HLA-DRB1*0404 is strongly associated with high titers of anti-cyclic citrullinated peptide antibodies in rheumatoid arthritis.

OBJECTIVE: To test whether the presence of RA associated HLA-DRB1*0101, HLA-DRB1*0401 and HLA-DRB1*0404 alleles individually influences anti-cyclic citrullinated peptide antibodies (anti-CCP) production. METHODS: The frequency of anti-CCP antibodies was calculated in the sera of 260 RA patients expressing either two (double dose genotypes SE+/SE+), one (single dose genotypes SE+/SE-) or no RA associated HLA-DR alleles (SE-/SE-). Anti-CCP antibodies titers were also determined. RESULTS: RA associated HLA-DR alleles are not mandatory for production of anti-CCP. We found that 68% of SE-/SE- patients were anti-CCP positive. There was no significant difference in anti-CCP between SE negative patient (SE-/SE-) and patients expressing at least one SE (SE+/SE+ and SE+/SE-) (p=0.140). We observed no statistical difference in anti-CCP between RA patients expressing one or two SE (82% vs. 77%, p=0.577). Among SE+/SE-patients, HLA-DRB1*0404 was associated with anti-CCP with a statistically significant difference compared with SE negative patients (90% anti-CCP positive, p=0.02). HLA-DRB1*0404 was also associated with high titers of anti CCP with a statistically significant difference compared with HLA-DRB1*0401 and HLA-DRB1*0101 patients (p=0.025). CONCLUSIONS: The RA-associated HLA-DRB1*0404 allele was the most strongly associated with the presence of anti-CCP in RA sera. Moreover, HLA-DRB1*0404 patients had higher titers of anti CCP than patients with other RA associated HLA-DR alleles.

Relevance of KIR gene matching in unrelated hematopoietic stem cell transplantations.

The aim of this collaborative study was to evaluate the impact of killer cell immunoglobulin-like receptor (KIR) gene disparities on unrelated hematopoietic stem cell transplantations (HSCT) outcome. To address this question, we have determined the presence or absence of 14 functional KIR genes in HLA-matched (n= 164) or HLA-mismatched (n= 100) donor/recipient pairs and investigated whether KIR gene disparities had an impact on both the occurrence of acute graft-vs-host-disease incidence and overall survival. In a univariate analysis, our preliminary results suggest a detrimental effect of a few KIR gene disparities on patient survival that should be avoided in unrelated HSCT.

Quantification of mixed chimerism by real time PCR on whole blood-impregnated FTA cards.

This study has investigated quantification of chimerism in sex-mismatched transplantations by quantitative real time PCR (RQ-PCR) using FTA paper for blood sampling. First, we demonstrate that the quantification of DNA from EDTA-blood which has been deposit on FTA card is accurate and reproducible. Secondly, we show that fraction of recipient cells detected by RQ-PCR was concordant between the FTA and salting-out method, reference DNA extraction method. Furthermore, the sensitivity of detection of recipient cells is relatively similar with the two methods. Our results show that this innovative method can be used for MC assessment by RQ-PCR.

HLA-DRB1 frequencies of the Comorian population and their genetic affinities with Sub-Saharan African and Indian Oceanian populations.

BACKGROUND: Ethnic-historic sources have considered the Comorian population to be the result of an amalgamation of African, Arabian and Southeast Asian groups. AIM: This study seeks to determine the genetic relationships and contributions from Sub-Saharan Africa and Indian Oceania and to reconstruct past migration events. SUBJECTS AND METHODS: The human leukocyte antigen (HLA) polymorphism of a Comorian population was described and analysed. RESULTS: Genetic distances and multidimensional scaling analyses showed complex patterns of genetic differentiation in the Indian Oceanian area as a result of continuous gene flow occurring within the past approximately 2500 years. Nevertheless, the Comorian genetic pool appears to be a mix of Bantu-speaking and Arab populations as testified to by admixture estimations of almost 50-60% and 27-33%, respectively. CONCLUSION: The Comorian population may represent the eastern limit of the recent and massive eastward Bantu expansion. In contrast to the population from Madagascar (Merina), only a restricted influence of Austronesian populations was found.

