HLA-DMA alleles are possible new markers of rheumatoid arthritis: study of a Corsican group.

The HLA-DMA gene, along with the HLA-DMB gene, encodes the not classical class II molecule. This molecule catalyzes the class-II-associated invariant-chain peptide (CLIP)-antigen peptide exchange in classical class II molecule peptide-binding groove. As such, the DM heterodimer is an antigen presentation regulator and may be linked to immune system deficiencies such as those observed in autoimmune diseases. The study of DMA gene polymorphism seems be a reasonable approach to provide an answer to this question. Thanks to PCR-derived methods, the relationship between DMA gene polymorphism and rheumatoid arthritis (RA) was demonstrated in the present study. The DMA*0101 allele was observed to confer a significant predisposition to RA while the DMA*0102 allele significantly protected from this disease. Polymorphism experiments with the HLA-DRB1 gene revealed that this relationship between DMA polymorphism and RA is not a consequence of a linkage disequilibrium with the HLA-DRB1 alleles implicated in this pathology. The study of the DMA gene could therefore prove to be very useful in the early diagnosis of RA.

TCR repertoire and cytokine profiles of cerebrospinal fluid- and peripheral blood-derived T lymphocytes from patients with multiple sclerosis.

The cerebrospinal fluid (CSF) represents an important source of T lymphocytes that could be involved in the inflammatory response occurring in the central nervous system in multiple sclerosis (MS). In order to investigate whether the Vbeta gene usage of CSF T lymphocytes is restricted, we analyzed the TCR Vbeta expression in twelve CSF expanded by in vitro culture compared to the paired in vitro-stimulated peripheral blood T lymphocytes. The overexpression of one or two Vbeta genes was demonstrated in ten CSF, but the type of Vbeta over expressed varied from one patient to another. For one patient, the Vbeta repertoire was also investigated by single cell cloning. High frequency of BV6S7-expressing T cell clones was observed in the CSF while no BV6S7 clone was derived from the peripheral blood T lymphocytes suggesting that these cells could be involved in the immunopathological process in the central nervous system (CNS). The cytokine patterns of the T cell clones derived from the CSF- and peripheral blood-T lymphocytes of this patient were determined. The CSF T cell clones produced higher levels of cytokines than the peripheral blood T cell clones. The high frequency of IL-4-producing-T cell clones observed in CSF demonstrate that T cells which could downregulate the inflammatory process are present in the CNS.

Implication of HLA-DMA alleles in corsican IDDM.

The HLA-DM molecule catalyses the CLIP/antigen peptide exchange in the classical class II peptide-binding groove. As such, DM is an antigen presentation regulator and may be linked to autoimmune diseases. Using PCR derived methods, a relationship was revealed between DM gene polymorphism and IDDM, in a Corsican population. The DMA*0101 allele was observed to confer a significant predisposition to this autoimmune disease while the DMA*0102 allele protected significantly. Experiments examining polymorphism of the HLA-DRB1 gene established that these relationships are not a consequence of linkage disequilibrium with HLA-DRB1 alleles implicated in this pathology. The study of the DMA gene could therefore be an additional tool for early IDDM diagnosis in the Corsican population.

Anti-pancreatic autoimmunity and Graves' disease: study of a cohort of 600 Caucasian patients.

The aim of this study was to investigate the frequencies of clinical diabetes and humoral markers of anti-pancreatic autoimmunity in a homogeneous population of 600 Caucasian patients with recently diagnosed Graves' disease (GD), in order to characterize the specific features of this group of endocrine patients among subjects at risk of diabetes. Ten were already diabetic at GD diagnosis. Among the 590 non-diabetic patients, 29 had islet cell antibodies (ICA), including 15 with low titre ICA and only 1 ICA-positive subject with a familial history of diabetes. Twenty-four patients had insulin autoantibodies, including three in association with ICA. Glutamic acid decarboxylase (GAD)/64 kDa antibodies were found in 16 of the 150 tested sera, including 13 of the 29 ICA-positive sera. Four ICA-positive patients displayed 37/40 kDa antibodies, including three in association with GAD/64 kDa antibodies. During follow-up, one of the ICA-positive patients developed insulin-dependent diabetes, 14 years after the GD diagnosis. To summarize, this anti-pancreatic autoimmunity study was focused on a large but specific and homogeneous group of subjects at risk for diabetes: recently diagnosed GD patients. This population was characterized by a high prevalence of GAD/64 kDa antibodies but also by a low frequency of evolution towards diabetes and the slowness of the process which could be due to the fact that only a minority of subjects possessed a sufficient combination of anti-pancreatic markers at the same time.

