Multiple transfusions for sickle cell disease in the Democratic Republic of Congo: the importance of the hepatitis C virus.

BACKGROUND AND OBJECTIVES: Improvement of transfusion security in sub-Saharan countries requires the determination of priorities taking into account the specific context. PATIENTS AND METHODS: One hundred and forty patients with sickle cell disease (SCD) from one clinical centre for SCD in Kisangani, DRC were tested for HBsAg, anti-HIV antibodies, anti-HCV antibodies and for alloantibodies against red blood cells and human leucocyte antigens (HLA). RESULTS: Thirteen patients had not been transfused and were free of HBV, HIV or HCV infection. HBV, HIV and HCV infections were detected in 2/127 (1.6%), 1/127 (0.9%) and 10/127 (7.9%) transfused patients, respectively. All ten cases of HCV infection were associated with patients who had transfusions prior to the introduction of HCV testing in 2004 (P=0.043). Red blood cells and HLA alloantibodies were detected in 13/127 (10%) and 2/127 (1.6%), respectively. CONCLUSION: HCV testing should be a priority. The rhesus (Rh) phenotype, mainly the RhD antigen and the Kell antigen should be assessed in SCD patients. Further extended phenotyping and deleucocytation should not be considered as priorities.

[Cord blood: from bench to bedside]. / Le sang de cordon: du laboratoire au lit du malade.

Since 1974, umbilical cord blood (CB) has been shown to contain haematopoietic stem cells similar to stem cells from the bone marrow. In 1988, E. Gluckman and her colleagues performed - successfully - the first familial CB transplantation and cured a 5 years old child suffering from Fanconi's anemia. Rapidly, CB banks were organised throughout in the world and thanks to this novel source of haematopoietic stem cells, we can now find a donor for 75 % of the patients requiring a "bone marrow" transplantation. The major benefit of CB as a source of hematopoietic stem cells is its easy access. CB also allows a more significant degree of HLA incompatibility and thus offers an opportunity of transplantation to ethnic minorities for whom no HLA identical donors are available. However, several studies have shown that the number of cells harvested in a CB was closely correlated with the engraftment post transplantation and today, a minimum of 3.7 x 10(7) mononucleated cells/kg is recommended. This required amount of cells is not always reached due to the small volume often harvested from a CB. Therefore, to apply CB transplantations to adults, different approaches are currently being investigated : coinfusion of haploidentical cells, mesenchymal cells, a second CB, or the addition of CB expanded ex-vivo. Among these approaches, double CB transplantation seems nowadays the most promising alternative and ongoing studies should soon inform us whether the duration of aplasia will be improved.

Earlier occurrence of severe alopecia areata in HLA-DRB1*11-positive patients.

BACKGROUND: Alopecia areata (AA) is a polygenic immune-mediated disorder affecting the hair follicle for which an association with human leukocyte antigen HLA-DRB1*11 has been described. OBJECTIVE: Two parameters including age of onset and extent of the disease (patchy AA and AT/AU forms) were correlated with the presence or absence of HLA-DRB1*11 and its alleles in 88 severe AA patients. METHODS: Patients and healthy controls were typed for HLA-DR and -DQ by molecular method. RESULTS: Among AA patients, 37.5% (a proportion rising to 72% when taking patients who began their first patch before the age of 20 years) were positive for HLA-DRB1*11 compared to 21.2% healthy controls (p = 0.004, RR = 2.1). DRB1*11-positive status was associated with earlier development of the first AA patch, at the mean age of 16 years compared to 27 years (p = 0.003) in DRB1*11-negative patients. Among the DRB1*11 alleles, the presence of DRB1*1104 was associated with the earliest occurrence of AA. CONCLUSION: Our data indicate that the HLA system largely through DRB1*1104 allele influences AA onset rather than extension considering patchy AA and AT/AU.

[The immunology-hematology-transfusion department]. / Le Service d'Immunologie-Hématologie-Transfusion.

New immunotherapies derived from biotechnology offer fascinating perspectives in different fields of medicine including anti-infectious vaccines, cancer, organ transplantation and autoimmune diseases. In this paper, we illustrate how the Department of Immunology can contribute to the development of these new treatments within a academic hospital such as the Erasme Hospital at the Université Libre de Bruxelles.

Isolation and infusion of donor CD34+ bone marrow cells in cadaver kidney transplantation.

BACKGROUND: Infusion of donor bone marrow cells induces tolerance in allograft models. CD34+ stem cells present in human bone marrow could be endowed with tolerogenic properties. METHODS: CD34+ stem cells were isolated from bone marrow extracted from vertebral bodies of cadaveric donors. Donor CD34+ cells (0.6-3.7 x 10(6)/kg) were infused during surgery in 10 kidney transplant recipients receiving OKT3 as induction therapy. Chimerism was investigated using nested PCR for donor-specific HLA alleles. RESULTS: The infusion of CD34+ stem cells was perfectly tolerated. Five patients remained free of acute rejection at follow-up, 47-325 days post-operatively. The five other patients underwent a single episode of corticosensitive acute rejection. Long-term chimerism was not induced in the seven patients investigated for the persistence of donor DNA. CONCLUSIONS: Infusion of donor CD34+ stem cells in kidney transplantation is safe. The clinical usefulness of the procedure remains to be established.

