A novel allele HLA-C*07:445 identified in a French hematopoietic stem cell donor.

The novel allele HLA-C*07:445 has 1 nucleotide change from HLA-C*07:01 at nucleotide 277 C>A in exon 2.

FY*X real-time polymerase chain reaction with melting curve analysis associated with a complete one-step real-time FY genotyping.

BACKGROUND AND OBJECTIVES: The Duffy (FY) blood group system is controlled by four major alleles: FY*A and FY*B, the Caucasian common alleles, encoding Fy(a) and Fy(b) antigens; FY*X allele responsible for a poorly expressed Fy(b) antigen, and FY*Fy a silent predominant allele among Black population. Despite the recent development of a real-time fluorescent polymerase chain reaction (PCR) method for FY genotyping FY*X genotyping has not been described by this method. This study focused on the real-time FY*X genotyping development associated with a complete, one-step real-time FY genotyping, based on fluorescence resonance energy transfer (FRET) technology. MATERIALS AND METHODS: Seventy-two blood samples from Fy(a+b-) Caucasian blood donors were studied by real-time PCR only. Forty-seven Caucasian and Black individual blood samples, referred to our laboratory, were studied by PCR-RFLP and real-time PCR. For each individual, the result of the genotype was compared to the known phenotype. RESULTS: The FY*X allele frequency calculated in an Fy(a+b-) Caucasian blood donors population was 0.014. With the Caucasian and Black patient samples we found a complete correlation between PCR-RFLP and the real-time PCR method whatever the alleles combination tested. When the known phenotype was not correlated to FY*X genotype, the presence of the Fy(b) antigen was always confirmed by adsorption-elution. CONCLUSION: The real-time technology method is rapid and accurate for FY genotyping. From now, we are able to detect the FY*X allele in all the alleles combinations studied. Regarding its significant frequency, the detection of the FY*X allele is useful for the correct typing of blood donors and recipients considering the therapeutic use of blood units and the preparation of test red blood cells for antibody screening.

[Identification strategy of RHCE gene variants at the National Blood Group Reference Laboratory: impact on transfusion safety]. / Stratégie d'identification des variants du gène RHCE au Centre national de référence pour les groupes sanguins: impact sur la sécurité transfusionnelle.

The RH blood group is the most polymorphic and immunogenic blood group system. The RH locus is composed of 2 highly homologous genes: the RHD gene encoding the D polypeptide; and the RHCE gene, encoding C or c together with either E or e polypeptides. Numerous variants exist for both RHD and RHCE genes. Among them we were interested in the serological and molecular definition of numerous pre-published RHCE variants encountered in different populations. The identification of these variants is crucial to ensure the transfusion safety for patients expressing these variants. Here we propose a procedure to identify some of them and discuss the adequate transfusion strategy. This procedure has enable us to identify new variants to be presented at the symposium.

[Annual Report 2004 - French National Reference Centre for Rare Blood Groups and Immunohaematology (CNRGS)]. / Rapport 2004 - Centre national de référence pour les groupes sanguins (CNRGS).

In 2004, the French Reference Centre for Rare Blood Groups and Immunohaematology (CNRGS) developed 7 types of activities: 1) Studies of complex Immunohaematology issues (IH), 2) Studies of rare blood phenotypes, 3) the transfusion of patients showing complex issues, 4) IH reactive control in consistency with the 98/79/CE European Directive, 5) European studies and expertise on reactives and techniques, 6) Biotechnologies applied to blood groups, in particular RH, KEL, FY, JK, DO and CO, 7) Implementation of allo-immunization research programs (cellular immunology and grafting issues). The CNRGS efficiency is based on the 'reference-research' link thanks to the Inserm partnership and direct applications to patients allowing to a better risk management and control.

[Cellular mechanisms implicated in anti-erythrocyte alloimmunization]. / L'allo-immunisation anti-érythrocytaire: mécanismes cellulaires.

