cells, platelets and neutrophils. Unfortunately, the response to ATG has not been shown to be beneficial in resolving acute GvHD. Bacigalupo and colleagues^ref [26] recently reported a new strategy, using pre-emptive therapy with ATG in patients at high risk for developing GvHD. High-risk patients were identified on day seven post-transplant, based on serum bilirubin and blood urea nitrogen levels. These patients received three doses of 1.25 mg/kg rabbit ATG on alternate days. The results were promising in that clinically significant GvHD developed in only 27% of the patients in the ATG-treated group, versus 55% for untreated controls.
• monoclonal antibodies have been developed to inhibit T-lymphocyte activity. These agents interact with T cells specifically and spare the B cells, neutrophils and platelets
• muromonab-CD3 (Orthoclone OKT®3, Ortho Biotech Products, Raritan, NJ, USA) is a synthetically derived all-murine anti-CD3 antibody approved for renal transplant rejection^ref [27]. Muromonab-CD3 binds to the CD3 complex on T lymphocytes, which blocks antigen recognition and leads to rapid disappearance of CD3+ T cells from the circulation. In Europe, muromonab-CD3 has been studied in the stem-cell transplant setting for use in steroid-refractory GvHD^ref [28]. In a study by Hebart and colleagues^ref [28], 65% of the patients with severe treatment-refractory acute GvHD responded to therapy with muromonab-CD3 and 25% of these patients were alive at one year post transplant
• daclizumab (Zenapax®, Roche Laboratories Inc., Nutley, NJ, USA) and basiliximab (Simulect®, Novartis Pharmaceutical Corporation, East Hanover, NJ, USA) are humanized antibodies that bind to the CD25 Tac receptor for IL-2. The IL-2 receptor is found on the surface of activated cytotoxic T cells and binding of the monoclonal antibody inhibits T-lymphocyte function. These agents have been used for the treatment, but not prevention, of acute GvHD. Daclizumab was studied in 43 patients with steroid-refractory GvHD^ref [29]. The first group of 24 patients was treated with 1 mg/kg doses on post-transplant days 1, 8, 15, 22 and 29. The complete response rate in this cohort was 29%. Complete response was defined as grade 0 GvHD in the organ system affected, as defined by the GvHD grading criteria. A second group of 19 patients was treated with the same dose but on post-transplant days 1, 4, 8, 15 and 22. The complete response rate in this group was 47%. There were no infusion-related reactions and no serious side effects. In a different study, Willenbacher and colleagues^ref [30] published their experience with daclizumab in 16 patients with acute and chronic GvHD. Response rates were similar to those reported by Przepiorka et al.^ref [29], although the rate of infection was much higher in this series. Of the 16 patients, 14 suffered from infection with daclizumab therapy and 3 patients died due to infection.
• infliximab (Remicade®, Centocor Inc., Malvern, PA, USA) is a chimeric human/mouse antibody that binds to soluble and membrane-bound forms of TNF?. This agent is approved by the US Food and Drug Administration for the treatment of Crohn's disease and rheumatoid arthritis. TNF? is an important cytokine in the inflammatory cascade of acute GvHD [31 and 32]. Infliximab has been used in the setting of steroid-refractory acute GvHD with encouraging results^ref [33]. The agent is generally well tolerated, with some patients experiencing infusion-related reactions. Serious infections may develop because of significant immunosuppression that occurs. Recently, infliximab therapy has been associated with an increased incidence of tuberculosis reactivation^ref [34]. Physicians should be aware of this potential complication and screen patients for latent tuberculosis infection or disease prior to beginning therapy with infliximab
• pentostatin (Nipent®, SuperGen Inc., San Ramon, CA, USA) is an immunosuppressive agent that inhibits adenosine deaminase, thus decreasing lymphocyte number and function. This agent has historically been used for treatment of a variety of malignancies including hairy-cell leukemia. Because of its significant immunosuppressive effect, it is currently being studied as an agent for the treatment of acute GvHD [35].
Chronic GvHD is a major long-term complication of survivors of allogeneic HSCT. This is an immunological complication that affects approximately one-half of transplant patients and can result in 25% mortality^ref [36]. Like acute GvHD, chronic GvHD is mediated by an immunological attack of donor T-cells on host tissue. In acute GvHD, the cytokine production is mainly by T_h1 cells. In chronic GvHD, T_h2 cytokines predominate^{ref1, ref2} [5 and 37]. The primary risk factor associated with chronic GvHD development is prior history of acute GvHD. Other factors that increase a patient's chance of developing chronic GvHD include transplantation of a non-T-cell-depleted graft, which increases HLA disparity, and older age of donor and recipient. Diagnosis and grading of chronic GvHD is difficult and subjective. Clinical manifestations of chronic GvHD are similar to autoimmune diseases and include oral mucosal ulcerations, xerostomia, intrahepatic obstructive liver disease, obstructive pulmonary disease and scleroderma^ref [38]. Immunodeficiency is the manifestation with the most significant consequences in chronic GvHD.
Treatment of cGvHD :
• There are no proven pharmacological agents to prevent the development of chronic GvHD. Corticosteroids remain the primary treatment for chronic GvHD, with or without another immunosuppressive agent such as cyclosporine or tacrolimus. Alternate-day cyclosporine and prednisone is commonly used in the treatment of chronic GvHD^ref [39]. In cases of steroid-refractory chronic GvHD, thalidomide has been used^{ref1, ref2} [40 and 41]. Although patients have a difficult time tolerating the side effects of this agent, its immunosuppressant action has been effective in treating some patients with chronic GvHD.
• Non-pharmacological approaches have also been used to treat chronic GvHD. In extracorporeal photopheresis, DNA-damage in peripheral blood mononuclear cells is caused by irradiation with ultraviolet A after exposing cells to the photosensitizing agent 8-methoxypsoralen (8-MOP)^{ref1, ref2} [42 and 43]. This therapy has improved cutaneous as well as visceral involvement of chronic GvHD. Psoralen and ultraviolet A irradiation (PUVA) has been used to treat refractory cutaneous chronic GvHD^ref [44]. Aubin and colleagues^ref [44] reported on a case series of 11 patients with cutaneous GvHD refractory to standard immunosuppressive treatment. 6 of these patients suffered from chronic GvHD while the remaining five patients had refractory acute GvHD. There was a 70% response rate in the chronic group and 75% response rate in the acute GvHD group. Although there have been some advancements in the treatment of chronic GvHD, this is an area that requires more clinical research. Unfortunately, some patients continue to suffer from chronic GvHD requiring maintenance dosing with agents such as prednisone or cyclosporine. With long-term treatment of GvHD, patients often experience complications such as aseptic necrosis and opportunistic infections. For these reasons it is important to improve the treatment of chronic GvHD.
