Evaluation of Iron Deficiency Using Reticulocyte Indices in Dogs Enrolled in a Blood Donor Program.

BACKGROUND: People donating blood more than twice annually are at risk of developing iron deficiency. Little is known about the iron status of dogs enrolled in blood donor programs. HYPOTHESIS: Dogs donating blood ≥6 times annually will show evidence of iron deficiency based on their reticulocyte indices. ANIMALS: Thirteen dogs enrolled in a blood donor program donating ≥6 times over the preceding 12 months and 20 healthy nondonor control dogs. METHODS: Prospective observational study. Mature red blood cell (RBC) indices, reticulocyte indices, serum iron, serum ferritin, and total iron-binding capacity (TIBC) were compared between groups. RESULTS: Packed cell volume (median 47%, range 40-52%, P < .01), hematocrit (median 46.4%, range 40.3-52.5%, P < .01), and reticulocyte count (median 16,000/µL, range 9,000-38,000/µL, P < .01) were significantly lower in the blood donor dogs. No statistically significant differences were noted in the mature RBC indices between groups. Both reticulocyte mean corpuscular volume (median 88.8 fL, range 83.4-95.5 fL, P = .03) and reticulocyte hemoglobin content (median 24.6 pg, range 23.1-26.6 pg, P < .01) were significantly lower in the blood donor group. Serum iron and ferritin were similar between groups; however, TIBC was significantly higher in the control group (median 403 µg/dL, range 225-493 µg/dL, P = .02). CONCLUSIONS: The findings in dogs donating ≥6 times annually suggest the presence of iron-deficient erythropoiesis in this population.

[Therapeutic monoclonal antibodics]. / Les anticorps monoclonaux thérapeutiques.

Less than ten years after the discovery of hybridomas by Kohler and Milstein in 1975, the first monoclonal antibody was injected in humans to prevent graft rejection. The antibodies used were targeted against the CD3 molecule involved in the transduction of T cell signals after recognition of the antigen. Paradoxically, because of pharmaceutical hesitations later found to be unwarranted, the therapeutic class developed slowly. It was not until the 1990s that other formulations emerged. In parallel, work was undertaken to modify the antibodies using genetic engineering techniques to humanize the molecules by changing the majority of the species determinants from the murine producing animals to the human recipients. It was even found possible to produce totally human antibodies using several different strategies based on in vitro phage display. This led to rapid development of new antibodies used for a wide range of indications: organ transplantation (anti-CD2, anti-CD4, antiCD25, antiCD40L), as well as anti-respirator syncytial virus (RSV) for respiratory infections, and anti cancer agents (Anti-CD20 antibodies and anti-tumor antibodies), as well as autoimmune diseases (with significant success with anti-TNF antibodies). It was found the monoclonal antibodies offer an exceptionally effective method in domains where conventional compounds have reached their limit. This specific action of monoclonal antibodies, with fine specificity for the targets (which can be varied to infinity), as well as their capacity to provide positive signals to cells whose functions are not often inhibited by small molecules. An illustration of this mode of action is the effect recently demonstrated by our team on restored self tolerance using anti-CD3 antibodies in newly diagnosed diabetics in whom sustained remission can be injected after less than one week's treatment.

A thymulin analogue peptide with powerful inhibitory effects on pain of neurogenic origin.

The effects of a synthetic peptide analog of thymulin (PAT) were tested on nociceptive behavior in two animal models for peripheral mononeuropathy and in another two models for capsaicin-induced hyperalgesia. Treatment with PAT (0.25-25 microg/rat, i.p.) produced significant reduction of the mechanical allodynia and heat hyperalgesia in rats subjected to either chronic constriction injury (CCI) or spared nerve injury (SNI) models for mononeuropathy. Cold allodynia was moderately reduced in the CCI model. The inhibition of neuropathic manifestations peaked at 1-2 h post-treatment and disappeared in 3-4 h. Daily treatment with PAT, however, produced progressive attenuation of all neuropathic manifestations in the SNI model. On the other hand, pretreatment with similar doses of PAT produced dose-dependent reduction of the hyperalgesia induced by intraplantar injection of capsaicin (10 microg in 50 microl). The highest dose of PAT (50 microg) produced significant reduction of abdominal aversive behavior induced by i.p injection of capsaicin (20 microg in 100 microl). Compared with the effects of treatment with morphine or meloxicam (injected at single doses known to produce analgesia), PAT exerted equal or stronger inhibitory effects on neuropathic manifestations. The reported results suggest a possible direct action of PAT on afferent nerve fibers but its mechanisms remain to be determined.