In many clinical situations patients are dependent on blood transfusions. Occurrence of alloimmunization to blood group antigens (BGA) complicates the transfusion strategy and may be involved in clinical transfusion stalemate situations. B cell differentiation into antibody-secreting plasma cells is triggered by antigen and requires helper T cells which produce cytokines. Although antibodies implicated in BGA alloimmunization have been studied for many years, little is known about helper T cell responses that drive their production. Few studies on BGA specific T cell responses have been published today. This review summarizes the new developments in the field of cellular mechanisms implicated into antibody production. The definition of immunodominant peptides derived from RhD and Jk(a) BGAs, the cytokine patterns induced and the HLA class II molecules implicated in their presentation are analyzed. A tolerogenic route for RhD immunodominant peptides is experimented. Identification of such immunodominant peptides, the cytokine patterns induced and the HLA class II molecules implicated in their presentation, would facilitate the design of new therapeutic strategies including the specific control of alloimmunization with peptide antigen tolerogens or the ex-vivo induction of regulatory T cells.

Serological studies of monoclonal RH antibodies with RH1 (D), RH2 (C), RH3 (E) and RH5 (e) variant RBCs.

One hundred and forty five Mabs against RH antigens were tested. In this paper, we chose to detail reactivity of MoAbs directed against variant RBCs of the CNRGS collection for which we studied the molecular background. Because we developed procedures to identify variants of the RhD, RhC, RhE and Rhe antigens, we were especially interested in finding new monoclonal antibodies that could help us to characterize more accurately these variants. Therefore, we drew parallels between our procedures and results obtained with the 2001 workshop antibodies.

[The French reference laboratory for rare blood groups: activities in 2001]. / Le Centre national de référence pour les groupes sanguins (CNRGS): activité 2001.

The French reference laboratory for rare blood groups (CNRGS) is working for all participants of the transfusion chain: from the donors to the recipients; from the French Establishment for Blood to medical laboratories; from hospital to the haemovigilance network; from governmental agencies to European structures. This laboratory is in charge of: (1) studies of complex problems of immunohaematology; (2) studies of rare blood group phenotypes; (3) reagents quality controls; (4) production of biological standards; (5) specific specimen banks; (6) molecular studies of blood group antigens and antibodies involved; (8) panels of reference cells or DNA; (9) international exchanges.

Two new alleles of the RHCE gene in Black individuals: the RHce allele ceMO and the RHcE allele cEMI.

Six unrelated individuals of Afro-Caribbean origin, whose red cells have a marked reduction of the Rhe antigen expression, have been identified. All exhibited the same serological profile with anti-e monoclonal antibodies and lacked expression of the high frequency e-related antigen hrS. Transcripts and genomic analysis showed that these phenotypes resulted from the presence of two new RHCE alleles, ceMO and cEMI. The ceMO allele corresponded to a RHce gene carrying a G667T mutation (exon 5) and was detected at the homozygous state in sample 1 and at the heterozygous state in samples 2-6. The G667T mutation resulted in a Val223Phe substitution on the Rhce polypeptide, in close proximity to Ala226 (e-antigen polymorphism), which might account for the altered expression of e. The ceMO allele is also associated with the lack of expression of the hrS antigen. The absence of the hrS antigen expression may have implications in transfusion as hrS-negative individuals may develop clinically significant antibodies. The cEMI allele corresponded to a silent RHE allele carrying a nine nucleotide deletion within exon 3 and was detected at the heterozygous state in sample 2. This deletion resulted in a shortened polypeptide of 414 residues (instead of 417) that was absent (or severely reduced) at the red cell surface, as the E antigen was undetectable using serology and Western blot analysis with anti-E reagents. In DNA-based polymerase chain reaction genotyping for RHE determination, the cEMI allele provided a false positive result as the cells carrying this allele are serologically phenotyped as E-negative. The incidence of this allele in the Black population is unknown but, as shown already for D genotyping, one must exercise caution when genotyping is performed to detect the e/E polymorphism.

Characterization of the B*5002-Cw*0602-DRB1*0406-DQB1*0402 Haplotype: impact on the unrelated-bone marrow donor search strategy.

Improvements in HLA-typing by DNA-based methods now allow accurate genotyping of unrelated bone marrow (UBM) donors and recipients and also definition of haplotypes. In this regard, B*5002 has been predicted in linkage disequilibrium with Cw*0602, DRB1*0406 and DQB1*0402 based on the frequency of allele coexistence. Here, we confirm this assumption by HLA genotyping of four informative families and three unrelated individuals. In the four families, the extended haplotype HLA-B*5002, -Cw*0602, -DRB1*0406, DQB1*0402 can be definitely assigned by the mode of heritance. Furthermore, this association of alleles was also found in the three B*5002 unrelated individuals. Knowledge of the frequent linkage disequilibrium of these rare alleles can improve UBM donor search strategies.