TAP gene transporter polymorphism in inflammatory bowel diseases.

BACKGROUND: Many studies suggest the implication of genetic factors in inflammatory bowel diseases. Despite some associations with HLA genes, the lack of definite data may be due to ethnic variations, clinical heterogeneity, or the involvement of additional susceptibility genes beside or within the major histocompatibility complex (MHC), such as TAP genes. The aim of this study was to analyze in patients with ulcerative colitis (UC) or Crohn's disease (CD) the polymorphism of TAP genes that encode the proteins necessary for the transfer of antigenic peptides through the endoplasmic reticulum membrane. METHODS: One hundred and one UC and 148 CD patients were compared with 173 unrelated healthy controls. Dimorphisms within the TAP1 and TAP2 alleles were analyzed by sequence-specific oligonucleotide typing. RESULTS: No difference was found between patient groups and controls. However, when CD patients were classified on the basis of their responsiveness to steroid therapy, a significant decrease of TAP2 AA (*0101/*0101) genotype was found in CD patients who did not respond to steroid therapy (22.9% versus 43.7% in steroid responder group; Pc < 0.05; odds ratio = 2.6; 95% confidence limits (CL) = 1.2-5.9). These data appear independent of the distribution of HLA DRB1*01 or DRB1*03 alleles despite a significant linkage disequilibrium between these alleles and TAP2A. CONCLUSIONS: This result suggests, despite the absence of arguments favoring a genetic susceptibility to CD, that the TAP2 gene or other genes located on chromosome 6 may be involved in the genetic heterogeneity of CD.

Longitudinal study of HIV-specific cytotoxic lymphocytes in HIV type 1-infected patients: relative balance between host immune response and the spread of HIV type 1 infection.

To evaluate the contribution of a specific cytotoxic response in the control of HIV infection in relation to clinical status, we performed serial analysis of anti-Env and anti-Gag cytotoxic activity in 13 infected individuals over a 6- to 10-year period, using cryopreserved peripheral blood mononuclear cells (PBMCs). Autologous EBV-transformed B cell lines infected in vitro with recombinant vaccinia viruses expressing HIV-1 env and gag genes were used as targets. Without any stimulation of the effector cells, we were able to show an anti-HIV cytotoxic activity in the PBMCs of 12 of 13 HIV-1-infected patients, consistent with chronic immune activation in HIV infection. Different patterns of HIV-specific cytotoxic activity were observed, and the extent of this cytotoxic response varied between the clinically defined groups of individuals. No direct relationship was observed with the number of CD4 and CD8 lymphocytes during the observation period. However, patients who remained asymptomatic had a more vigorous cytotoxic response than patients with clinical deterioration during the observation period, and a significant difference was observed for HIV Gag-specific CTL activity. From these data, we suggest that the HIV-specific cytotoxic response has a protective role in the course of HIV infection.

Epoetin alfa facilitates presurgical autologous blood donation in non-anaemic patients scheduled for orthopaedic or cardiovascular surgery.

Patients expected to need at least three units of blood for their elective cardiovascular or orthopaedic surgery, were allocated randomly to receive intravenous (i.v.) Epoetin alfa 600 IU kg-1 (n = 27), 300 IU kg-1 (n = 30) or placebo (n = 23), on days 1, 4 and 7. Provided haemoglobin > or = 11 g dL-1, one unit of blood was collected on days 1, 4, 7, 11 and 14. Iron supplementation was given throughout the study. Surgery was scheduled between days 18 and 21. Significantly more patients treated with Epoetin alfa (100% for 600 IU kg-1; 97% for 300 IU kg-1) were able to donate > or = 4 units of blood compared with placebo (78%) (P = 0.011 and P = 0.032). No significant differences were seen in total patient exposure to homologous blood (7.4%, 3.3% and 17.4%, respectively). Mean red cell volume donated (P = 0.005 for 600 IU kg-1; P = 0.158 for 300 IU kg-1 both vs. placebo) and production (P < 0.001 and P = 0.012, respectively) were dose related. Twenty-four patients became iron deficient. No differences in the incidence of adverse events were seen between the groups.