Therapy :
• stage I-II : 6-methylprednisolone 2 mg/kg for 5-7 days (stage I usually not treated); TRM = 16%-27%^ref; 5-yr OS = 53%^ref; if no response or progression (29%) =>
• stage III-IV : steroid 5-10 mg/kg
• 6-methylprednisolone 5 mg/kg per day for 10 days + ATG 6.25 mg/kg in 10 days does not improve patient outcome (response, TRM, and survival), compared with 6MPred alone. 1 month after randomization, 26% had a CR; 23%, a PR; 33%, stable GvHD; 10%, worsened; and 8%, died.
=> steroid refractory (TRM = 46%^ref-49%^ref; 5-yr OS = 35%^ref)
• gut stage >= II : infliximab
• gut stage = I : investigational mAbs
• ATG^{ref1, ref2, ref3, ref4, ref5} : ORR = 43% (CR = 15%; skin = 59-72%, gut = 27-38%, liver = 14-38%); alive at 6-months : 23%
• anti-T lymphocytes : ORR = 62% (CR = 22%; alive = 28%; skin = 73-100%, gut = 45-100%, liver = 28-100%)
• OKT-3^ref
• BC3^ref
• H65^ref
• visilizumab^{ref1, ref2}
• anti-IL2-R mAbs : ORR = 62% (CR and alive = 36%; skin = 56-83%, gut = 50-75%, liver = 20-25%)
• BT563^ref
• B-B10^ref
• 2A3^ref
• TAC^ref
• basiliximab
• daclizumab^{ref1, ref2}
• denileukin diftitox
• anti-TNF-a :
• infliximab 10 mg/kg/wk for 2-8 infusions : CR = 20%, PR = 40%, no response = 40%, skin = 100%, bowel = 80%, liver = 18%, survival = 37% among responders, 0% among nonresponders^{ref1, ref2, ref3}
• etanercept
• alefacept^ref
• combination mAbs
• infliximab (10 mg/kg weekly, 4 doses) and daclizumab (1 mg/kg, days 1, 4, 8, 15, and 22) were given over 1 month.^ref
• combination chemoimmunotherapy :
• prednisone + daclizumab worse than prednisone alone^ref
• prednisone + BT563^ref
• prednisone + anti-CD5 immunotoxin^ref
• prednisone + ATG^ref
• prednisone + infliximab^ref

	CMV	IFI	EBV and lymphomas	deaths
ATG	12%	24%	1%	56%
anti-CD3	8%	11%	21% (pre-treated with rituximab)	41%
anti-IL-2	21%	6%	4%	35%

• lung => extracorporeal photochemotherapy (ECP) / photopheresis^{ref1, ref2}
• MSC in 8 patients with steroid-refractory grades III-IV GVHD and 1 who had extensive cGVHD. The MSC dose was median 1.0 (0.7-9 x10⁶/kg). No acute side-effects occurred after the MSC infusions. 6 patients were treated once and three patients twice. 2 patients received MSC from HLA-identical siblings, 6 from haplo-identical family donors and 4 from unrelated mismatched donors. aGVHD disappeared completely in 6 of 8 patients. One of these developed CMV gastroenteritis. Complete resolution was seen in gut (6), liver (1) and skin (1). 2 died soon after MSC treatment with no obvious response. One of them had MSC donor DNA in the colon and a lymph node. 5 patients are still alive between 2 months and 3 years after the transplantation. Their survival rate was significantly better than that of 16 patients with steroid-resistant biopsy-proven gastrointestinal GVHD, not treated with MSC during the same period (P = 0.03). One patient treated for extensive cGVHD showed a transient response in the liver, but not in the skin and he died of EBV lymphoma. MSC is a very promising treatment for severe steroid-resistant acute GVHD^ref.
• chronic GvHD (cGVHD)  > 100 days), which can result in the misclassification of patients with clinical features of acute GVHD occurring after day 100 as having chronic GVHD^ref : cGvHD classically arises in about 30% of recipients and rarely > 500 days after transplantation, involving :
• skin (ranging from a maculopapular exanthema to epidermal necrosis)
• major and minor salivary glands
• intestine (diarrhea, malabsorption, and abdominal pain)
• eyes
• neuromuscular apparatus
• liver (caused by cholestatic hepatitis or veno-occlusive disease of liver and marked by serum enzyme abnormalities)
• bronchiolitis obliterans responds infrequently to therapy and is associated with a dismal prognosis.
• central nervous system GvHD remains a controversial entity in HSCT. Animal studies^{ref1, ref2} based on the expression of myosin heavy chain molecules in the CNS support the possibility of brain being a target for the development of GVHD. CNS-GVHD has also been reported in autopsy reports^{ref1, ref2, ref3}. However, diagnostic substantiation of CNS-GVHD is difficult even in the autopsy setting. The very first report that described CNS-GVHD in 1988^ref based on focal lymphohistiocytic aggregates was later found to be EBV related lymphoproliferative disorder as delineated by positive EBER 10 years later^ref. It is particularly important to exclude CNS- PTLD as similar to CNS-GVHD, it may present with perivascular lymphocytic infiltrates. However, most cases of CNS-PTLD are of B-cell phenotype and are associated with EBV infection^ref (Heslop HE. Biology and treatment of Epstein–Barr virus-associated non-Hodgkin lymphomas. Hematology (Am Soc Hematol Educ Program) 2005, 260–266). The accurate distinction of the two entities is critical as clinical management is distinctly different. CNS-GVHD may be considered only if there is no evidence of other diseases with overlapping features, radiographic characteristics of CNS involvement, response to immunosuppressive therapy and adequate documentation of perivascular T-cell infiltrate by histological and immunophenotypic evaluation. The presence of donor T cells in the brain biopsy specimen, although supportive of the diagnosis, should be interpreted cautiously and cannot be used as the sole evidence establishing the diagnosis of CNS-GVHD. CNS-GVHD may present with a variety of clinical manifestations often associated with systemic GVHD. Histologically, 3 distinct pattern of brain involvement is described: one with vasculitis presenting with CNS angiitis^{ref1, ref2} a the second with focal lymphocytic encephalitis and microglia activation^{ref1, ref2}, and a third with leptomeningeal involvement^ref. The finding of lymphocyte populations composed of different subsets in our patients (one case with cytotoxic T cells phenotype and the other with helper T-cell phenotype) of T cells is of interest and may represent different stages of CNS-GVHD in these two cases. Of note, in systemic GVHD CD4 infiltration may predominate in some cases and CD8 in other cases. Usually the CD4 appears earlier and is followed by CD8 but it also depends on the T-cell composition infused and in experimental situations both CD4 and CD8 can behave in a cytotoxic fashion or mobilize cytokines. Of the 10 patients with possible CNS-GVHD described in the English literature^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8, ref9}, histologic interpretation is available in only 5 cases^{ref1, ref2, ref3, ref4}; and ante-mortem histologic diagnosis was established in only one^ref. 2 more cases have extensive immunophenotype and chimeric studies^ref
• endothelium : rarefaction of microvessels caused by the infiltration of alloreactive cytotoxic T lymphocytes. Furthermore, late cardiovascular accidents are more likely to occur in patients treated with allogeneic than autologous HCT, suggesting that an immunological mechanism is involved in the development of atherosclerosis^ref.