In vivo blockade of the Fas-Fas ligand pathway inhibits cyclophosphamide-induced diabetes in NOD mice.

There is compelling evidence to show that insulin dependent diabetes ensues from selective apoptosis of pancreatic beta-cells mediated by autoreactive T-lymphocytes. The respective implication in this phenomenon of the various apoptotic pathways driven by Fas, perforin, or tumor necrosis factor is still ill- defined. Here we took advantage of the cyclophosphamide-induced model of accelerated diabetes in NOD mice to explore the physiopathological role of the Fas-Fas Ligand pathway. A single injection of cyclophosphamide (200 mg/kg) to 7-8 week-old prediabetic NOD mice triggered diabetes within 10-15 days in 85-100% of the animals. Cyclophosphamide also induced a significant decrease in spleen T cells, that was most evident by days 6-10 after treatment, and selectively affected the CD3(+)CD62L(+)compartment that includes immunoregulatory T cells. To block the in vivo Fas-Fas ligand (Fas L) interaction we administered a biologically active recombinant fusion protein coupling mouse Fas to the Fc portion of human IgG1 (FAS-Fc). Mice treated with FAS-Fc (10 doses iv of 15 microg) starting on the day of cyclophosphamide injection up to day 22, were fully protected from disease. Unexpectedly this protective effect was not due to blockade of Fas-FasL-mediated beta-cell apoptosis but rather to the inhibition of the cyclophosphamide effect on T cells. Indeed FAS-Fc treatment prevented the drug-induced T cell depletion in general and that of immunoregulatory T cells in particular. Additionally, FAS-Fc administration limited to the phase of beta-cell destruction did not afford any protection.

Anti-titin antibodies in myasthenia gravis: tight association with thymoma and heterogeneity of nonthymoma patients.

BACKGROUND: Titin is the major autoantigen recognized by anti-striated muscle antibodies, which are characteristic of generalized myasthenia gravis (MG). OBJECTIVE: To seek a correlation between anti-titin antibodies and other features of MG patients, including histopathology, age at diagnosis, anti-acetylcholine receptor (anti-AChR), autoantibody titers, and clinical severity. METHODS: A novel, highly specific radioligand assay was performed on a large group of 398 patients with generalized MG. RESULTS: Among thymectomized patients, anti-titin antibodies were present in most patients with thymoma (56/70 [80%]), contrasting with only a minority of patients with thymus atrophy or hyperplasia (17/165 [10%]). They were also present in 64 (41%) of 155 nonthymectomized patients who had a radiologically normal thymus. In these patients and in those who had a histologically normal thymus, anti-titin antibodies were associated with a later age at onset of disease and with intermediate titers of anti-AChR antibodies. After controlling for these 2 variables, disease severity was not significantly influenced by anti-titin antibodies. CONCLUSIONS: Anti-titin antibodies are a sensitive marker of thymoma associated with MG in patients 60 years and younger, justifying the insistent search for a thymoma in MG patients of this age group who have these antibodies. In nonthymoma patients, anti-titin antibodies represent an interesting marker complementary to the anti-AChR antibody titer, identifying a restricted subset of patients. These clinical correlations should prompt further studies to examine the mechanisms leading to the production of anti-titin antibodies.

Protective role of infections and vaccinations on autoimmune diseases.

Infectious agents may induce autoimmune disease through several mechanisms, notably antigen mimicry and inflammation of the target organ; conversely, infections may protect from autoimmune diseases. This paradoxical effect has been demonstrated for a number of bacteria, viruses and parasites on a variety of spontaneous or experimentally induced animal models of autoimmune diseases (e.g. experimental allergic encephalomyelitis, lupus mice, non-obese diabetic mice). The mechanisms of the protection are still ill-defined, and probably vary according to models. Stimulation of immunoregulatory CD4 T cells has been shown to play a central role in several major models. The role of superantigens is also important, like that of Toll-like receptors. Antigen competition is another major mechanism, itself open to several interpretations. Epidemiological data support a protective role of infections on human allergic and autoimmune diseases. These diseases are much more common in countries with high socio-economic development (typically Northern countries in Europe). The reason for this cannot be fully explained by genetic differences because migrating populations develop these diseases with the same incidence of the adoptive country rather than that of the country of origin. It is interesting that the frequency of these diseases has been increasing in developed countries over the last 20 years but not in undeveloped ones.