Intestinal pseudo-obstruction and acute pandysautonomia associated with Epstein-Barr virus infection.

We report the association of neurological and intestinal disorders with the reactivation of Epstein-Barr virus (EBV) in a child. This previously healthy 13-yr-old boy presented with pharyngitis and acute abdominal ileus. Laparotomy excluded a mechanical obstruction. Postoperatively, he suffered from prolonged intestinal obstruction, pandysautonomia, and encephalomyelitis. Histological examination of the appendix and a rectal biopsy taken 3 months after the onset showed an absence of ganglion cells (appendix) and hypoganglionosis (rectum), with a mononucleate inflammatory infiltrate in close contact with the myenteric neural plexuses. EBV-PCR was positive in the blood and cerebrospinal fluid, and in situ hybridization with the Epstein-Barr virus encoded RNA probe showed positive cells throughout the appendix wall including the myenteric area, in a mesenteric lymph node, and in the gastric biopsies. EBV spontaneous lymphocytic proliferation was noted in the blood. The serology for EBV showed previous infection but anti-early antigen antibodies were present. No immunodeficiency was found. Neurological and GI recovery occurred after 6 months of parenteral nutrition and bethanechol. The omnipresence of EBV associated with the neurointestinal symptoms suggest that the virus was the causal agent. This is the first documented case of acquired hypoganglionnosis due to EBV reactivation.

Naive CD4+ T cell subset is the predominant population involved in cytolysis through Fas-ligand in human at birth.

Here we show that Fas ligand, a death factor that plays a key role in peripheral T cell tolerance and homeostasis, can be induced in peripheral blood mononuclear cells from both newborns (cord) and adults. Indeed, stimulation of both populations by ionomycin and phorbol 12-myristate 13-acetate or through the T cell receptor (anti-CD3) leads to a selective lysis of targets transfected with Fas but not of the parental cell line. This lysis clearly involves a Fas-based mechanism inasmuch as it was correlated with the appearance of Fas ligand transcripts and competitively inhibited by a Fas-Fc fusion protein. Yet, the use of separated T cell populations revealed that both CD4+ and CD8+ T cells express this function in adults whereas the CD4 subset is primarily involved in lysis by a Fas-based mechanism in cord. Altogether these results suggest that distinct T cell subsets might be recruited to exert Fas-based lysis during T cell ontogeny. Furthermore, it is tempting to speculate that CD4+ T cells are primarily involved in peripheral T cell homeostasis and tolerance in early life because they are committed to exert Fas-based lysis before any antigen-priming. Finally, the model of Fas ligand exploration described herein might be of great help for the identification of Fas ligand dysregulation in various diseases in infants, including those patients with autoimmune lymphoproliferative syndrome in which Fas is functional.

High prevalence of low femoral bone mineral density in elderly women living in nursing homes or community-dwelling: a plausible role of increased parathyroid hormone secretion.

The present study was designed to visit elderly women living in nursing homes and to compare their femoral neck bone mineral density (BMD) and circulating levels of parathyroid hormone (PTH) and 25-OH vitamin D (25-OHD) with those of subjects living at home, in the immediate vicinity of the nursing homes. Of 1483 women, aged 70 years and older, who were selected, 993 agreed to participate in this trial. Their femoral neck BMD (n = 993) was measured by dual-energy X-ray absorptiometry, with a specific device installed in a mobile truck. The circulating levels of 25-OHD and PTH were assessed after an overnight fast (n = 748). After stratification for age, there were no significant differences in mean femoral neck BMD values, prevalence of femoral neck osteoporosis, mean serum 25-OHD and prevalence of absolute or relative 25-OHD deficiency between the two groups. Serum levels of PTH were significantly higher in women over 80 years old living in nursing homes, compared with the community-dwelling women. After adjustment for age, a significant relation was found between femoral neck BMD and PTH levels in the whole population (p = 0.004) and in community-dwelling subjects (p = 0.039). When stratifying our population by quartiles of serum PTH values, the odds ratios for femoral neck osteoporosis were significantly increased for the top two quartiles compared with the lowest one both before (p = 0.00146) and after (p = 0.0013) adjustment for age and type of housing. From this study we conclude that femoral osteoporosis is largely underestimated in European women. Living in a nursing home is not, per se, a risk factor for decreased femoral BMD, and circulating PTH levels are a key determinant of low femoral bone density and osteoporosis.