Distribution of killer-cell immunoglobulin-like receptor (KIR) in Comoros and Southeast France.

Killer-cell immunoglobulin-like receptors (KIRs) expressed by natural killer cells are cell surface molecules able to recognize groups of HLA class I alleles. The number and distribution of KIR genes vary among individuals and populations. The aim of this study is to analyse the KIR gene content in a Comorian population in order to investigate genetic relationships with other populations and to reconstruct past migration events. The Comorian population consisted of 54 unrelated immigrants living in France and a control population consisted of 38 individuals from Southeast France. We investigated the presence or absence of 15 KIR genes, two pseudogenes expressed and non-expressed forms of KIR2DL5 and the two major subtype full-length and deleted forms of KIR2DS4. All individuals were typed positive for the framework genes, i.e. KIR2DL4, KIR3DL2 and KIR3DL3, and the two pseudogenes KIR3DP1 and KIR2DP1. The frequencies of full-length KIR2DS4 (*00101/00102/002) were lower in the French population (F = 29%) than in the Comorian population (F = 72%) (P(c) < 0.05). No significant differences were found for other KIR genes. A total of 11 genotypes were identified in the Southeast French population and 22 genotypes in the Comorian population. The most common genotype (2DL1, 2DL3, 2DL4, 3DL1, 3DL2, 3DL3 and 2DS4) accounted for 41% in the Comorian population and 34% in the Southeast French population. Principal component analysis using KIR gene data from 20 populations was performed to determine genetic differences and relations between populations. The Comorian population exhibited closest kinship with Africans and Asians. As KIR gene content is heterogeneous among ethnic groups, it can probably be used to assess the genetic relationships among populations from different geographic areas.

TAP1 and TAP2 gene polymorphism in rheumatoid arthritis in a population in eastern France.

The 'transporter associated with antigen processing' (TAP) gene products are involved in the processing of endogenous peptides that bind to class I molecules. Polymorphism within these genes could alter the level of the immune response, a phenomenon relevant to the development of autoimmune diseases. In this study, we examined the polymorphism of TAP1 and TAP2 genes in patients with rheumatoid arthritis (RA). TAP1 and TAP2 typing was performed for 138 Caucasian RA patients and 100 healthy controls, all originating from eastern France. TAP1 polymorphic residues at positions 333 and 637 and amino acid variants 379, 565, 651 and 665 in the TAP2 gene were found using amplification refractory mutation system-polymerase chain reaction (ARMS-PCR). This method enabled us to determine four TAP1 alleles (TAP1A to TAP1D) and eight TAP2 alleles (TAP2A to TAP2H). All patients and controls had been HLA-DRB1* genotyped. The polymorphic residues TAP1333 and TAP1637 did not show any difference in their distribution between patients and controls. Similar findings were obtained for TAP2379 and TAP2665. However, we found an increased frequency of Thr homozygosity and heterozygosity at position 565 in the TAP2 gene in RA patients (RA vs. controls: 25.3 vs. 14%; P = 0.032; OR = 2.09; CI = 1.01-4.38). Similarly, the prevalence of subjects who were homozygote and heterozygote for Cys651 was increased in the RA group (RA vs. controls: 36.8 vs. 11%; P = 0.02). The dimorphic site TAP2565 defines TAP2D and TAP2E alleles, while the site at position 651 characterizes TAP2F. Thus, we found that TAP2D and TAP2E alleles were more prevalent in RA, but not significantly so (RA vs. controls: TAP2D: 10 vs. 3.6%; P = 0.24; TAP2E: 3.6 vs. 0%; P = 0.19). Similarly, the frequency of TAP2F was higher in RA patients (24.5%) than in controls (11.3%), but this was not significant after correction (P = 0.029; Pcorr = 0.17). Finally, we found no linkage disequilibrium between DRB1* RA-associated alleles and amino acid substitution Thr565 or TAP2D and TAP2E alleles, whereas Cys651 (and TAP2F) was not independent of DRB1*04, a strongly RA-associated allele. Finally, Thr at position 565 in the TAP2 gene was associated with manifestations of disease severity in only a few patients. Examination of TAP1 and TAP2 gene polymorphisms in RA patients revealed an association between a particular amino acid residue, namely Thr565 in the TAP2 gene, and RA. This association was found to be weak and did not seem to be a predictor for the severity of the disease.