The activity of immunoregulatory T cells mediating active tolerance is potentiated in nonobese diabetic mice by an IL-4-based retroviral gene therapy.

Splenocytes from nonobese diabetic mice overexpressing murine IL (mIL)-4 upon recombinant retrovirus infection lose their capacity to transfer diabetes to nonobese diabetic-scid recipients. Diabetes appeared in 0-20% of mice injected with mIL-4-transduced cells vs 80-100% of controls injected with beta-galactosidase-transduced cells. Protected mice showed a majority of islets (60%) presenting with noninvasive peri-insulitis at variance with beta-galactosidase controls that exhibited invasive/destructive insulitis. Importantly, in all recipients, the transduced proteins were detected within islet infiltrates. Infiltrating lymphocytes from recipients of mIL-4-transduced cells produced high levels of mIL-4, as assessed by ELISA. In recipients of beta-galactosidase-transduced cells, approximately 60% of TCRalphabeta(+) islet-infiltrating cells expressed beta-galactosidase, as assessed by flow cytometry. The protection from disease transfer is due to a direct effect of mIL-4 gene therapy on immunoregulatory T cells rather than on diabetogenic cells. mIL-4-transduced purified CD62L(-) effector cells or transgenic BDC2.5 diabetogenic T cells still transferred disease efficiently. Conversely, mIL-4 transduction up-regulated the capacity of purified immunoregulatory CD62L(+) cells to inhibit disease transfer. These data open new perspectives for gene therapy in insulin-dependent diabetes using T cells devoid of any intrinsic diabetogenic potential.

Genetic and physical mapping of a type 1 diabetes susceptibility gene (IDDM12) to a 100-kb phagemid artificial chromosome clone containing D2S72-CTLA4-D2S105 on chromosome 2q33.

Polymorphic markers within the CTLA4 gene on chromosome 2q33 have been shown to be associated with type 1 diabetes. Therefore, a gene responsible for the disease (IDDM12) most likely lies within a region of <1-2 cM of CTLA4. To define more precisely the IDDM12 interval, we genotyped a multiethnic (U.S. Caucasian, Mexican-American, French, Spanish, Korean, and Chinese) collection of 178 simplex and 350 multiplex families for 10 polymorphic markers within a genomic interval of approximately 300 kb, which contains the candidate genes CTLA4 and CD28. The order of these markers (D2S346, CD28, GGAA19E07, D2S307, D2S72, CTLA4, D2S105, and GATA52A04) was determined by sequence tagged site content mapping of bacterial artificial chromosome (BAC) and yeast artificial chromosome (YAC) clones. The transmission disequilibrium test (TDT) analyses of our data revealed significant association/linkage with three markers within CTLA4 and two immediate flanking markers (D2S72 and D2S105) on each side of CTLA4 but not with more distant markers including the candidate gene CD28. Tsp analyses revealed significant association only with the three polymorphic markers within the CTLA4 gene. The markers linked and associated with type 1 diabetes are contained within a phagemid artificial chromosome clone of 100 kb, suggesting that the IDDM12 locus is either CTLA4 or an unknown gene in very close proximity.

Quantitative radioligand assays using de novo-synthesized recombinant autoantigens in connective tissue diseases: new tools to approach the pathogenic significance of anti-RNP antibodies in rheumatic diseases.