Impaired leucocyte functions in diabetic patients.

This study evaluates polymorphonuclear neutrophil (PMN) cell performance in 61 diabetic patients free of infection (40 Type 1, 21 Type 2), using tests that explore all the functional steps of PMN: (1) adherence: expression of adhesion molecules, CD 11a, CD 11b, CD 11c; nylon fiber adherence test; (2) chemotaxis under agarose towards the bacterial oligopeptide FMLP and complement fractions, used as attracting agents; (3) phagocytosis of opsonized latex microbeads; (4) bactericidal activity: chemiluminescence assessment of the oxidative killing potential before and after stimulation by opsonized zymosan and PMA; nitroblue tetrazolium reduction test. Results were analysed according to potentially influential factors: metabolic control (HbA1C, glycaemia), age of patient, type of diabetes, disease duration, and existence of vascular complications. PMN chemotaxis was significantly lower in patients than in healthy controls (p < 0.001) and associated with spontaneous adherence and increased expression of adhesion molecules (CD 11b, CD 11c). The increased response to chemiluminescence reflects spontaneous activation of PMN cells and increased free radical production; after stimulation, response was lower than in controls. The type of diabetes, the age of patients, HbA1C level and disease duration did not affect the responses. Chemotaxis and chemiluminescence were further reduced in patients with vascular complications and hyperglycaemia. We conclude that all steps of PMN functioning are altered in diabetic patients, which may increase the risk of vascular complications and infectious episodes.

Interaction between certain major histocompatibility complex class II and T-cell receptor V beta alleles promotes the antibody production to extractable nuclear antigen-related peptides.

Our objective was to study the interaction between major histocompatibility complex (MHC) class II and T-cell receptor (TCR) alleles in the recognition of extractable nuclear antigen-derived peptides in 32 patients with systemic lupus erythematosus and 173 of their family members. MHC genes were analyzed using sequence specific oligonucleotides, and TCR beta-chain gene polymorphism using restriction fragment-length polymorphism. One dominant peptide (as defined by enzyme-linked immunosorbent assay autoantibody reactivity) was identified in each antigen studied: peptide 1-20 in Sm-D, peptide 35-58 in U1-RNP-A, and peptide 304-324 in the Ro/SSA 60 Kd protein. None of the MHC class II and TCR beta haplotypes was directly associated with any of the autoantibodies. Twenty-six subjects had antibodies to the peptide Sm-D1-20; nine of them were DRB1*0101/DQB1*0501. Among subjects with this haplotype, the number of responders was higher (p < 0.028, p corrected, pc = 0.336) in those with the 2-25-9 TCR beta haplotype than in the remainder. Conversely, the number of DRB1*04/DQB1*0302 responders was lower (p < 0.030, pc = 0.360) among subjects with the 23-20-9 TCR beta haplotype than in those without. The odds ratios (OR) were 4.23 and 0.21, respectively. Of the 54 subjects positive for anti-U1-RNP-A 35-38, 13 were DRB1*0101/DQB1*0501 and eight DRB1*04/DQB1*0302. The percentage of responders was higher (p < 0.041, pc = 0.492, OR = 3.48) in the former group of subjects with the 2-25-9 TCR beta haplotype, and lower (p < 0.02, pc = 0.024, OR = 0.09) in the latter with the 23-20-9 TCR beta haplotype. Three of the 12 anti Ro/SSA 60Kd 304-324-positive subjects were DRB1*0101/DQB1*0501. All had the 2-25-9 TCR beta haplotype (p < 0.046, pc = 0.552, OR = 6.29) and none the 23-20-9 (p < 0.031, pc = 0.372, OR = 0.10). The same combinations of genes were associated with high/low response toward the three peptides. These data provide evidence for an interplay of the MHC class II and TCR beta alleles in the control of specific autoantibody response to well-defined nuclear Ag peptides.