Lung rejection occurs in lung transplant recipients with blood chimerism.

BACKGROUND: It has been postulated that chimerism after transplantation might promote graft acceptance. In the present study, we prospectively assessed blood chimerism in 10 lung transplant recipients during the first posttransplant year and investigated whether chimerism was associated with an immunologically stable situation of the graft. METHODS: The recipients' peripheral blood mononuclear cells were obtained before transplantation and at various time points during the first postoperative year. Donor cells were detected using nested polymerase chain reaction amplification of a donor-specific HLA-DRB1 allele. Clinical graft acceptance was determined by the number of rejection episodes. RESULTS: The incidence of blood chimerism was high during the first 3 postoperative months and then decreased over time. All patients experienced at least one acute rejection episode, and three patients developed chronic rejection. CONCLUSION: We, thus, conclude that rejection of the lung allograft may occur in the presence of blood chimerism.

Optimal control of interferon-gamma and tumor necrosis factor-alpha by interleukin-10 produced in response to one HLA-DR mismatch during the primary mixed lymphocyte reaction.

Interleukin (IL)-10 is an immunosuppressive cytokine potentially involved in the control of the allogeneic response. Several studies failed to detect it in mixed lymphocyte reaction supernatants. However, experiments using IL-10-specific antibodies, revealing its inhibitory action on interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha, provided indirect evidence that endogenous IL-10 was produced. The aim of the present work is to elucidate the role of IL-10 during mixed lymphocyte reaction and to investigate the influence of HLA-DR antigens on its production and on the regulatory loop involving TNF-alpha and IFN-gamma. Using a highly sensitive ELISA, a significant (P < 0.0001) but low IL-10 release could be detected (33.7 +/- 3.6 pg/ml) in response to HLA-DR disparities. However, IL-10 release was not graded as 1 DR mismatch (MM)-induced maximal secretion (32.3 +/- 5.1 pg/ml). This contrasted with TNF-alpha and IFN-gamma productions, which significantly increased in 2 DR MM pairs. Addition to IL-10-specific antibodies resulted in higher enhancement of INF-gamma (235 +/- 38% vs. 122 +/- 39%, P = 0.02) and, to a lesser extent, TNF-alpha (147 +/- 56% vs. 112 +/- 20%, NS) in 1 compared with 2 DR MM pairs. We conclude that the 1 DR MM setting is associated with optimal IL-10 secretion and more efficient inhibition of IFN-gamma and TNF-alpha compared with the 2 DR MM configuration. Although promoting enhanced IFN-gamma and TNF-alpha release, introduction of an additional DR MM does not result in increased IL-10 production. These data indicating that the IL-10 regulatory feedback loop is more effective in 1 DR rather than complete DR incompatibility could have an impact on matching policies for planned transfusion.

Interleukin 12 unmasks HLA class I differences during mixed lymphocyte reaction induced interferon gamma production.

We investigated the genetic control of IFN-gamma release during MLR and its relationship with TNF-alpha and IL-12. Blocking experiments demonstrated the IFN-gamma dependence of TNF-alpha production and the significant contribution of IL-12 to IFN-gamma secretion. We studied informative pairs allowing the evaluation of the relative importance of HLA class I and class II antigens. Maximal IFN-gamma secretion allowing discrimination between fully HLA different and identical subjects required 5 days. In class I different but DRB1 identical pairs, a moderate but discriminant IFN-gamma release was found. Exogenous IL-12 addition after 24 hours of preactivation by MLR resulted in a marked enhancement of IFN-gamma production at day 2. In pairs differing only by class I antigens, the discriminating capacity was significantly increased as compared to values obtained in absence of IL-12 at day 2 (p < 0.004) and at day 5 (p < 0.004). The crucial role of class I antigens on IFN-gamma release was further substantiated by the blocking action of the W6/32 mAb directed against a monomorphic epitope common to all HLA-A, -B, and -C antigens. We conclude that IFN-gamma production during MLR is under the control of class I antigens. Furthermore, exogenous IL-12 strongly amplifies their influence.

Influence of donor-recipient HLA-DR mismatches and OKT3 prophylaxis on cadaver kidney graft survival.