Defective IL2 gene expression in newborn is accompanied with impaired tyrosine-phosphorylation in T cells.

Here we confirmed that IL2 mRNA expression in CD3-stimulated T cells is defective at birth. Because protein-tyrosine phosphorylation is an important part of signaling through CD3 and plays a key role in IL2 transcription, we further investigated whether impaired IL2 response to CD3 in newborns would be accompanied with an alteration of tyrosine phosphorylation. In this purpose, CD3-induced tyrosine phosphorylation was evaluated comparatively in newborn and adult cells by immunoblotting of total cellular extract with an antiphosphotyrosine antibody. Results show that, in both peripheral lymphocytes or purified CD4 T cells from both cord and adult, CD3 stimulation could induce small even significant tyrosine-phosphorylation. Tyrosine phosphorylation occurs as soon as 2' following CD3 ligation and was still evident up to 15-20'. Yet, by using a highly sensitive method to analyze CD3-induced accumulation of phosphorylated substrates, which consisted in adding pervanadate, an inhibitor of phosphatases, during the last 2 min of CD3 stimulation, we showed that the intensity of tyrosine phosphorylation was clearly decreased in cord cells. From these results, it is tempting to speculate that suboptimal capacities of cord T cells to up-regulate tyrosine phosphorylation might contribute to defective IL2 production in neonates.

Reproducibility and diagnostic sensitivity of ultrasonometry of the phalanges to assess osteoporosis.

OBJECTIVE: The present study was designed to assess the reproducibility and the diagnostic sensitivity of the amplitude-dependent speed of sound (SoS) at the distal metaphysis of the proximal phalanges. METHOD: Fourteen presumably healthy volunteers were repeatedly measured every 6 weeks for approximately 6 months in order to assess the reproducibility of the SoS of the phalanges. We recruited 91 post-menopausal women, aged 55-75 years, who were divided in three groups according to their lumbar bone mineral density (BMD) and the existence of prevalent vertebral fractures. The objective was to evaluate the diagnostic sensitivity of SoS measurements. We used DBM Sonic 1200 equipment, and assessed the velocity at which US cross the phalanx in a lateral-medial direction. In post-menopausal women, BMD was measured by dual energy X-ray absorptiometry (DXA) at the level of the lumbar spine, the total zone of the non-dominant hip and the femoral neck zone of the non-dominant hip. RESULTS: The precision of the SoS measurements was 0.71+/-0.05% (mean+/-S.E.M) whereas the reproducibility was 0.95+/-0.06%. Subjects with low BMD or prevalent fractures had significantly lower values of SoS (P < 0.001) than the controls. ROC curve analysis applied to the study population confirmed that SoS was able to discriminate between the controls and osteoporotic subjects (area under the ROC curves were 0.82 (low bone mineral density) and 0.85 (prevalent fractures), respectively). Hip BMD was found to be the most significant variable when comparing the controls and the low density patients by stepwise discrimination and SoS significantly improved the discrimination between the groups when added to the hip BMD. The hip BMD was again the most discriminant variable when applying the same techniques to controls and patients with prevalent fractures, followed by SoS and lumbar BMD. A cut-off value of 1881 m/s is defined for SoS by logistic discrimination and likelihood ratio function. With this value, the sensitivity and the specificity for SoS used in the diagnosis of established osteoporosis were, 81.5% and 79.3%, respectively. Sensitivity and specificity were significantly improved when combining ultrasonometry and densitometry. CONCLUSION: Measurement of ultrasound velocity at the phalanges appears to be a precise and reproducible technique. SoS discriminates between normal post-menopausal women and patients with either low lumbar BMD or prevalent fractures to the same extent as BMD measurements.

Cytokine mRNA and protein expression in a mixed leukocyte reaction before and after allogeneic transfusions. Groupe Coopératif de Transplantation d'Ile de France.