OBJECTIVE: To describe new assays for the detection and quantification of antibodies to RNPs in rheumatic diseases, using soluble nuclear antigens synthesized de novo in reticulocyte lysates. METHODS: Sera from 381 patients with various rheumatic diseases, including 212 patients with systemic lupus erythematosus (SLE), were analyzed in order to evaluate the sensitivity and specificity of serum autoantibody reactivities to several recombinant soluble autoantigens: U1-A RNP, Sm-B, SSA/Ro 52 and SSA/Ro 60, SSB/La, and Ku. Radioligand assays (RLAs) were performed following the in vitro transcription and translation of each autoantigen from the corresponding complementary DNA, labeled with 35S-methionine. The radiolabeled protein was then bound by the specific serum autoantibody, forming immune complexes that were captured by protein A-Sepharose beads and quantified by counting the radioactivity. RESULTS: Among the SLE patients, 44% were positive for anti-U1-A RNP activity, 34% for anti-Sm-B, 44% for anti-SSA (32% for Ro 52 and 46% for Ro 60), 32% for anti-SSB/La, and 11% for anti-Ku reactivities. SSA antibodies had a high frequency in patients with mixed connective tissue disease (MCTD) (80%); 65% of these patient sera reacted with Ro 52, 45% with Ro 60, and 45% with U1-A RNP. Twenty percent of the MCTD patients also exhibited antibodies to Sm-B and Ku. In patients with Sjögren's syndrome, anti-SSA was the main anti-RNP antibody (63%), together with SSB/La antibodies (44%). Among patients with inflammatory myopathy, only antibodies against Ro 52 (36%) and Ro 60 (36%) were present. These new RLA allowed observation of a strong correlation (P < 0.0001) between Sm-B antibody levels and the severity of SLE (as measured by the SLE Disease Activity Index), and establishment of a correlation between anti-U1-A RNP antibodies and the occurrence of SLE nephritis (P < 0.02). All RLAs were highly specific for the antigen tested and displayed, in the disease groups studied, a higher sensitivity than conventional immunodiffusion assays. CONCLUSION: These highly sensitive, specific, and quantitative RLAs represent new tools for the detection of autoantibodies to RNP antigens in rheumatic diseases, and may be useful for (differential) diagnosis in clinical practice.

Presence of antinucleosome autoantibodies in a restricted set of connective tissue diseases: antinucleosome antibodies of the IgG3 subclass are markers of renal pathogenicity in systemic lupus erythematosus.

OBJECTIVE: To study the frequency and disease specificity of antinucleosome antibody reactivity in diverse connective tissue diseases (CTD), and to determine factors, such as antibody subclass, that may influence the pathogenicity of these antibodies in relation to disease activity. METHODS: IgG and IgM antinucleosome activities on nucleosome core particles from 496 patients with 13 different CTD and 100 patients with hepatitis C were measured by enzyme-linked immunosorbent assay (ELISA). Of the patients with CTD, 120 had systemic lupus erythematosus (SLE), 37 had scleroderma (systemic sclerosis; SSc), 20 had mixed connective tissue disease (MCTD), and 319 had other CTD, including Sjögren's syndrome, inflammatory myopathy, rheumatoid arthritis, primary antiphospholipid syndrome, Wegener's granulomatosis, Takayasu arteritis, giant cell arteritis, relapsing polychondritis, Behçet's syndrome, and sarcoidosis. Antinucleosome-positive sera were further analyzed, by isotype-specific ELISA, for antinucleosome and anti-double-stranded DNA (anti-dsDNA) IgG subclasses. RESULTS: SLE, SSc, and MCTD were the only 3 CTD in which antinucleosome IgG were detected (71.7%, 45.9%, and 45.0% of patients, respectively). Antinucleosomes of the IgG3 subclass were present at high levels in patients with active SLE and were virtually absent in those with SSc, MCTD, or inactive SLE, and their levels showed a positive correlation with SLE disease activity. Of note, an increase in levels of antinucleosome of the IgG3 isotype was observed during SLE flares, and this increase was found to be closely associated with active nephritis. Levels of antinucleosome of the IgG1 subclass showed a trend toward an inverse correlation with SLE disease activity. No significant fluctuation in the anti-dsDNA isotype profile was observed in relation to SLE severity or clinical signs. CONCLUSION: Our data suggest that IgG antinucleosome is a new marker that may help in the differential diagnosis of CTD; antinucleosome of the IgG3 isotype might constitute a selective biologic marker of active SLE, in particular, of lupus nephritis.

CD3 antibody-induced IL-10 in renal allograft recipients: an in vivo and in vitro analysis.