A novel mechanism of antitumor response involving the expansion of CD3+/CD56+ large granular lymphocytes triggered by a tumor-expressed activating ligand.

We describe a patient with acute myeloid leukemia (AML) who developed polyclonal large granular lymphocyte (LGL) proliferation. The reciprocal evolution of AML and LGLs suggested that these LGLs had an anti-tumor activity. The patient's LGLs killed autologous leukemia cells in a different way to classical T lymphocyte-mediated cytotoxicity since it did not rely on the recognition of target antigens presented by major histocompatibility complex (MHC) class I molecules by the CD3/TcRalphabeta complex. This killing was also different from natural killer (NK)-mediated cytotoxicity, which depends on the absence of MHC class I molecule recognition by NK inhibitory receptors. The LGLs were polyclonal, had a CD3+/CD8+/CD56+ phenotype, and did not express the natural killer cell receptors (NKRs) for MHC class I molecules. The LGLs did not express the NK-specific activating natural cytotoxicity receptors but expressed the 2B4 non-MHC restricted triggering receptor, whose ligand CD48 was expressed by leukemic cells and normal bone marrow cells. The 2B4 receptor participated in the ability of LGLs to lyse patient's leukemia. This represents a novel function for 2B4 in man, since this molecule, at variance with the murine system, was considered not to have direct effects on CD8+ T cell-mediated cytotoxicity. This case report allowed us to describe a novel T lymphocyte-mediated anti-tumor mechanism which relied on (1) the abnormal expansion of the rare 2B4-positive CD3+/CD8+/CD56+ T lymphocyte subset, (2) an as yet undescribed cytotoxicity mechanism in man which depended on 2B4 molecule. The relevance of this observation in human cancer immunotherapy has to be further investigated.

Influence of shared epitope-negative HLA-DRB1 alleles on genetic susceptibility to rheumatoid arthritis.

OBJECTIVE: Most patients with rheumatoid arthritis (RA) express the shared epitope (SE). It is not known whether SE-negative HLA-DRB1 alleles influence the development of RA. This study examined the influence of SE-negative HLA-DR alleles (DRB1*X) on the development of RA in 3 different French populations. METHODS: HLA-DRB1 alleles were defined by polymerase chain reaction with sequence-specific oligonucleotide hybridization or sequence-specific primers. SE-negative alleles were classified according to the electric charge of their P4 pocket. HLA-DRB1 alleles *0103, *0402, *07, *08, *11 (except *1107), *12, and *13 have a neutral or negative P4 charge and are called DRB1*XP4n. HLA-DRB1*03, *0403, *0406, *0407, *0901, *1107, *14, *15, and *16 have a positive P4 charge and are called DRB1*XP4p. RESULTS: Among the SE-negative subjects, DRB1 genotypes with 1 or 2 DRB1*XP4n alleles were significantly overrepresented in the control subjects compared with the RA patients, whereas DRB1*XP4p/XP4p genotypes were equally represented in the patients and controls. In single-dose SE-positive subjects, SE/XP4n genotypes were equally represented in the patients and controls. However, SE/XP4p genotypes were significantly overrepresented in the RA patients. CONCLUSION: The DRB1*X allele polymorphism influences susceptibility to RA. Alleles that have a neutral or negative electric charge in their P4 pocket (DRB1*XP4n), such as DRB1*0103, *0402, *07, *08, *11 (except *1107), *12, and *13, protect against RA. Alleles that have a positive electric charge in their P4 pocket (DRB1*XP4p), such as DRB1*03, *0403, *0406, *0407, *0901, *1107, *14, *15, and *16, have no influence on the predisposition to RA.

