[Banking and indications for cord blood grafts]. / Conservation et indications des greffons de sang placentaire.

A cord blood graft is now frequently used in the context of allogeneic hematopoietic transplantation. In 2007, 27% of unrelated allotransplantations performed in France used a cord blood graft. In comparison to other sources of hematopoietic grafts, cord blood has a number of advantages (immediate availability of the graft, possibility of a lesser HLA compatibility between donor and recipient...) and inconveniences (a limited number of stem cells, increased risk of infectious complications...). Recently, the possibility of combining two cord blood grafts has been demonstrated. In 2007, 55% of unrelated cord blood grafts transplantation were performed as such. Results of multicentric studies published as of now, as well as considerable potential associated with cord blood transplantation requires important efforts to increase both the quantity and quality of cord blood grafts made available for transplantation.

T-cell flow-cytometry crossmatch and long-term renal graft survival.

Flow cytometry crossmatch (FCXM) is a more sensitive technique than classical complement-dependent cytotoxicity (CDC) for the detection of donor-directed antibody before renal transplantation. Nevertheless, the role of FCXM in predicting long-term survival of kidney grafts is still unclear. The purpose of our study was to evaluate the impact of a positive T-cell FCXM (T-FCXM) on long-term kidney allografts outcome. Of the 184 consecutive kidney transplantations performed in our center between 1 January1991 and 15 November 1996 a FCXM, performed concurrently to the pre-transplant CDCXM, was available for 170 patients. The CDCXM was negative in all recipients. Among these recipients, 12 (7.1%) had a positive T-FCXM. These patients were not different from patients with a negative T-FCXM for donor and recipient age, sex, frequency of second transplantation, number of human leukocyte antigen matches or mismatches. Frequency of immunized patients was higher in kidney recipients with a positive FCXM (58.3% vs. 24.7%; p=0.02, chi-square test). Survival analysis revealed that kidney graft outcome was better in negative T-FCXM recipients (p=0.03), while patient survival was not statistically different. Our results suggest that a positive pre-transplant T-FCXM despite a negative CDCXM is associated with an impaired long-term graft survival in renal allotransplantation.

Polymorphisms of the TAP1 and TAP2 genes in human alveolar echinococcosis.

We postulated that TAP genes may influence the susceptibility of some individuals to Echinococcus multilocularis infection. Six coding region variants (codons 333 and 637 in TAP1, and 379, 565, 651 and 665 in TAP2) were typed in 94 patients and 100 controls. Thr/Thr homozygosity at TAP2/665 was more prevalent in patients than in controls [64% vs. 45%, respectively; odds ratio (OR) = 2.1 (95% confidence interval (CI) 1.1; 2.7)] and Thr/Ala heterozygozity was less prevalent (32% vs. 50%, respectively) (P = 0.014). Of the 38 patients with progressive lesions, 76% were Thr/Thr, as compared with 55% of patients without progressive lesions and 45% of controls (P = 0.058 and 0.02, respectively), independent of HLA status. To determine whether this association is functionally relevant, functional analyses and/or confirmation in distinct populations of patients with alveolar echinococcosis would be required.

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.

Flow-PRA evaluation for antibody screening in patients awaiting kidney transplantation.

Flow-PRA is a flow cytometric method for both anti-HLA class I and class II antibody (Ab) detection. We evaluated this technique for Ab screening in patients awaiting kidney transplantation. After having established a rigorous threshold for positivity, a three-dilution difference in sensitivity between Flow-PRA and complement-dependent cytotoxicity (CDC) persisted. The sensitivity of the method was satisfactory since all CDC-positive sera were also found to be positive with the Flow-PRA method. Discrimination between anti-HLA class I and class II Abs was excellent. Furthermore, all sera responsible for a positive flow cytometry crossmatch (FCXM) and a negative CDC-crossmatch (CDCXM) at the time of a putative transplant were found to be positive with Flow-PRA beads. The specificity was excellent for anti-class I Ab detection since no false positive serum was found. On the other hand, the specificity was lower for anti-class II detection, since 8.3% (2/24) false positive results were detected among all the negative sera tested. Overall, our results suggested that Flow-PRA should be of value for anti-HLA Ab screening prior to kidney transplantation.