BACKGROUND: The first administration of CD3 monoclonal antibodies, such as anti-human CD3 (OKT3), induces a massive release of several cytokines, including tumor necrosis factor alpha (TNF-alpha), interferon (IFN)-gamma, interleukin (IL)-2, IL-3, IL-6, and granulocyte-macrophage colony-stimulating factor. METHODS: Cytokine levels in patient's sera were measured by specific ELISA. In vitro cultures were performed using OKT3-stimulated peripheral blood mononuclear cells and/or whole blood from patients and normal controls. RESULTS: Here we describe that OKT3 administration to human renal allograft recipients also leads to a significant release of IL-10. Contrasting with most OKT3-induced cytokines, such as TNF-alpha whose release is transient, IL-10 levels show a more progressive increase, they peak only by 4-8 hr after the first OKT3 injection and persist longer. Thus, significant IL-10 levels are still detectable at the time of the second and the third OKT3 injection. Administration of corticosteroids, 1 hr before the first OKT3 injection, significantly reduced both TNF-alpha and IL-10 release. Experiments were performed to evaluate the source(s) of IL-10 and its (their) influence on the initial T-cell activation. When stimulated in culture with soluble OKT3, the production of IL-10 was dependent on the cooperation between T lymphocytes and monocytes. It is important that, as assessed through the use of a specific neutralizing antibody, the endogenous IL-10 produced in the co-culture system exerted a negative feed-back on the release of the other pro-inflammatory CD3-induced cytokines, which was reproducible. CONCLUSION: These results are supportive of a major role of IL-10 in the down-modulation of the OKT3-triggered T-cell activation cascade.

Truncating mutations in CCM1, encoding KRIT1, cause hereditary cavernous angiomas.

Cavernous angiomas are vascular malformations mostly located in the central nervous system and characterized by enlarged capillary cavities without intervening brain parenchyma. Clinical symptoms include seizures, haemorrhage and focal neurological deficits. Cavernous angiomas prevalence is close to 0.5% in the general population. They may be inherited as an autosomal dominant condition in as much as 50% of cases. Cerebral cavernous malformations (CCM) loci were previously identified on 7q, 7p and 3q (refs 4,5). A strong founder effect was observed in the Hispano-American population, all families being linked to CCM1 on 7q (refs 4,7). CCM1 locus assignment was refined to a 4-cM interval bracketed by D7S2410 and D7S689 (ref. 8). Here we report a physical and transcriptional map of this interval and that CCM1, a gene whose protein product, KRIT1, interacts with RAP1A (also known as KREV1; ref. 9), a member of the RAS family of GTPases, is mutated in CCM1 families. Our data suggest the involvement of the RAP1A signal transduction pathway in vasculogenesis or angiogenesis.

Soluble tumor necrosis factor receptors in chronic hepatitis C: a correlation with histological fibrosis and activity.

BACKGROUND/AIMS: Tumor necrosis factor-alpha (TNF) is a mediator of inflammation and cellular immune response. Soluble TNF receptors (sTNFR) sTNF-R55 and sTNF-R75, which compete with cellular receptors for the binding of TNF, have been detected at high levels in infectious diseases including human immunodeficiency virus and HBV infection. In order to investigate the activation of the TNF system in HCV infection, we have analyzed the balance between TNF and sTNF-R in 60 HCV-infected subjects according to their clinical, biological, virological and histological characteristics. METHODS: Serum TNF, sTNF-R55 and sTNF-R75 levels were determined by ELISA before any therapy and were compared to a control group of 60 healthy subjects and a group of 34 HBV-infected patients. RESULTS: Mean TNF levels were 50.5+/-4.5 pg/ml in HCV patients, and undetectable (<5 pg/ml) in the control subjects. sTNF-R55 and sTNF-R75 levels were significantly higher in HCV-infected patients than in the controls: 2.88+/-0.14 ng/ml vs. 1.30+/-0.05, (p = 0.0001), and 9.54+/-0.58 ng/ml vs. 4.19+/-016, (p = 0.0001), respectively. sTNF-R55 and TNF-alpha levels in HCV patients were not significantly different from levels in HBV patients. sTNF-R75 levels were slightly lower than in HBV patients (9.54+/-0.58 vs. 11.4+/-0.79 ng/ml, p = 0.03). In contrast to other infectious diseases, there was no correlation between levels of sTNF-R and TNF. sTNF-R75 but not TNF levels were correlated with aminotransferases levels (p = 0.0001 and p = 0.0015 for aspartate and alanine aminotransferase, respectively), while sTNF-R55 levels were significantly correlated only with aspartate aminotransferase levels (p = 0.003). sTNF-R75 levels were significantly correlated with the Metavir activity index (p = 0.01), and sTNF-R55 and sTNF-R75 levels were significantly higher in patients with vs. without cirrhosis (3.22+/-0.21 vs. 2.54+/-0.17 ng/ml (p<0.02) and 11.6+/-0.86 vs. 7.5+/-0.53 ng/ml (p<0.001), respectively). sTNF-R55, sTNF-R75 and TNF levels were not correlated with viral load, genotype or response to interferon therapy. CONCLUSIONS: Levels of soluble TNF receptors, and particularly sTNF-R75, are significantly correlated with the severity of the disease but not with virological parameters such as quantitative viremia and genotype. High TNF-R production could thus suggest that HCV-related liver disease involves immunological mechanisms, including activation of the TNF system.