Risk factors :
• prior acute GVHD
• increasing patient age
• use of a parous female donor for a male recipient^{ref1, ref2, ref3}
• addition of buffy coat to the marrow inoculum, used successfully to reduce the incidence of graft failure in transfused patients with aplastic anemia, resulted in a significant increase in the incidence of cGVHD^ref
• donor CD152 / CTLA-4 +49 A/G*GG genotype^ref
Pathogenesis : it has not yet been determined if donor postthymic or de novo graft-derived T cells predominate in this aberrant immune response. Impaired thymic negative selection of the recipients will permit the emergence of pathogenic donor-derived T cells that cause cGVHD : thymectomy prevented chronic GVHD^ref. Whether the inciting antigens represent different allelic forms of donor and recipient molecules (alloantigens) or nonallelic endogenous molecules (autoantigens) is also not known. Recent clinical studies have shown that antibody responses against documented allogeneic targets correlate with the development and severity of cGVHD^ref. Since alloantibodies are not thought to play a role in aGVHD, these data further suggest that distinct pathologies underlie these syndromes. cGvHD is in part, T_h2 mediated, and involves thymic damage, resulting in a syndrome of immunodeficiency and an autoimmune disorder : it is associated with increased numbers of peripheral blood CD4⁺CD25^hi regulatory T lymphocytes. The most important risk factor for chronic GVHD is prior history of aGVHD and strategies that prevent aGVHD also decrease the risk of ccGVHD. Other important risk factors are the use of a non-T cell-depleted (TCD) graft, and older age of donor and recipient.
• limited
• extensive
• runting syndrome : GvHD characterized by diarrhea, maculopapular exanthema, hepatosplenomegaly, hemolytic anemia, and pancytopenia
Prevention : prolonged cyclosporin prophylaxis may decrease the occurrence of cGVHD^ref
Therapy : slower and alloreactive B-cell recovery (normally 1 year after allogeneic HSCT)
• steroid 1 mg/kg, but only a minority of patients durably responds to them => sirolimus in combination with tacrolimus and corticosteroids^ref
• limited cGvHD : MMF => extracorporeal photochemotherapy (ECP) / photopheresis^ref => rituximab^{ref1, ref2} : ORR = 50-80%; liver (50%), skin (50-80%), lung (50%), mouth (50-80%), thrombocytopenia (100%), no response on eye and oral cavity mucosae (due to drop in IgA ?), genitalia and muscles
• extended cGvHD :
• skin, mucosae, eyes, liver => extracorporeal photochemotherapy (ECP) / photopheresis^{ref1, ref2} is currently indicated and FDA approved for the treatment of skin manifestations of cutaneous T-cell lymphoma (CTCL), which is a clonally derived skin malignancy of CD4⁺ cells with the phenotype of mature helper T cells. CTCL responds to biological response modification^ref, and ECP produces a high clinical response rate^{ref1, ref2, ref3}. It is felt that this therapy not only augments the function of monocytes but also induces the malignant T cells to undergo a high rate of apoptosis, exerting an antitumor effect through cytokine modulation and modification^{ref1, ref2}. In cGVHD, ECP has been tested quite extensively in small cohorts of patients, and responses were observed in skin, liver, gastrointestinal (GI) tract, mouth, eye, and lung^{ref1, ref2, ref3, ref4} (Sniecinski IPP, Dagis A, Smith B. Extracorporeal photopheresis (EP) is effective treatment for chronic refractory graft versus host disease. Blood. 1998;92: 454a. Abstract 1877; Abhyankar S, Bishop M. Adjunctive treatment of resistant chronic graft-versus-host disease with extracorporeal photopheresis, using UVADEX Sterile Solution [abstract]. Blood. 1998;92: 454a. Abstract 1876). Unfortunately, the literature is difficult to interpret due to the heterogeneity of treatment schedules and diagnostic and response assessment criteria. Greinix et al^ref treated 15 patients with extensive cGVHD failing corticosteroids and reported responses of up to 80% in skin, 70% in liver, and all of the patients with involvement of the oral mucosa. Cases with skin GVHD included sclerodermatous forms with improvement in contractures. Responses in ulcerative cGVHD of the oral mucosa showed resolution in 100% of patients. The procedure was well tolerated, and no major toxicities were reported in this study. Apisarnthanarax et al^ref reported on 32 heavily pretreated patients with cGVHD of the skin, with responses in both lichenoid and sclerodermal forms in about half of the patients (CR 22%, PR 34%). The procedure was also well tolerated in this group of patients^ref. There are several other reports on the efficacy of ECP for the treatment of chronic GVHD in small groups of patients, with overall response rates of 50% and higher in skin, oral, eye, liver, GI, and also lung GVHD^ref (Sniecinski IPP, Dagis A, Smith B. Extracorporeal photopheresis (EP) is effective treatment for chronic refractory graft versus host disease. Blood. 1998;92: 454a. Abstract 1877; Abhyankar S, Bishop M. Adjunctive treatment of resistant chronic graft-versus-host disease with extracorporeal photopheresis, using UVADEX Sterile Solution [abstract]. Blood. 1998;92: 454a. Abstract 1876). A similar response rate and tolerability was observed in children with skin and visceral GVHD^{ref1, ref2}. All of these reports included patients who had received at least 1 line of therapy, in most cases steroids, prior to initiation of ECP. The interpretation of these results is complicated by different factors, including the small number and heterogeneity of patients treated in all of these reports, the diversity of schedules and duration of treatment, the possibility of "delayed" responses to concurrent immunosuppression (eg, steroids), or spontaneous improvement over time. Finally, although a variety of different mechanisms have been proposed to explain the efficacy of ECP and ultraviolet (UV) light in the treatment of GVHD^{ref1, ref2, ref3, ref4, ref5, ref6,ref7, ref8}, we are still in the process of understanding the fundamental pathophysiology underlying cGVHD as well as the biologic effects of ECP on this process. Massive induction of lymphocyte apoptosis^{ref1, ref2}, changes in DC differentiation and function^ref, induction of regulatory T-cell subsets synthesizing IL-10^ref, and, in the long term, restoration of the DC1/DC2 and T_h1/T_h2 balance in favor of DC2/T_h2^{ref1, ref2} in the course of the disease are some of the proposed mechanisms of action of ECP. In 71 patients with severe cGVHD treated with ECP, response rate was 61% (n = 43), and 14 patients had CRs. The best responses were observed in skin, liver, oral mucosa, and eye. Factors affecting outcomes were assessed in the less heavily pretreated subgroup (n = 63). Thrombocytopenia was associated with a lower response rate, and there was a trend toward a higher response rate in de novo cGVHD. At 6 months, a total of 27 (69%) of 39 patients who were alive continued to have a sustained response (CR 4 [10%] of 39, and PR 23 [59%] of 39). The cumulative incidence of steroid discontinuation at 1 year was 22%. The overall survival since initiation of therapy was 53% at 1 year. Response to ECP and platelet count at initiation of therapy were the strongest predictors of nonrelapse mortality (NRM) on univariate analysis. Objective responses were observed in a substantial number of patients with both skin and visceral cGVHD failing corticosteroids and other immunosuppression^ref.