DRB1 alleles in polymyalgia rheumatica and rheumatoid arthritis in southern France.

To investigate the association of HLA-DRB1 alleles with polymyalgia rheumatica (PMR) and rheumatoid arthritis (RA), 55 patients with PMR without giant cell arteritis, 203 patients with RA and 230 controls, all from the European population of Marseille, were HLA-DRB1 genotyped by PCR-SSO. HLA-DRB1*01 was significantly increased in both the PMR and RA groups compared to controls (35% versus 17%, P(c) < 0.05, and 41% versus 17%, P(c) < 0.001, respectively). HLA-DRB1*04 was significantly increased in the RA group compared to controls (48% versus 23%, P(c) < 0.001) but not in the PMR group. HLA-DRB1*04 subtype frequencies were significantly different between PMR patients and RA patients. Shared epitope-positive HLA-DRB1*04 alleles (DRB1*0401, 0404, 0405, 0408) were significantly overrepresented in RA patients compared to PMR patients and shared epitope-negative HLA-DRB1*04 alleles were overrepresented in PMR patients compared to RA patients. In conclusion, in the Mediterranean population studied, HLA-DRB1*01 is associated with RA and PMR whereas HLA-DRB1*04 is associated with RA only.

Modeling the HLA component in rheumatoid arthritis: sensitivity to DRB1 allele frequencies.

Rheumatoid arthritis is an inflammatory disease for which positive associations have been described with some HLA-DRB1 alleles. The associated alleles share a similar amino acid sequence in the third hypervariable region, the shared epitope, but differ at position 71 and 86. It has been suggested that HLA susceptibility to rheumatoid arthritis could be due not only to the shared epitope but could also be influenced by specific amino acids at positions 71 and 86. In this study, we investigated the role of these amino acids in rheumatoid arthritis on 203 unrelated patients. An involvement of amino acid 71 was detected but no conclusion was possible regarding amino acid 86. A study of the sensitivity of the conclusions to marker allele frequencies was performed. We showed that the results obtained for amino acid 71 are not very sensitive to allele frequencies but those obtained at position 86 are highly sensitive. This emphasizes the importance of studying the robustness of results to variations in allele frequencies before conclusions are drawn.

HLA-DRB1 and DQB1 polymorphisms in southern France and genetic relationships with other Mediterranean populations.

This study presents the results of HLA-DRB1 and DQB1 sequence-specific oligonucleotide probe (SSOP) typing for a population sample of 181 individuals originating from southern France. On the basis of allele and haplotype frequencies, we compared our population with others from the Mediterranean area. Allele frequencies are comparable to those found in other western European populations (France, Portugal, Spain) and indicate neighboring exchanges. The haplotype frequencies showed relationships with North Africans and Jewish populations, as well as the common origin of Moroccan and Lebanese Jews. Therefore, allele frequencies seem to be more able to show recent exchanges while haplotype frequencies might show ancestral relationships. These results may serve as references for future studies of HLA and disease in southern France.

Immune reconstitution and outcome after unrelated cord blood transplantation: a single paediatric institution experience.

We report the outcome of 12 children who underwent unrelated cord blood transplant (U-CBT) in a single institution between February 1997 and July 1998. The 1 year event-free survival was 67% (95% CI of 26%). Four children died with infectious complication as cause of death in three cases. Immune reconstitution was studied during first year post transplant by assaying total lymphocyte counts, B cells, NK cells and T cell subsets in the eight disease-free surviving patients. We observed a prompt recovery of CD19+ cell number which was greater than 500/microl at 9 months for all patients except the one with severe cGVHD. B cells constituted the predominant lymphocyte subset at 6 and 9 months post transplant with normal or elevated B cell numbers according to normal paediatric range. We noted normal serum immunoglobulin levels at 6 months post transplant for IgA and IgM and at 9 months for IgG. The CD3+ cell count and particularly the CD3+CD8+ T cell subset remained depressed until 12 months post transplant. Six months after unrelated CBT, seven out of eight patients had less than 100 CD3+CD8+ cells/microl. CD3+CD4+ cell recovery was less impaired with all children achieving an absolute count of CD3+CD4+ cells greater than 200/microl during the first year in a median of 5 months. The percentage of NK cells was elevated during the first 6 months after CBT but their absolute count remained within the normal range. Bone Marrow Transplantation (2000) 25, 53-57.