Are there unique autoantigens triggering autoimmune diseases?

Using three reference disease models--insulin-dependent diabetes mellitus (IDDM) as a prototype of T-cell mediated organ-specific autoimmune disease, myasthenia gravis (MG) as a prototype of autoantibody-mediated organ-specific autoimmune disease and systemic lupus erythematosus (SLE) as a prototype of non-organ-specific autoimmune disease--we have reached several conclusions: 1) All three diseases are associated with the presence of multiple autoantibodies and/or autoreactive T cells that recognize a large number of antigenic molecules. The apparent predominant role of certain antibodies in some diseases could relate to their functional properties such as acetylcholine receptor (AChR) blockade for anti-AChR autoantibodies in MG or anti-dsDNA in SLE. 2) Major target antigens are clustered in the target cell affected by organ-specific autoimmune diseases: beta cells in IDDM, striated-muscle cells in MG, or apoptotic cells in the case of SLE. 3) Antibodies and T cells recognize multiple epitopes in these molecules. 4) The most evident explanation for the observed clustering and diversity is autoantigen spreading. Spreading probably involves T cells secreting proinflammatory cytokines but also possibly antibodies as in the case of nucleosome autoantibodies in SLE. 5) The counterpart of antigen spreading is bystander suppression in which regulatory cytokines deviate the immune response towards a protective response. 6) The mechanisms underlying the initiation of the autoimmune response and antigen spreading are still undetermined. They could imply a direct abnormality of the target cell in the case of organ-specific autoimmune diseases (e.g. infection with a virus showing a selective tropism for the target cell in organ-specific autoimmune diseases, or loss of physiological regulation of major histocompatibility complex molecule expression) or could be consequence of a ubiquitous cell abnormality such as increased apoptosis in SLE. The respective roles of genetic and environmental factors in these triggering events remain to be determined.

N-terminus cleavage of bcl-2 by a novel cellular non-ICE cysteine proteinase.

The bcl-2 protein plays an essential role in preventing cell death. Its activity is regulated through association with bcl-2 homologous and nonhomologous proteins and also by serine phosphorylation. We now report that bcl-2 can be proteolytically cleaved towards its N-terminus by a cysteine proteinase present in RL-7 lymphoma cell lysates, yielding a major product of apparent MW 20 kDa, different from the products of bcl-2 cleavage by HIV protease. Moreover, bcl-2 proteins mutated for Asp residues at positions 31 and 34 were efficiently cleaved by RL-7 cell lysates, indicating that this proteolytic activity is distinct from the caspase-3 that cleaves bcl-2 at Asp 34. This bcl-2 cleaving activity is inhibited by E-64 and is therefore distinct from the proteinases of the ICE/Ced-3 family (caspases), whereas reciprocally, ICE (caspase-1) is unable to cleave bcl-2. It is optimally active at pH 5, a feature distinguishing it from calpain, another non-ICE cysteine proteinase which has been associated with apoptosis. This novel bcl-2 cleaving protease, although constitutively present in RL-7 cells and resting peripheral blood lymphocytes (PBL) was upregulated following induction of apoptosis in RL-7 cells or mitogen activation in PBL. The N-terminus of bcl-2 which contains the BH4 domain that binds the kinase Raf-1 and the phosphatase calcineurin is essential for anti-apoptotic activity. Its cleavage might provide a novel post-translational mechanism for regulating bcl-2 function and could amplify ongoing programmed cell death.

Identification of mimicry peptides based on sequential motifs of epitopes derived from 65-kDa glutamic acid decarboxylase.

Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease with a predominantly non-hereditary etiology that results in a destruction of pancreatic beta cells by autoaggressive T lymphocytes. Neither the mechanism of initial stimulation of these T cells nor the nature of the environmental factors implicated in the disease have so far been identified. However, both issues are taken into account by the hypothesis of initial T cell activation by viral or bacterial mimicry peptides with sequence similarities to pancreatic self antigens. We determined sequential epitope motifs to search for mimicry peptides stimulating T cell lines specific for two epitopes derived from the IDDM autoantigen 65-kDa glutamic acid decarboxylase (GAD65). These were GAD65 (88-99), presented by HLA-DRB1*0101, and GAD65 (248-257), presented by HLA-DRB5*0101. T cell stimulation by peptides with substitutions in HLA anchor or T cell contact positions was analyzed to establish degenerate epitope motifs for database searching. Out of 28 tested candidate mimicry peptides derived from bacterial, viral and human proteins, 3 stimulated T cell lines and a T cell clone specific for epitope GAD65 (248-257). Our results demonstrate that mono- and polyclonal GAD65-specific T cells from IDDM patients can be stimulated by viral and bacterial peptides with little apparent sequence homology with autoantigenic epitopes. Moreover, in a synopsis with related published studies, our findings suggest that simple degenerate search motifs comprising principal T cell contacts plus HLA class II binding motifs may suffice to identify most mimicry peptides.

Down-regulation of Fc alpha receptors on blood cells of IgA nephropathy patients: evidence for a negative regulatory role of serum IgA.

IgA nephropathy (IgAN) is associated with increased serum IgA1 and IgA1-immune complexes (IC). As Fc alpha receptors (Fc alpha R) are candidate molecules to regulate IgA levels, increased receptor occupation by IgA1 prompted us to study the expression of Fc alpha R on blood cells of IgAN patients. Surface and cytoplasmic Fc alpha R expression were markedly decreased on monocytes, despite normal levels of transcripts. Fc alpha R expression on patients' neutrophils was slightly decreased, exclusively at the cell surface. However, when autologous plasma was removed from the cells Fc alpha R was up-regulated. This observation led us to search for circulating regulatory factors. In vitro experiments revealed that Fc alpha R was down-regulated on normal monocytes following long-term culture with control or patient purified serum IgA at high concentrations (5 mg/ml). Moreover, polymeric myeloma IgA1 induced stronger down-regulation than monomeric IgA1. These results point to a negative regulatory role of serum IgA on surface Fc alpha R expression. This is also supported by a negative correlation between levels of Fc alpha F on blood cells and serum IgA. On the other hand, endogenous IgA bound to IgAN cells was significantly higher than IgA bound to control cells pre-incubated with patients' plasma, suggesting abnormalities in the receptor-ligand interaction. Patient Fc alpha R had a higher Mr (60 to 85 kDa) than those of controls (55 to 75 kDa) and a decreased binding to a sialic acid-specific lectin on blots, indicating post-translational modifications with impaired sialylation of surface Fc alpha R molecules that might be involved in enhanced IgA binding. Continuous Fc alpha R occupation by IgA, associated with receptor down-regulation, might contribute to the enhancement of circulating IgA1 and IgA1-IC by impairing their binding and degradation. Finally, increased receptor occupation by IgA on monocytes was linked to mesangial proliferation and glomerular sclerosis, suggesting a role for IgA-bound cells in the pathogenesis of mesangial damage.

Resistance of T-cells to apoptosis in autoimmune diabetic (NOD) mice is increased early in life and is associated with dysregulated expression of Bcl-x.

Activated lymphocytes of autoimmune non-obese diabetic (NOD) mice exhibit an increased resistance to programmed cell death (PCD) following withdrawal of interleukin-2 (IL-2). In the present study, we found that resistance of NOD T lymphocytes to PCD was increased as early as 1 week of age, hence several weeks before the invasion of the pancreas by inflammatory cells, which is compatible with a role of the NOD apoptotic phenotype in the autoimmune susceptibility of this strain. In the thymus, mature single positive but not double positive or double negative thymocytes were more resistant to PCD in NOD compared to B6 mice. Moreover, in both NOD and B6 mice, CD4+ T cells were more resistant to PCD induced by IL-2 deprivation than CD8+ cells. As a result, NOD CD4+ T cells were remarkably resistant to cell death induced in this manner. In relation with this increased resistance to apoptosis, expression of the anti-apoptotic Bcl-x protein was upregulated in activated T cells of NOD mice, most notably after 24 h of IL-2 deprivation. These results should help us to understand the relationship of the NOD apoptotic phenotype to the emergence of the NOD mouse autoimmune disease.