A novel HLA-B null allele (B*4022N) generated by a nonsense codon in the alpha1 domain.

We describe in this work a novel HLA-B null allele designated B*4022N. This new variant was found in a Caucasian individual who was serologically typed for one HLA-B allele as a B-blank, Bw-blank. Retrospective DNA typing by polymerase chain reaction using sequence-specific primers (PCR-SSP) has established the correspondence of this blank allele with the classical HLA-B*4001 allele. Nucleotide sequence analysis of exon 2 and 3 has revealed the presence of two adjacent point mutations at position 170 and 171 of exon 2 (GG to TT). While the first difference is silent, the second leads to the creation of a nonsense codon at position 58 of the alpha1 domain, providing the most likely mechanism underlying the observed null phenotype.

The association of HLA-DM genes with rheumatoid arthritis in Eastern France.

In this study, the polymorphisms of the HLA DMA and DMB genes in patients with rheumatoid arthritis (RA) were examined.DMA and DMB typing was performed in 120 white RA patients from eastern France and 100 healthy controls, using PCR-SSO (sequence specific oligonucleotide probes) method for DMA determination and PCR-RFLP (restriction fragment length polymorphism) method for DMB typing. All patients and controls had been HLA DRB1* genotyped.DMA*0103 was found significantly increased in RA patients (RA vs. controls: 18.3% vs. 4%) (p(corr) = 0.004; OR: 5.39; CI: 1.67-19.23). A decreased frequency of DMA*0102 was also observed in the RA group (RA vs. controls: 18.3% vs. 31%), but not significantly. There were no differences in the prevalence of DMB alleles between RA and controls. The patients and the controls were then stratified according to the expression of the HLA DRB1* RA-linked alleles (DRB1*01 and *04) and this allowed us to find no linkage disequilibrium between DMA*0103 and DRB1*01 or *04 alleles. Finally, most DMA*0103 patients were positive for rheumatoid factors and had extraarticular involvement such as subcutaneous nodules. Thus, our results suggest that DMA*0103 could be an additional genetic factor for RA susceptibility in French whites.

HLA-DRB1 alleles and shared amino acid sequences in disease susceptibility and severity in patients from eastern France with rheumatoid arthritis.

OBJECTIVE: To examine the effects of HLA-DRB1 alleles and amino acid sequences that carry the shared epitope (SE) upon rheumatoid arthritis (RA) susceptibility and disease severity in patients from Eastern France. METHODS: HLA-DRB1 alleles were determined in 120 patients and 104 healthy controls by polymerase chain reaction/sequence specific oligonucleotide probes. Subtyping of DRB1*01 and *04 were performed using sequence specific primers. Patients were retrospectively evaluated for disease duration, age at disease onset, presence of rheumatoid factors, subcutaneous nodules, vasculitis and other extraarticular diseases, for the need for arthroplasty and immunosuppressive/immunoregulatory agents, and for radiographic damage. RESULTS: The prevalence of HLA-DRB1*04 was significantly higher in patients (46.6%) than in controls (17.3%) (Pcorr = 0.000003). HLA-DRB1*0101 and *0401 were the most prominently associated subtypes in patients with RA (33.3%, Pcorr = 0.011, and 28.3%, Pcorr = 0.00008, respectively). A significant fraction of patients (72.5%) expressed one or 2 copies of the SE (p < 0.0000001; OR 4.77, CI 2.61-8.78). The presence of double SE was associated with a higher risk of developing RA (OR 4.83, CI 1.91-12.71; p = 0.0001). No significant differences in the clinical records among patients expressing no RA linked alleles, one and 2 copies of the SE, were observed. However, analyzing the specific effect of each amino acid sequence, we observed a significant association of the QKRAA motif with vasculitis (p = 0.03) and history of joint replacement surgery (p = 0.05), suggesting a role for lysine in position 71 of the shared sequence. CONCLUSION: These findings differ from those of previous HLA-DRB1 allele studies in patients with RA from other regions of France. Thus, the heterogeneity in both the expression of DRB1 alleles and the association of these alleles with disease severity could be relevant within a country such as France.