[Polymorphism of the microsatellites and tumor necrosis factor genes in chronic inflammatory bowel diseases]. / Etude du polymorphisme des microsatellites et des gènes du tumor necrosis factor (TNF) au cours des maladies inflammatoires chroniques de l'intestin.

OBJECTIVES: Multiplex family studies have excluded chromosome 6 as a candidate gene of susceptibility to inflammatory bowel disease. However, one recent study suggested that a gene involved in the pathogenesis of Crohn's disease is located on chromosome 6 confering to a microsatellite allelic combination (a2, b1, c2, d4, e1) a strong genetic risk factor in Crohn's disease. The aim of our study was to determine simultaneously the polymorphisms of the TNF microsatellites and of the genes coding for TNF synthesis in patients with inflammatory bowel disease. PATIENTS AND METHODS: Sixty patients with ulcerative colitis, 100 patients with Crohn's disease were compared to 64 healthy ethnically matched controls. Five TNF microsatellite loci (a, b, c, d, e) were typed using polymerase chain reaction PCR, and two dimorphisms of TNF alpha and TNF beta (intron 1) were studied by restriction fragment length polymorphism (RFLP). RESULTS: Allelic frequencies of TNF microsatellites and of TNF alpha and beta genes were similar in Crohn's disease, ulcerative colitis and controls. Five loci microsatellite haplotypes, especially a2 b1 c2 d4 e1 allelic combination, were not more frequent in Crohn's disease (25%) compared to ulcerative colitis (27%) or controls (20%). Subgroups stratification according to clinical characteristics did not modify haplotype frequencies. Analysis of our data taking simultaneously into account the MHC alleles (DRB*01 or DRB1*04) did not modify our data; however, it suggested that extended haplotype on short arm of chromosome 6 differed between patients and controls. Linkage disequilibrium (delta = -360.10(-4); P < 0.01) between a2, b1, c2, d4, e1 allelic combination and DRB1*04 allele was observed only in Crohn's disease. CONCLUSION: Percentages of patients with Crohn's disease or ulcerative colitis carrying TNF microsatellite or TNF alpha and beta gene haplotypes were similar to those of healthy controls. These data argue against involvement of the TNF locus without exclusion of short arm of chromosome 6 implication in Crohn's disease pathogenesis.

Clinical validity of intercellular adhesion molecule-1 (ICAM-1) and TSH receptor antibodies in sera from patients with Graves' disease.

We compared the concentrations of soluble intercellular adhesion molecule-1 (sICAM-1) and the activities of thyroid-stimulating antibodies (TSAb) and thyrotropin-receptor antibodies (TBIAb) as measured with a commercial kit (TRAK). Sera were obtained from patients with Graves' disease (GD) before, during and after therapy with carbimazole (1-methyl-2-thio-3-carbethoxyimidazole). In all the situations, TSAb method was more sensitive than TBIAb. These two parameters dropped during therapy and were not correlated at any stage of measurement. sICAM-1 levels increased in 56.4% of patients before treatment, remained elevated at the beginning of treatment and decreased after twelve months of therapy. TSAb levels were significantly different between patients in relapse (78%) and those in remission (18%) (Z = -2.250, P = 0.025), with a relapse rate depending on the TSAb positivity (chi 2 = 7.103, P = 0.0077). Positive sICAM-1 values were found in 3 of the 9 (33.3%) patients who relapsed after discontinuing the drug but were negative in all the patients remaining in remission with a significant difference (Z = -1.982, P = 0.0475). The relapse rate was also dependent on positive sICAM-1 values (chi 2 = 3.958, P = 0.0466). No correlation was found between sICAM-1 levels and anti-TSH receptor antibodies TSAb or TBIAb. We conclude that the TBIAb technique is too insensitive to explore GD. TSAb and sICAM-1 assays in patients with GD are good markers of immune process after treatment withdrawal. Because of its rapid implementation, the sICAM-1 assay may advantageously replace TSAb measurement for forming a prognosis of GD.

Are HLA-DR or TAP genes genetic markers of severity in ulcerative colitis?