CD36 deficiency is frequent and can cause platelet immunization in Africans.

BACKGROUND: CD36 is expressed on several cell lineages. About 5 to 10 percent of Asians lack platelet membrane CD36 (pCD36), but the frequency of pCD36 deficiency in other ethnic groups is not known. Persons who are pCD36-negative are apparently healthy but can develop CD36 isoimmunization. STUDY DESIGN AND METHODS: The pCD36 phenotype was studied in 1885 subjects belonging either to a group of 1127 healthy French blood donors (almost all of whom were white Europeans) or to a group of 758 patients of known ethnic origin. RESULTS: No pCD36-negative persons were found among the blood donors. Only 1 of the 301 white European patients was pCD36-negative. In contrast, 16 of the 206 sub-Saharan Africans was pCD36-negative, a proportion higher than that among that black Caribbeans (1/148, p<0.01). The frequency of pCD36-negative patients was similar in blacks with and without sickle cell disease. Monocyte CD36 (mCD36) expression was studied in 15 of 22 pCD36-negative individuals: it was <10 percent in 7 subjects (type I deficiency) and between 12 and 100 percent in 8 others (type II deficiency). Thirteen pCD36-negative individuals had risk factors for immunization, and 4 had anti-CD36. Some had a history resembling posttransfusion purpura (n = 2), platelet transfusion refractoriness (n = 1), and recurrent miscarriage (n = 1). No correlation was found between immunization and the amount of mCD36. Anti-CD36 from an immunized type II-deficient woman reacted with monocytes from normal controls but not with monocytes from type I- or type II-deficient individuals, and thus it is postulated that mCD36 could be structurally different in normal and type II CD36-deficient individuals. CONCLUSION: CD36 deficiency is frequent in sub-Saharan Africans; development of anti-CD36 can lead to serious complications in multiply transfused patients, such as those with sicke cell disease.

Relationship between HLA-DQ and -DR genotypes and clinical characteristics in a French population of Type 1 diabetic patients.

AIMS: The present study was designed to look for a heterogeneity in the association between Type 1 diabetes mellitus (DM) and class II alleles of major histocompatibility complex (MHC) according to clinical presentation and C-peptide secretion during the first year of the disease, in a population living in south of France. METHODS: HLA DRB1 and DQB1 genotypes were determined in 129 Caucasoid patients with Type 1 DM and compared to a control group (n = 88). In a subgroup of 46 young adult diabetic patients, basal and postglucagon C-peptide secretion was followed during the first year of the disease (at 0, 1, 3, 6, 9 and 12 months). RESULTS: The two main haplotypes associated with Type 1 DM were DRB1*04DQB1*0302 and DRB1*03DQB1*02. The genotypes DRB1* 04DQB1 *0302/DRB1*04DQB1*0302 and DRB1 *03DQB1*02/DRB1*04DQB1* 0302 were associated with an early onset of diabetes, while homozygosity for DRB1*03DQB1*02 was characterized by later onset. Levels of residual insulin secretion in patients genotyped DRB1*03DQA1*0501DQB1* 02/DRB1* 04DQA1*0301DQB1*0302 were higher than in patients genotyped DRB1* 3DQA1*0501DQB1*02/DRB1*XDQA1*XDQB1*X or DRB1* XDQA1* XDQB1*X/DRB1*XDQA1*XDQB1*X. CONCLUSIONS: This study confirms some clinical heterogeneity of Type 1 DM linked to HLA DR and DQ genotypes, and leads to a paradoxical finding: DQB1*02/ DQB1*0302 combination predisposes to an early onset in the whole population but residual secretion of insulin disappears more slowly in a subgroup of young adults with recently diagnosed diabetes. These data suggest that interrelations between MHC genotype and diabetogenic process could be different at various ages of life.