Impaired renal graft survival after a positive B-cell flow-cytometry crossmatch.

BACKGROUND: The clinical and immunological relevance of a positive B-cell flow-cytometry (B-FCXM) crossmatch in renal transplantation is still controversial. METHODS: We retrospectively analysed 145 consecutive cadaveric renal transplantations performed from May 1991 to September 1995 in our institution. All grafts were transplanted following a negative IgG T-cell complement-dependent cytotoxicity crossmatch (T-CDCXM). Concomitantly to CDCXM, B-cell and T-cell FCXM were performed and results were expressed as a mean fluorescence index (FI). Two groups were compared: 116 recipients grafted with a negative B-FCXM vs a group of 19 patients grafted with a positive B-FCXM. RESULTS: The two groups were similar for length of cold ischaemia, donor and recipient's age and degree of HLA mismatching. The proportion of patients with pre-transplant anti-HLA class I antibodies or a retransplantation was significantly increased in the positive B-FCXM group vs the negative B-FCXM group. Recipient survival at 48 months was not significantly different in the two groups. However, graft survival at 12 and 48 months was significantly poorer in the positive B-FCXM than in negative B-FCXM (68% vs 90% at 12 months: P = 0.007, and 57% vs 79% at 48 months: P = 0.02). Within the positive B-FCXM group, no differences were found in pre-transplant anti-HLA class I or II alloimmunization as well as retransplantation frequency between the patients who lost their graft and the patients who did not. CONCLUSION: Our results suggest that a pretransplant positive B-FCXM is associated with an impaired long-term graft survival in renal allotransplantation.

Fortuitous detection of uniparental isodisomy of chromosome 6.

Uniparental isodisomy is defined as the inheritance of two copies of the same parental chromosome and can result in defects when it produces homozygosity for a recessive mutation or in the presence of imprinting. We describe the detection of a chromosome 6 uniparental isodisomy in a 9 year old girl, discovered during a search for an HLA identical sib. HLA typing, erythrocyte phenotyping, and genotypes of microsatellite polymorphisms were compatible with a paternal isodisomy of chromosome 6, with normal biparental origin of the other chromosomes. Paternal cells were not responsive to the patient's cells in mixed lymphocyte cultures. This fortuitous detection of a chromosome 6 isodisomy suggests that cases of chromosome 6 UPD may not be deleterious and may therefore go undetected.

Variability of the alloreactive T-cell response to human leukemic blasts.

Clinical and experimental data suggest a role for the immune response in preventing leukemic relapses after allogeneic bone marrow transplantation (BMT): the graft-versus-leukemia (GVL) effect. In this report, we have evaluated the response of normal donor lymphocytes against allogeneic leukemic cells as an in vitro model of the GVL effect. We used a limiting dilution technique in order to determine the frequency of cytotoxic T-lymphocyte precursors (pre-CTL) against allogeneic leukemic blasts among normal donor lymphocytes. We demonstrate a considerable variability of CTL precursor frequency. This variability depended on leukemic populations since, for a given leukemia, the pre-CTL frequency was comparable among our tested normal allogeneic donors. Moreover, when HLA-DR negative leukemias were used as allostimulators, the pre-CTL frequencies were extremely low. In order to verify the impact of leukemic DR expression on the stimulatory capacity of leukemic cells, we selected and analyzed in mixed lymphocyte tumor cell culture (MLTC), a panel of myelogenous and lymphoblastic leukemias with variable levels of DR expression, each against different allogeneic responders. Our results demonstrated a close correlation (r = 0.953, p < 0.0001) between the proliferative response of alloactivated lymphocytes and the percentage of stimulatory leukemic cells expressing HLA-DR molecules. Anti-MHC class II monoclonal antibodies inhibited the lymphocyte proliferation in the MLTC, confirming the preponderant role of DR in the generation of this response. Overall, our results demonstrate the extreme variability of leukemic cells in their allostimulatory capacity and the central role of DR expression in determining leukemic allo-recognition. In the setting of a clinical protocol, our data suggest that the infusion of allogeneic T lymphocytes in a DR negative leukemia will not lead to an alloreactive T-cell anti-tumor effect.