The pathogeny of ulcerative colitis (UC) is not yet elucidated, but some arguments suggest the implication of genetic factors. Among the candidate genes, those encoding for HLA class II genotypes have been extensively studied in UC; however, discordant data may be imputable to heterogeneity, characterized by immunological markers such as atypical ANCA (p-ANCA), or to inclusion of more or less intractable UC. The aim of our study is to evaluate the interest of HLA class II and TAP genetic markers to identify different clinical forms of UC, according to p-ANCA status. Unrelated patients with a history of UC (n = 91) and healthy control subjects with no personal or family history of inflammatory bowel diseases (IBD) (n = 200) were included. HLA-DRB1*03 was less frequent in UC patients than in healthy controls (8% vs 28%, PC < 0.03). No association was found with any TAP genotypes. Moreover, there was no association with the HLA-DR2 specificity, either in the entire group of UC patients (38% vs 28%) or in the p-ANCA-positive subgroup of patients (30%). The most consistent finding in the present study is that some genetic markers may characterize intractability in UC patients. HLA-DR2 was associated with poor prognosis, regardless of p-ANCA status. In HLA-DR2 and non-HLA-DR2 groups, colectomy was done in 55% and 27% of patients, respectively, (PC < 0.05). Furthermore, in non-HLA-DR2 patients, p-ANCA could be of interest to characterize those with more severe prognosis. Our results confirm the interest of genetic studies to define UC genetic susceptibility, taking into account intractability of the disease. They do not support the hypothesis that p-ANCA is a subclinical marker of genetic susceptibility to UC.

Genetic heterogeneity between type 1a and type 1b insulin-dependent diabetes mellitus: HLA class II and TAP gene analysis.

The aim of this study was to compare the genetic susceptibility linked to the HLA Class II region genes of the Major Histocompatibility Complex in isolated insulin-dependent diabetes mellitus (1a-IDDM) and insulin-dependent diabetes mellitus associated with another autoimmune endocrinopathy (1b-IDDM). HLA genes DRB1, DQA1 and DQB1 were studied at the genomic level, as well as genes TAP1 and TAP2. One hundred and seventy-nine 1a-IDDM diabetic patients were compared with 83 1b-IDDM patients. While it appeared that common genetic traits characterize diabetes regardless of the subtype (1a or 1b), certain features differentiate the two forms of IDDM. Extending the analysis of risk haplotypes DRB1*03 and DRB1*04 to TAP genes elicited a difference between 1a-IDDM and 1b-IDDM patients. Haplo-type DRB1*03 was thus characterized in 1a-IDDM patients by a lower frequency of alleles TAP1-B (13.5%) and TAP2-B (16.2%), not found in 1b-IDDM patients (33.3% for each allele). Likewise, haplotype DRB1*04 is characterized in 1b-IDDM patients by a lower frequency of alleles TAP1-C (4.0%) and TAP2-B (8.0%) than in 1a-IDDM patients (22.2% and 25.9%, respectively). In total, this study showed that extending the characterization of HLA Class II haplotypes to TAP genes discriminates between the forms of diabetes restricted to a specific pancreatic affection and those reflecting a wider autoimmune disorder affecting several organs.

Genetic susceptibility and anti-human platelet antigen 5b alloimmunization role of HLA class II and TAP genes.

Platelet alloimmunization may result in post-transfusion purpura, and during pregnancy may cause neonatal alloimmune thrombocytopenia (NAIT), with a frequency estimated at 1.3 per 1000 live births. The risk of morbidity is significant: 20% of affected infants have neurologic sequelae and the death rate is about 10%. A better understanding of the immune response to platelet alloantigens would allow for a better definition, and thus better management of pregnant women at high risk. Limited data are available on the immune response against HPA-5b, the second most frequent antigen, after HPA-1a, implicated in NAIT. We studied HLA class II and TAP gene polymorphism in 50 women immunized against HPA-5 system antigens. Our results suggest a strong association of alloimmunization with a cluster of HLA DR molecules sharing a particular polymorphic amino acid sequence at position 69-70 (Glu-Asp encoded by GAA-GAC nucleotide sequence) of the DR beta 1 chain (RR = 2.95, RR = 5.70 when patients were homozygous for this sequence), and a negative association with the DRB1*0301 allele (2.1% vs. 28%; RR = 0.08). Furthermore, increased frequency of a TAP2 dimorphism at position 379 was observed in immunized women against the HPA-5 antigens (RR = 4.7).