 + MMF => rituximab
• lung : rituximab +/- azathioprine
Prognosis : chronic GVHD has a negative impact on the morbidity and quality of life, as well as in nonrelapse mortality (NRM)^{ref1, ref2}. Despite immunosuppressive treatment, approximately 20-50% of patients who develop cGVHD die from complications associated with this disease^ref. 3 risk factors (RFs) at diagnosis of cGVHD were significantly associated with increased nonrelapse mortality :
• extensive skin involvement (ESI)
• thrombocytopenia (TP)
• progressive type of onset (PTO)
The hazard ratio (HR) for mortality of the patients with prognostic score (PS) between 0 and 2 (intermediate-risk; 1 RF) compared to those with PS 0 (favorable-risk; 0 RF) was 3.7 (95% CI, 1.4, 9.3); the HR for patients with PS equal to or more than 2 (high-risk; > 1 RF) compared with intermediate-risk group was 6.9 (3.8, 12.4). A rare presentation of TP and PTO without ESI yielded a PS of 1.8 (intermediate-risk).
Experimental animal models : few animal models exist for the study of cGVHD. Some of the best-studied models use strain combinations that differ at the mouse MHC equivalent of the human HLA, called H-2^{ref1, ref2}. In humans, HLA disparities certainly increase the likelihood of developing cGVHD. However, approximately 40% of recipients of HLA-matched sibling grafts develop the syndrome. Thus, in the search for ways to accurately model the cGVHD syndrome, investigators are now more frequently turning to the use of donor/recipient strain combinations that are matched at H-2 but differ at other genetic loci (minor alloantigens)^ref. A new mouse model of cGVHD is based on a donor/recipient pair that has not been previously reported, and disease is induced in recipients given the equivalent of a mouse nonmyeloablative transplant. DBA/2 and BALB/c mice are matched at H-2 but have multiple minor antigenic differences. When sublethally irradiated BALB/c mice receive transplants of large numbers of DBA/2 spleen cells (which contain progenitor and mature immune cells), the mice develop a syndrome with features resembling human cGVHD within 20 to 50 days after HCT. These features include sclerodermatous skin changes (more pronounced than in an existing model) and lupuslike abnormalities including serum antibodies to double-stranded DNA and glomerulonephritis. Removal of the thymus in some recipients did not change disease outcome, suggesting that donor postthymic T cells cause this syndrome. More importantly, mature donor B lymphocytes and not de novo B cells that arise after transplantation are associated with disease development. The investigators also address the role of donor T-cell subsets in mediating disease, including the activities of regulatory CD4⁺CD25⁺ cells. Development of cGVHD in this model is dependent upon CD4⁺ and not CD8⁺ T cells, and CD4⁺CD25^- cells are required for cGVHD induction, where as CD4⁺CD25⁺ cells suppress the disease^ref.
Prevention :
• either donor or host APCs can initiate CD4-mediated GVHD in a model that has features of cGVHD. Both donor and host APCs must provide CD80/86-dependent costimulation to elicit maximal cGVHD, and there is no GVHD when both donor and host lack CD80/86. Although either donor or host APCs are sufficient to stimulate skin cGVHD, donor APCs play a dominant role in intestinal cGVHD. Both CD40 and CD80/86 are critical for donor APC function in intestinal cGVHD, but only CD80/86 is required for skin cGVHD. Thus, there are target-tissue-specific differences in APC requirements^ref
• prevention of HBV reactivation : lamivudine if anti-C antibodies and low-titer anti-HBsAg antibodies are detected, even when HBV-viremia is negative
Prevention / therapy strategies :
• T cell depletion (TCD) efficiently prevents GvHD, but leads to :
• impaired engraftment : one approach to overcoming immune rejection of incompatible HSCs rigorously depleted of T cells made use initially of increased doses of TCD bone marrow in mice^{ref1, ref2, ref3, ref4} and rats^ref. Subsequently the cell-dose escalation concept was also shown with purified stem cells^{ref1, ref2, ref3, ref4}. However, although this modality has become a clinical reality in the treatment of patients with leukemia conditioned by intensive chemotherapy, it has been suggested in studies in mice^ref and nonhuman primates (X. Yao, unpublished data, July 2001) that the number of hematopoietic precursors required to overcome the immune barrier in hosts pretreated with sublethal regimens cannot be attained with the state-of-the-art technology for stem cell mobilization. Rachamim et al^ref demonstrated that when purified CD34⁺ cells were added to bulk mixed-lymphocyte reaction (MLR) they suppressed CTLs against matched stimulators but not against stimulators from a third party^ref. These results strongly indicated that cells within the human CD34⁺ population are endowed with potent veto activity. Clearly, the limitations of CD34⁺ collection in humans might be overcome if it was possible to supplement these cells with other veto cells provided the latter also lack GVH reactivity. Despite their remarkable potent veto activity, CD8 CTLs could not be used for tolerance induction in allogeneic stem cell transplantation due to their marked GVH reactivity. To address this problem we developed a new approach for the generation of host-nonreactive CTLs, based on stimulation of donor CD8 T cells against third-party stimulators under IL-2 deprivation. This approach is based on the observation that only the activated CTLs are capable of surviving the IL-2 starvation in the primary culture^ref. The conditions that afford large numbers of anti–third-party CTLs under IL-2 deprivation were optimized,, and the efficacy of such cells was tested to induce specific transplantation tolerance without GVHD (Bachar-Lustig E, Reich-Zeliger S, Reismen Y. Synergism between rapamycin and host-nonreactive veto CTLs in murine models for BM allograft rejection: a new approach to facilitate engraftment of T cell depleted allogeneic BMT under reduced intensity conditioning. Blood. 