BACKGROUND: The precise mechanism by which pretransplant blood transfusions may favorably influence the graft outcome in human transplantation remains unknown. Here, we explored whether the mechanism might be related to an alteration of cytokine response to transplantation antigens. METHODS: Eight patients awaiting kidney transplantation were selected to receive a single planned pretransplant blood transfusion. Before transfusion and 7 days after transfusion, peripheral blood mononuclear cells from these patients were isolated and in vitro stimulated in a one-way mixed leukocyte reaction (MLR) by using allogeneic fixed Epstein Barr virus-transformed cells as stimulators. RESULTS: The use of a semiquantitative reverse-transcriptase polymerase chain reaction cycle technique to analyze cytokine mRNAs revealed that allostimulation by donor cells clearly induced accumulation of interleukin (IL)-2, IL-4, interferon (IFN)-gamma, and IL-10 mRNA in peripheral blood mononuclear cells collected both before and after transfusion (eight of eight patients). However, both T helper 1 (IFN-gamma) and T helper 2 (IL-4) cytokine responses were more elevated after transfusion in eight of eight patients, as were IL-2 responses in five of eight patients. Such up-regulation of cytokine responses by transfusion was mostly directed against blood donor cells. Indeed, after stimulation by third-party cells, this up-regulation was both inconstant (two of three patients) and of less intensity, and no change was detected after stimulation by autologous cells (three of three patients). CONCLUSIONS: That IL-2, IL-4, and IFN-gamma responses to donor cells were increased by transfusion was further supported by results on cytokine secretion showing increased levels of IL-2 (P < 0.05), IFN-gamma (P = 0.054), and IL-4 (P < 0.05) proteins in supernatants of posttransfusion MLR as compared with pretransfusion MLR. In contrast, transfusion-induced changes in the amount of IL-10 mRNAs were not obvious and were quite variable from one patient to another.

Contribution of p56lck to the upregulation of cytokine production and T cell proliferation by IL-2 in human CD3-stimulated T cell clones.

Interaction of the interleukin 2 receptor (IL-2R) beta chain with the lymphocyte-specific protein tyrosine kinase (PTK), p56lck, has led to the speculation that p56lck participates in growth signal transduction. Although activation of T cells with interleukin 2 (IL-2) results in the activation of p56lck, accumulating data support the notion that Lck does not play an essential role in mitogenic signal delivery from the IL-2R. Since this src-related PTK has been shown to enhance TCR/CD3-mediated T cell responsiveness, here we investigated whether activation of Lck by IL-2 could contribute to enhance TCR/CD3-mediated T cell functions. This was achieved by using human CD4(+)-cloned T cells and comparing the effects of IL-2 on p56lck kinase activation and cytokine production. Results show that p56lck kinase activity increased as early as 1 min, reached a maximum within 5 min and decreased within 60 min after IL-2 stimulation. Such treatment with IL-2 also resulted in enhancing T cell responsiveness to CD3+PMA stimulation, as assessed by IL-2 and IFN-gamma secretion and by T cell proliferation. This increase of T cell functions was correlated with IL-2-induced p56lck activation in both dose-response and time-course experiments. Taken together these results strongly suggest that activation of Lck by IL-2 may play a role in regulating CD3-mediated T cell functions.

Contribution of tyrosine kinases to the selective orientation of human CD4+ bifunctional cloned T cells toward proliferation or cytolytic function.

We show that T cell activation of human CD4+ cloned T cells through the CD2 molecule can induce either autocrine proliferation or cytolysis, depending on the pair of anti-CD2 mAbs used for stimulation, that is, D66/T11(1) or GT2/T11(1), respectively. As the earliest biochemical event after CD2 stimulation is likely the induction of tyrosine phosphorylation of various proteins, we investigated whether differential activation of protein tyrosine kinases (PTKs) could contribute to the selective induction of each function. Results show that herbimycin A, a potent PTK inhibitor, markedly decreased the induction of both proliferation and cytolysis. This implies a regulatory role for tyrosine phosphorylation in the induction of each function by CD2. However, that PTKs are differentially activated upon induction of proliferation by D66/T11(1) or cytotoxic function by GT2/T11(1) emanated from two different approaches. First, immunoblotting total cellular extracts with an anti-phosphotyrosine mAb showed different patterns of tyrosine phosphorylation depending on the pair of CD2 mAbs used for stimulation. Second, a differential activation of p56lck, a src-related PTK, was observed after stimulation with D66/T11, and GT2/T11(1). Although induction of proliferation by D66/T11(1) was correlated with increased Lck activity, this was not observed when cells were triggered to lyse by GT2/T11(1). Thus, by providing striking correlative evidences linking differences in PTK activation with induction of different functions in bifunctional cloned T cells, our results strongly suggest that PTKs may contribute to the selective orientation of T cell functions at a single-cell level.