Investigation of the association of major histocompatibility complex genes, including HLA class I, class II and TAP genes, with clinical forms of Crohn's disease.

The aim of this study was to determine immunogenetic markers of susceptibility in Crohn's disease (CD), taking the different features of the clinical course of the disease into account. HLA class I, HLA class II and TAP transporter gene polymorphisms were studied using DNA typing methods. Gene and antigen frequencies were analysed and compared in a group of 102 CD patients and 200 unrelated healthy controls from the same area. Analysis of the whole CD patient population revealed no definite association with either HLA or TAP gene alleles, with the exception of an association with DRB1*1302 (Pc < 0.05). However, when clinical subgroups of patients were considered, specific associations with some genetic markers were found. The most definitive results involved a genetic association in the group of patients who did not respond to glucocorticoid therapy. This group was characterized by a high frequency of HLA-DRB1*04 (P < 0.05). Conversely, a positive association with the TAP2-A allele was found in cortico-responder patients (Pc < 0.03). Furthermore, analysis of the distribution of HLA class II alleles in relation to the presence of extra-intestinal manifestations revealed an association with the DQB1*0501 or *0503 suballele of DQ5 (P < 0.05). Finally, patients with lesions in the small bowel were more frequently HLA DRB1*07 (P < 0.05). The present study supports the concept of clinical heterogeneity in Crohn's disease associated with a background of genetic heterogeneity.

The role of HLA-DR-DR and HLA-DR-DP interactions in genetic susceptibility to rheumatoid arthritis.

In order to analyze the relationships between the DR and DP loci in the genetic susceptibility to RA, HLA-DRB1 and -DPB1 polymorphism was studied in 155 RA patients compared to 150 controls, using a reverse dot-blot analysis. Our data were consistent with the involvement of the amino acid in position 71 of the third hypervariable region of the DR beta 1 chain in susceptibility to the disease. The higher risk for RA was observed in patients who carried the association of a lysine (K), characterizing the DRB1* 0401 susceptibility allele, with an arginine (R), observed in all the other DRB1* susceptibility alleles (21.9% vs 0.6%, p(c) < 10(-6), OR = 42) In the absence of arginine, the presence of lysine was still associated with the disease (33% vs 19%, p(c) < 0.03, OR = 2). In contrast, in the absence of lysine, the frequency of arginine in position 71 was similar in patients and controls (30% vs 26%, p = NS). On another hand, the analysis of the HLA-DPB1 locus showed that the DPB1 *0401 allele frequency was significantly increased in the RA patient group (n = 47) who expressed only arginine at the position 71 of the beta 1 chain (82% vs 56% in controls, p < 0.008), with role of HLA-DR--DR and -DR-DP interactions in the genetic susceptibility to RA.

Distinct pattern of IL-2 and IFN-gamma gene expression in CD4 and CD8 T cells: cytofluorometric analysis at a single cell level using non-radioactive probes.