2002;100: 211a). Furthermore, considering that previous studies documented the role of the immunosuppressive drug rapamycin in GVHD prevention^ref and in overcoming rejection^ref, the potential synergy between rapamycin and veto CTLs was evaluated. Anti–third-party CTLs generated under IL-2 deprivation afford a suitable source for effective veto cells that can enhance BM allografting without GVHD. In particular, these cells are effective if applied in conjunction with rapamycin. This synergism is compatible with the recent demonstration that veto CTLs operate via Fas-FasL triggering upstream of rapamycin inhibition of IL-2R signaling. Thus, rapamycin does not block the veto activity of CTLs but rather enhances their tolerizing effect so that effector T cells that might have escaped deletion by the veto cells could still be eliminated by rapamycin. To analyze the relative contribution of each agent to BM allografting we used a murine model that enables distinguishing between T-cell–mediated immune rejection and stem cell competition. In this model, rejection of T-cell–depleted BM allografts from Nude donors is induced in lethally irradiated mice by adoptive transfer of graduated numbers of purified host-type T cells. Stem cell competition is minimal under this conditioning and, in the absence of T-cell infusion, the host mice uniformly accept the allogeneic BM without GVHD. Thus, the observed graft rejection can be attributed to the adoptive transfer of purified host T cells. It could be argued that the facilitating activity of the anti–third-party CTLs is associated with a possible contamination of host-reactive T-cell clones, which may not be sufficient to induce GVHD but could potentially eliminate the infused host-type T cells. This possibility is ruled out by 2 experiments: (1) anti–third-party CTLs that are not of donor origin, prepared by the same procedure used to generate the CTLs of donor origin, do not enhance engraftment of BM allografts; and (2) anti–third-party CTLs of (host x donor) F₁ origin, lacking alloreactivity due to their genetic background, enhance BM allografting similarly to the CTLs generated from the donor genetic background. The effective number of the veto CTL that was found to synergize with rapamycin in the graft rejection model is rather high. Although state-of-the-art technology enables large-scale expansion of T cells, it is likely that the potential synergy between the combined administration of veto CTLs and other tolerizing cells such as CD4⁺CD25⁺ and/or Sca-1⁺Lin^– cells along with the use of maximal tolerable doses of rapamycin might further reduce the effective CTL dose. An alternative approach to allow the use of anti–third-party CTLs has been suggested recently by Fowler et al^ref41 who showed that TC2 CTLs are more effective veto cells compared with TC1 CTLs, and the former cells are also depleted of GVH reactivity^ref.42 While we favor depletion of host-reactive CTL-p by IL-2 starvation, the 2 approaches might be combined in the future to further enhance veto potency and reduce host reactivity of anti–third-party CTLs. As could be anticipated from the studies in the graft rejection model, marked synergism between rapamycin and veto CTLs was also observed upon transplantation of Nude Balb/c BM into sublethally irradiated C3H hosts. In this model we have previously shown that purified Sca-1⁺Lin^– stem cell transplants can overcome rejection if very large numbers are administered. Thus, in evaluating the results in this model, it is important to note the contribution of veto cells within the stem cell compartment or other facilitating cells within the non–T-cell compartment. Indeed, while administration of both rapamycin and veto CTLs was required to secure engraftment upon transplantation of 4 x 10⁶ Nude BM cells, a high level of chimerism could be obtained by using CTLs without rapamycin when the BM cell dose was increased by 20%. These results suggest a quantitative relationship between the residual host antidonor CTL-p pool on one hand and the BM veto CTLs and rapamycin on the other. Preliminary results using (host x donor) F₁ CTLs to distinguish between the infused CTLs and host- or naive donor–derived T cells, suggest that the infused CTLs survive for at least a few weeks after transplantation. In principle, when an adequate number of CTLs are infused and engraftment of donor cells prevails, it can be assumed that the majority of alloreactive host CTL-p has been eliminated. If the latter cells are not eliminated and are allowed to mature, graft rejection is more likely to take place. This quantitative relationship between BM dose, veto CTLs, and rapamycin could have immediate implications to allogeneic BM transplantation (BMT), not only under reduced-intensity conditioning but also in the treatment of leukemia patients undergoing full myeloablation. Presently, the conditioning of hosts of T-cell–depleted BM or of purified CD34 cells, includes antithymocyte globulin (ATG), the serum levels of which remain high for a prolonged period of time. While this agent might reduce graft rejection and potential GVHD it can also interfere with immune reconstitution during the early period after transplantation. Considering the documented effect of short-term treatment with rapamycin on GVHD^ref and on graft rejection^ref in murine models, we anticipate that the combination of anti–third-party CTLs with rapamycin might successfully replace ATG in the conditioning of leukemia patients, even in the context of haploidentical transplants in which the risk for graft rejection and GVHD is significantly higher compared with HLA-identical transplants. Clearly, achieving engraftment under reduced-intensity conditioning represents a major challenge due to the relatively large number of residual host T cells that might survive the conditioning. At present, such transplantations are carried out predominantly in elderly patients who have an HLA-identical sibling or an unrelated donor. Engraftment in these patients is greatly enhanced by the presence of a large number of alloreactive T cells in the graft, which are also associated with > 20% GVHD lethality. The latter problem is even more pronounced in elderly patients with MM or with B-CLL. Thus, the use of purified CD34 cells in conjunction with anti–third-party CTLs and rapamycin may prove beneficial for such patients. If this approach will indeed prove to be associated with low transplant-related mortality, it could be used as a prelude for cell therapy with donor lymphocyte infusions or with cell lines or clones directed against specific host-type minor hematopoietic antigens^{ref1, ref2, ref3} or against tumor-specific antigens^{ref1, ref2}. In this context, it is assumed that the CTLs are depleted of GVL reactivity and they are predominantly used to induce tolerance for donor cells and, subsequently, other more specific cell lines or clones might be needed to eradicate disease. However, it is possible that antitumor or antiviral clones within the infused CD8 cells are not completely diminished and might be expended upon proper stimulation in vivo. Further quantitative studies using the TcLandscape method developed by Guillet et al^ref to define the repertoire of these anti–third-party CTLs are in progress. In addition, we found recently that anti–third-party human CTLs generated by a similar approach are endowed with an effective capacity to eradicate tumor cells from patients with B-CLL. This TCR-independent recognition between veto CTLs directed against third-party HLA and the B-CLL cells was shown to be mediated by LFA1–ICAM1 (F. Arditti and Y. L., unpublished results, 2003). Finally, if successful, this approach could be further extended to treat diseases that are lethal in the course of years but do not present an immediate threat, such as nonmalignant hematologic diseases or in the induction of tolerance for organ transplantation.
• prolonged immune deficiency => increased rate of infections and post-transplant lymphoproliferative disorder (PTLD) (unless also B cells are depleted)
• impaired GvL => increased frequency of leukemia relapse
Recipients who have undergone a TCD BMT to achieve mixed haematopoietic chimerism, followed by delayed administration of donor lymphocyte infusions (DLIs), achieve GVL effects from the DLIs, without GvHD. Anyway administration of DLI is often precluded by low-grade acute GvHD^ref. The delay in administration of DLIs allows conditioning-induced inflammation in epithelial tissues - that are the target of GvHD - to subside, so that the GvHD-mediating T cells are not drawn from the lymphohematopoietic tissues to these target tissues. The problem with this approach is that leukemias can often progress rapidly, so it would be better to enhance the GVL effect closer to the time of transplant. In murine models of allogeneic BMT, MHC-mismatched recipients given a delayed DLI at 21 days posttransplant develop significant GvHD whereas MHC-matched recipients do not : thymus-derived, Thy1⁺abTcR⁺ donor cells (CD4⁺CD8^- subpopulation and a CD4^-CD8^- subpopulation) generated early after allogeneic BMT suppress the graft-vs-host reactivity of T cells given as DLI. These cells may mediate dominant peripheral tolerance after allogeneic BMT^ref.
The current options for patients who relapse after an alloSCT include
• a second transplant : attempts to perform a second allogeneic HSCT have been successful in a minority of patients, primarily children, but these transplants have been associated with an extremely high treatment-related mortality^ref
• removal of immunosuppression
• IFN-a in selected cases of CML
• donor lymphocyte infusions (DLIs)^{ref1, ref2} has been successfully used in both children and adults^ref. However, DLI has severe limitations, including a decreased activity in patients with acute leukemias or bulky disease (eg, NHL), a delayed response (weeks to months), and potentially severe toxicity in the form of GVHD, which is observed in more than 50% of patients^{ref1, ref2, ref3}. DLI has been associated with relatively high response rates in patients with chronic myelogenous leukemia (CML) in chronic phase^{ref1, ref2, ref3}, but it has been less effective for patients with more advanced CML and other hematologic malignancies^ref. In addition, the response to DLI can take several months, and DLI may be associated with severe marrow aplasia and a high incidence of cGVHD
• filgrastim may be effective in treating leukemic relapses after alloSCT in selected cases^{ref1, ref2, ref3}. These observations were made in patients with both chronic and acute leukemias who received filgrastim to treat cytopenias that had occurred with relapse^ref. Filgrastim for relapse offers potential advantages over DLI. First, it is well tolerated; the primary acute side effects being those typically associated with filgrastim administration, including mild arthralgias and low-grade fevers. It may, however, be associated with the development of GVHD, both acute and chronic, which was observed in approximately 54% of patients in this analysis. Second, the response is relatively rapid; by a week after the completion of therapy (ie, 28 days), responses could be reasonably assessed. This may provide adequate time to make arrangements to secure donor leukocytes in the event that the donor is not readily available. Most importantly, marrow aplasia, which is observed in a significant number of patients receiving DLI, was not observed with filgrastim^{ref1, ref2}. Similar to results with DLI, filgrastim administration had limited efficacy in patients with overt hematologic relapse of their acute leukemias. In this situation, cytoreduction of the leukemia may be necessary, either with conventional chemotherapy or a nonmyeloablative preparative regimen, which seems to have success in patients with sensitive disease^ref. The primary question is why would this therapy work at all? There are several potential explanations. The first is that filgrastim had little or no effect, and the observed responses were a result of the removal of immunosuppression, permitting a GvL effect^ref. This may not provide an adequate explanation, however, because 2 of the observed responses occurred in patients receiving no immunosuppression at the time of their relapse. This clinical situation is not significantly different than for DLI where immunosuppression is also routinely withdrawn^{ref1, ref2}. A second possible explanation is that the filgrastim provides a competitive advantage for normal hematopoiesis, potentially increasing the numbers of effector cells^ref. Another related explanation is that the increased hematopoiesis after filgrastim dilutes the leukemic cells present in the marrow, and the observed results were actually a false-positive. This seems highly unlikely given the high sensitivity of FISH and the continued maintenance of response long after the acute effects of filgrastim had subsided. A last possible explanation for the efficacy of filgrastim is a direct effect on the immune system. The administration of filgrastim is known to cause a shift of T cells from T_h1 to T_h2 cytokine pattern and a reduction of cytotoxic T-cell activity^ref. These effects would seem contradictory to an enhanced antileukemic response; however, it has been demonstrated that cytotoxic T-cell activity is preserved through a perforin-dependent pathway after filgrastim exposure^ref. The administration of filgrastim, either alone or in conjunction with discontinuation of immunosuppression if present, can result in rapid, complete cytogenetic remissions for patients who relapse after alloSCT. Further studies are required to substantiate these results and to provide further understanding of this treatment. However, given the ease of administration, minimal related complications, and the relatively rapid assessment of response, it seems reasonable to consider filgrastim as an initial treatment option for relapse after alloSCT. It should be considered as an adjunct and not a replacement to other treatments, particularly DLI. A practical strategy could include the use of filgrastim as initial management for relapse, permitting time to make arrangements for donor leukocyte collection. In the event there was no response to filgrastim, DLI could then be used with little predicted loss in efficacy. A more interesting alternative would be a direct comparison of filgrastim administration with DLI. The administration of filgrastim seems to be a practical and effective option for cytogenetic or molecular leukemic relapses after alloSCT
• EDG6 / S1P₄ agonistscan trap lymphocytes in the lymph nodes and prevent the GvHD-mediating T lymphocytes from reaching the target tissues, they might be beneficial in reducing GvHD even around the time of BMT when tissue inflammation is present and could alow the administration of DLIs at the time of haplo-identical BMT
• enriching the donor bone marrow with CD4⁺25⁺ T_reg cells
• infusion of backup autologous peripheral blood HSCs^ref
• anti-CD20 monoclonal antibodies
• single-agent
• indolent lymphomas^{ref1, ref2, ref3, ref4}  : pneumonia (0.6%)
• B-CLL^{ref1, ref2, ref3} : pneumonia (3.9%), sepsis (0.8%), HSV/VZV (0.8%), URTI (11%)
• relapsed or refractory indolent lymphomas^{ref1, ref2, ref3, ref4, ref5} : pneumonia/sepsis (4.2%), HHV (4.2%), URTI (13.8%)