IL-2 and IFN-gamma gene expression was analyzed using an original method for in situ hybridization (ISH) with non-isotopic probes and flow cytometric analysis (FC). This method permits rapid detection of mRNA at a single cell level among in vitro activated human peripheral blood mononuclear cells (PBMC) and purified CD4 and CD8 T cell subsets. After stimulation with PMA and ionomycin (PMA+Io), cells were fixed at different times and hybridized with digoxigenin (DIG)-labelled RNA antisense or sense probes specific for IL-2 and IFN-gamma. The level of cytokine gene expression in individual cells was visualized using FITC-conjugated anti-DIG antibodies and the fluorescent signal was analyzed by flow cytometry. Specific hybridization with IL-2 and IFN-gamma antisense probes was detected among activated PBMC within lymphoid cells identified by their light scattering properties. Kinetic analysis of the frequency of mRNA producing cells exhibited a biphasic pattern with an early peak at 6-8 hrs. when percentages of IL-2 and IFN-gamma expressing cells reached 35 +/- 7% and 18 +/- 4%, respectively. Similar data were obtained by enzymatic detection on cell smears using AP-conjugated anti-DIG. Combination of ISH with FC was applied to the comparison of the pattern of cytokine gene expression between CD4 and CD8 T cell subsets isolated by negative selection using immunobeads and magnetic separation. IL-2 was expressed by activated CD8 T cells (25-35%), but CD4 T cells were the major producers of IL-2 as assessed by the high frequency of mRNA expressing cells (60%) and the large amount of mRNA per cell relative to the mean fluorescence intensity. In contrast, IFN-gamma mRNA was preferentially expressed by CD8 T cells (27-37%) and a minority of CD4 T cells (17-23%). Despite quantitative differences, kinetic analysis of IL-2 gene expression in CD4 and CD8 T cells showed similar profiles with an early peak at 6-8 hrs. Upregulation of IL-2 gene expression in CD4 T cells by CD28 co-stimulation increases the amount of IL-2 mRNA per cell as visualized by mean fluorescence intensity. In addition the effect of CD28 co-stimulation on IL-2 mRNA stabilisation was demonstrated by the maintenance of a high frequency of IL-2 expressing CD4 T cells and an elevated level of mRNA per cell for prolonged period after PMA+Io stimulation. By contrast CD28 co-stimulation had no obvious effect on IFN-gamma expression.

HLA-associated heterogeneity of the humoral response to islet antigens in insulin-dependent diabetes.

Insulin-dependent diabetes mellitus (IDDM) is associated with susceptibility HLA class II alleles. Islet cell antibodies (ICA), detected by indirect immunofluorescence on pancreas sections, represent the best marker of the disease. Autoantibodies to glutamic acid decarboxylase (GADA), one major islet antigen, do not totally account for ICA reactivity, suggesting heterogeneity of the anti-islet humoral response. In 97 patients with IDDM we have correlated ICA heterogeneity with clinical markers and DR and DQ alleles. ICA were found in 81% of the patients, and in 33% the serum blocked the binding to islet cells of reference sera with a granular fluorescence pattern. GADA were found in 62% of cases. Patients with high GADA titers and blocking sera were older at onset and less often had a family history of IDDM, suggesting that these antibodies might be a marker of slow progression to IDDM. ICAs were not associated with particular HLA DR or DQ alleles. Conversely, GADA were less frequent than ICA in DR4 subjects but not in the other groups. Moreover, among DR4 non-DR3 patients, GADA were found almost exclusively in DRB1*0401 patients but not in other DR4 subtypes. There was an association of GADA with DQ alleles but it was secondary to linkage disequilibrium between DR and DQ loci. In conclusion, the heterogeneity of the humoral response in IDDM is controlled by HLA class II genes and correlates with clinical heterogeneity.

Relevance of 10 Caucasian HLA haplotypes in searches for unrelated bone marrow donors for 100 patients from a single center.

Unrelated donor searches for 100 Caucasian patients were referred to France Greffe de Moëlle Registry (FGM) from September 1987 (24,600 donors) to December 1993 (71,500 donors, 61% DR typed). After DR typing of HLA-A,B matched donors, unsuccessful searches were extended to other European Registries for 36 patients. Twenty two patients had a donor (FGM: 19, other Registries: 3) selected on: (1) HLA-A,B and DRB,DQB1 split identity; and (2) unidirectional relative response < 5% in MLR performed twice. Estimated probability of finding a compatible donor at 9 months in FGM was 12% (s.e. +/- 4%) and 25% at 2 years (s.e. +/- 6%). This probability was stringently dependent on a phenoidentity to one very common HLA-A,B,DR or B,DR haplotype (25% at 9 months when present, representing 19 of 19 patients with a compatible donor). Without this phenoidentity, the probability was zero per cent (P = 0.0001) in FGM searches and < 4% (n = 1) in extended searches. The MLR test was shown to be insensitive for screening for DPB1 mismatches. Clinical status influenced the probability of finding a compatible donor at one year ranging from 9% +/- 9% for ALL to 23% +/- 8% for CML (NS). Disregarding DPB1 mismatches is the most efficient way of increasing search efficiency.