• + CHOP/MACOP^{ref1, ref2, ref3, ref4, ref5} : death (1.2%), pneumonia/sepsis (19.5%), fungal infections (1.2%), but due almost all to CHOP alone^{ref1, ref2}
• + fludarabine^{ref1, ref2, ref3, ref4} + cyclophosphamide^{ref1, ref2}, but due almost all to fludarabine
• persistent parvovirus infection : pure red cell aplasia resolved with IVIg 0.5 g/kg/weekly
• + CHOP^{ref1, ref2}
• + fludarabine and cyclophosphamide^ref
• anti-CD52 monoclonal antibody : 6 months after therapy are required to reachieve a 200 T cells/ml level^ref
• single agent in refractory CLL : death (6%), pneumonia/sepsis (16.8%), fungal infections (7.8%), HSV/VZV (5%), CMV (12.4%), similar to allogeneic HSCT !^{ref1, ref2}
• + fludarabine^{ref1, ref2} (Rai ASH 2002) : CMV (23%), death (3.9%)

Prevention : acyclovir, cotrimoxazole, fluconazole and CMV antigenemia measurements for 6 months
• + rituximab^ref : pneumonia/sepsis (29%), CMV (27%)
• GvHD prophylaxis^{ref1, ref2} :
• in vitro on donor cells (TCD)
• in vivo 20 mg : "in the bag", no wash, also in the recipient; no increase in infections and rapid immune reconsitution^{ref1, ref2}
• conditioning (100 mg) : late CMV reactivation (78.9%; plateau at month 9) => disease (4%)^{ref1, ref2} (Rizzieri ASH 2001), respiratory viruses (parainfluenza 46%; RSV 76%)^{ref1, ref2}, URTI/pneumonia
• BEAM
• Flu/Mel
• Flu/TBI450
• Flu/Cy
• Flu/Bu
• TBI200
Prevention : acyclovir 10 mg/kg 3 times a day (max 750-800 mg/day), cotrimoxazole and CMV PCR for > 1 year
aGvHD risk is related to number of monocytes^{ref1, ref2}. Alemtuzumab is more effective than ATG because host^{ref1, ref2, ref3, ref4} or donor^ref DCs (which express CD52 and are rapidly depleted by alemtuzumab^ref) initiate GvHD.
Post-allogeneic HSCT reconstitution : 6 months for B lymphocytes (naive recovering faster than memory) and T lymphocyte, 1 year for both myeloid and plasmacytoid DCs. Addition of alemtuzumab delays recovery of T lymphocytes to 1 year, B lymphocytes to ?, while DCs recover normally^ref!
Microsatellites are highly polymorphic repetitive sequences (1-6 nucleotides) of unknown function that are scattered throughout the genome^ref. They are inherited stably and are unique to each individual which makes them ideal tools for forensic purposes as well as for documentation of engraftment following allogeneic hematopoietic cell transplantation (HCT)^{ref1, ref2}. Ever since microsatellite length alterations were detected in hereditary nonpolyposis colorectal carcinoma and sporadic cancers, especially squamous carcinomas, microsatellite instability (MSI) has gained substantial attention and has been used as an indicator of genomic instability^{ref1, ref2}. Although typical in cancers, MSI has more recently also been found in non-neoplastic, chronically inflamed tissues from patients with ulcerative colitis and rheumatoid arthritis^{ref1, ref2, ref3}. The exact mechanism through which chronic inflammation produces MSI remains to be elucidated. To maintain genome integrity cells are equipped with several distinct but not completely independent DNA repair systems such as mismatch repair (MMR), base excision repeat (BER), and nucleotide excision repair (NER)^ref. Existing hypotheses postulate that either oxidative stress in inflamed tissues overwhelms the capacity for DNA repair, or that overproduction of free radicals inactivates the DNA repair pathways directly^{ref1, ref2, ref3, ref4}. The biologic meaning of the MSI found in chronic inflammatory settings is not yet clearly defined. Unlike ulcerative colitis, in which MSI has been associated with progression to cancer, MSI in rheumatoid arthritis does not indicate an increased cancer risk^{ref1, ref2}. After allogeneic HCT, epithelial tissues become injured through the preparative regimen and are then potentially attacked by alloreactive T cells. Graft-versus-host (GvH) reactions are complex processes involving release of cytokines, recruitment of inflammatory cells, cellular cytotoxicity, and production of free radicals, among others^ref. The net effect of these alloantigenic reactions is tissue stress which we recognize clinically as GvH disease (GvHD), and this most frequently occurs in colon, skin, mouth, and liver. In addition, various delayed posttransplantation clinical syndromes such as pulmonary or musculoskeletal disease as well as secondary malignancies have been linked to chronic GvHD^ref. On the basis of the concept that chronic tissue stress may cause genomic alterations, we examined genomic instability in epithelium after allogeneic HCT by MSI. Microsatellite analysis performed on nonmalignant epithelial tissues found frequent microsatellite alterations in the colon and oral mucosa of patients who underwent allogeneic HCT. Eexamination of similar tissues from both patients who did not receive a transplant and patients who received an autograft performed under exactly the same conditions did not result in any aberrant bands. Endler et al^ref examined mouthwashes of allograft recipients and found a surprisingly frequent chimeric pattern, even after magnetic separation of epithelial cells. Furthermore, additional bands which were of neither host nor donor origin were found by gel electrophoresis and were explained by the researchers as being heteroduplexes generated during PCR between donor and recipient DNA. What does the MSI found in non-neoplastic tissues after transplantation point out? Replication errors during DNA synthesis occur with a certain probability in all cells and may result in a change of the length of microsatellite loci, which are sequences particularly prone to DNA polymerase slippage because of their repetitive nature^{ref1, ref2}. The MMR system recognizes and corrects these genomic alterations^ref. Whenever the DNA error cannot be repaired, apoptosis pathways are activated^ref. Thus, cells with microsatellite alterations which escaped repair will not replicate and therefore should not become apparent among the large excess of the surrounding normal cells^ref. However, if deficient DNA repair is coupled with a failure to elicit an apoptotic response, this association may result in a growth advantage sufficient to generate a detectable clonal population of cells that share genetic alterations. Therefore, the detection of MSI in a clinical sample, as are the ones in our study, demonstrates the presence of populations which were first induced to change the length of their microsatellites, then escaped DNA polymerase proofreading and finally selected for clonal outgrowth^{ref1, ref2, ref3, ref4}.  Why does MSI occur? We found MSI in non-neoplastic epithelial tissues obtained from patients treated with myeloablative or reduced-intensity chemotherapy and allogeneic HCT but not from patients after myelosuppressive chemotherapy or myeloablative chemotherapy combined with autologous HCT. In addition, MSI in buccal cells was not evident at short intervals after myelosuppressive or myeloablative chemotherapy but was found many years after conditioning for HCT. Therefore, although chemotherapeutic agents alone may induce microsatellite alterations in individual cells in vitro^ref, it is very likely that an "alloantigenic" effect is the driving force in producing detectable MSI in the patients who received an allograft. Additional work is needed to characterize in detail the nature of the alloantigenic stimulation that is operative in MSI generation. The observation that all colon biopsies with histologically detectable GvHD but one were MSI+, indicates a relation between organ GvHD and MSI. However, no GvHD signs were evident in one MSI+ colon biopsy or in the oral mucosa of any of the 10 subjects with MSI+ buccal smears. The time of sampling when GvH reactions have just appeared or already been censored or the presence of only subclinical GvH signs are only some of the reasons that could explain this lack of correlation. Analysis of further tissues which, unlike the colonic and the buccal mucosa, are not typical targets of GvHD might be informative in this regard. Interestingly, MSI in buccal smears was found significantly more frequently at later time points after transplantation, which would suggest that the duration of alloantigenic reactions might be of importance in generation of MSI. How does MSI occur? Studies in chronic inflammatory diseases postulate that oxidative stress may lead to accumulation of genomic alterations either by overwhelming the capacity for DNA repair or by directly inactivating DNA repair pathways through epigenetic gene silencing or through damaging of DNA repair proteins^{ref1, ref2, ref3}. Although the MMR system is responsible for correcting DNA strand loops typical for MSI, studies suggest that the different DNA repair systems do not act independently and that MSI may be attributable to alterations in DNA repair pathways distinct from MMR^ref. Immunostaining of colon sections after allogeneic HCT for key proteins of 3 different DNA repair systems (MMR, BER, NER) showed widespread expression comparable with the one found in colon sections from healthy individuals who did not receive a transplant. Therefore, silencing of DNA repair genes is very unlikely to be the underlying mechanism explaining the MSI found here, although components further downstream of the repair systems (eg, polymerases) should also be evaluated^ref. NO produced by cytokine-stimulated (eg, TNF-a) epithelial cells has been found to participate in animal GvHD and enhanced plasma NO levels have been associated with GvHD severity in humans^{ref1, ref2}. NO has been found to inhibit DNA repair through protein nitrosylation and inactivation^ref. Although we did not find increased levels of nitrosylated proteins in the colon biopsies after allogeneic transplantation, we cannot exclude that NO is a link between chronic alloantigenic stimulation, relaxation of DNA repair mechanisms, and MSI. Alterations in noncoding microsatellites are unlikely to provide any growth advantage, ultimately leading to clonal outgrowth of the microsatellite-altered cells, which is required to detect MSI. How these clonal cells with novel repeat lengths are selected is unclear. Inactivation of p53 and the failure of cellular demise has been suggested as playing a mechanistic role in the occurrence of MSI in sporadic tumors and in chronically inflamed synovial tissue^{ref1, ref2, ref3, ref4}. Another hypothesis requiring further study is the role of the immune system in the selection of clones with microsatellite alterations. In hereditary nonpolyposis colorectal carcinoma, it has been suggested that cells with frameshift mutations avoid elimination by the immune system through abolishing expression of MHC proteins^ref. If this is so, the donor-derived immune system in an allogeneic setting might attack the recipient-derived, normal epithelial cells, yet spare cells with an MSI phenotype, providing in this way a selective growth advantage for the cells with novel repeat lengths. This is clearly an area that requires additional investigation. What does the MSI found in non-neoplastic epithelial tissues after allogeneic HCT mean? MSI has been mainly associated with cancer^ref or cancer risk in chronic inflammatory diseases^ref. Classical MSI in hereditary nonpolyposis colorectal carcinoma caused by inherited defects in MMR genes is characterized by mutations especially at mononucleotide and dinucleotide repeats, leading to the typical "ladder" effect in PCR microsatellite analysis. Interestingly, the form of MSI found in our study, namely tetranucleotide alterations detected as single aberrant sequences, is similar to the one frequently seen in sporadic squamous cancers with intact MMR systems and termed EMAST^{ref1, ref2, ref3}. Furthermore, squamous cell carcinomas are the most common secondary tumors after allogeneic HCT^ref. However, our findings do not show an association between MSI phenotype and the risk of posttransplantation malignancy. First, the percentage of subjects with MSI phenotype we found is much higher than the expected incidence of posttransplantation malignancies. Second, MSI was frequently found in colon, but colon carcinoma is a very uncommon secondary malignancy. Third, the frameshift alterations found in posttransplantation tumors differed from those in the associated nonmalignant tissues. Despite this lack of correlation, it is quite possible that multiple MSI lesions could arise in a single subject, few, if any, of which ever accumulate the additional genetic abnormalities required to progress to a malignancy. Impaired DNA repair may indeed be the link between the MSI phenotype and pathogenesis of posttransplantation solid tumors. MSI has been shown to be an indicator of genomic instability^ref. It is very likely that mutations occur not only in nontranscribed microsatellites but also in coding regions of the genome^ref. Mutations in coding genes might be responsible for the protean posttransplantation clinical syndromes and phenotypes such as scleroderma, Sjögren syndrome, musculoskeletal or pulmonary disease, and many others. In addition to serving as an indicator for genomic instability, mutations of microsatellites may directly contribute to evolution of posttransplantation diseases. A number of transcribed genes have been found to contain microsatellites within their coding regions, and changes in the lengths of these repetitive sequences have been shown to result in gene inactivation or modification of function^{ref1, ref2}. Examination of a larger number of patients, refinement of the selection of microsatellite markers, and correlation with clinical data may reveal important clinical associations.
Donor veto cytotoxic T lymphocytes (CTLs) can facilitate the induction of donor type chimerism in sublethally irradiated recipients without causing GvHD if they are effectively depleted of alloreactivity against host cells by means of stimulation against a third party. Human CTLs were prepared in 2 major steps:
• primary culture in the absence of IL-2, leading to death by neglect of antihost clones
• addition of IL-2 and subsequent dilution of antihost clones as a consequence of the expansion of the anti-third-party clones.
CTLs prepared in this way specifically suppress host cytotoxic T cells directed against antigens of the donor, but not against fourth-party antigens, as demonstrated in a standard ⁵¹Cr release assay. Human anti-third-party CTLs afford a new source of veto cells that are depleted of potential graft-versus-host-reactive clones. The cells generated by this approach could potentially be used to facilitate engraftment of allogeneic hematopoietic stem cells^ref.
Web resources : Immunobiology of Transplantation
• Web resources
• Cell injury and adaptation by James H.Harrison
• The lightning hypertext of disease by Pathology Informatics, Inc.
• The Pathguy
• Thesaurus of Cytopathology at the Internet College of Cytology
• Usenet sci.med.pathology
• WebPath : The Internet Pathology Laboratory for Medical Education