Previous studies from our center have shown that donor-recipient HLA-DR mismatches (MM), characterized by the presence of the DR antigen in the donor but not in the recipient or vice versa, are associated with differential effects on graft survival (GS): some of them are beneficial (BEN) with results similar to those of HLA-DR identical or compatible pairs (85% 18 months GS) and some are detrimental (DET) (64% 18 months GS), whereas the other MM, neither BEN nor DET (neutral [NEU]) yield intermediate results (78% 18 months GS). The aim of the present study was to update the results at a longer follow-up time and to assess whether they are influenced or not by prophylactic administration of anti-CD3 mAb (OKT3). The analysis of 234 transplantations performed from 1980 to 1994 with only 1 HLA-DR MM confirmed the BEN effects of HLA-DR5 in either the donor or the recipient and the DET effects of HLA-DR1 or -DR2 in the donor and of HLA-DR2 or -DRW6 in the recipient. These effects were independent of those exerted by other, HLA-DR not related, prognostic factors. The transplants with 1 HLA-DR MM were then compared with those with zero HLA-DR MM (n = 378) and 4 groups were formed according to 2 levels of HLA-DR MM (zero or BEN MM vs. NEU or DET MM) and immunosuppression (with vs. without OKT3 prophylaxis). GS at 5 years was 63% in the group with either zero or BEN MM as compared with 41% in the group with either NEU or DET MM in the absence of OKT3 prophylaxis (P < 0.02); in comparison, with OKT3 prophylaxis, GS at 5 years was 73% in the group with either zero or BEN MM as compared with 58% in the group with either NEU or DET MM (P = 0.07). We conclude that the differential effects of HLA-DR MM on GS are still observed under OKT3 prophylaxis, that the effects of HLA-DR and immunosuppression on graft outcome are additive, and that OKT3 induction therapy is superior to therapy without OKT3. These observations could have important implications for the allocation policy and management of renal transplants.

Detection of blood chimerism after lung and heart-lung transplantation. The superiority of nested as compared with standard polymerase chain reaction amplification.

Migration of donor cells from the graft to various tissues of the recipient has been demonstrated after different types of solid organ transplants. Currently, the detection of donor cells in the recipient's tissues is most simply performed by polymerase chain reaction (PCR) amplification of a donor-specific gene. In the present study, we first determined in vitro the sensitivity of standard and nested PCR amplification with sequence-specific primers (PCR-SSP) of a donor-specific allele of the HLA-DRB1 gene and then used this technique to assess prospectively blood chimerism in two single-lung (SLT) and one heart-lung (HLT) transplant recipients. Standard PCR-SSP consisted in a single amplification round with sequence-specific primers for the donor-specific DRB1 allele. Nested PCR-SSP consisted in a first round of generic amplification of exon 2 of the DRB1 gene, followed by a second amplification round with primers specific for the donor allele. In vitro, nested PCR-SSP of the donor-specific allele was 1000-fold more sensitive than standard PCR-SSP and allowed the detection of 1 donor cell in 10(5) recipient cells. In vivo, standard PCR-SSP detected donor cells among the recipients' peripheral blood mononuclear cells (PBMCs) only during the first postoperative days, whereas nested PCR-SSP demonstrated their presence until the end of the first postoperative month in patients 1 and 2 and until 3 months after transplantation in patient 3. We conclude that donor cells can be detected in the peripheral blood of SLT and HLT recipients during the first postoperative months and that nested PCR-SSP amplification of a donor-specific HLA-DRB1 allele is much more sensitive than standard PCR-SSP to demonstrate such chimerism.

Tumor necrosis factor-alpha and interleukin-6 production induced by variations of DR4 polymorphism during the primary mixed lymphocyte reaction.

Serologically defined MHC class II differences provoke release of TNF-alpha and IL-6 during MLR. In order to assess the influence of micropolymorphism defined at the genomic level, we selected informative donors pairs within DR2 DR4 serologically defined unrelated subjects by combining those differing only by DR4 alleles, as assessed by PCR-SSOP (DRB1*0401 to 07). Two groups of MLR combinations were tested including DRB1-identical (group 1, n = 12) and one DRB1 difference (group 2, n = 16). Pairs of HLA-identical siblings (n = 4) and of unrelated subjects differing by two major DR incompatibilities detected by serology (n = 27) were used as controls. We further investigated whether DP and DQ differences contributed to the observed CK production. Comparison of group 2 with group 1 showed that one DRB1 difference had a marked influence on CK production at day 3 (TNF-alpha: 401.8 +/- 85 pg/ml vs. 128.7 +/- 34.5 pg/ml, P = 0.001; SI = 2.97 +/- 0.23 vs. 1.27 +/- 0.09, P < 0.0001; IL-6: 317.6 +/- 44.8 pg/ml vs. 108 +/- 13 pg/ml, P = 0.003; SI = 2.53 +/- 0.37 vs. 1.11 +/- 0.05, P < 0.0001). However, CK release in group 2 was significantly lower than that observed in subjects with two serologically defined DR differences (TNF-alpha: 515.1 +/- 61.4 pg/ml, P = 0.05; SI = 5.61 +/- 0.48, P < 0.0001; IL-6: 545.9 +/- 75.8 pg/ml, P = 0.03; SI = 4.75 +/- 0.58, P < 0.0004). Addition of LPS after one day of MLR resulted in discriminant production of CK in group 2 as compared with group 1. Neither DP nor DQ differences affected CK production. In conclusion, DR subtypic differences induce significant CK release during primary MLR. This in vitro study demonstrates the immunodominance of the DR system in eliciting strong inflammatory mediators release.

T-cell-epitope mapping of the major secreted mycobacterial antigen Ag85A in tuberculosis and leprosy.

Lymphoproliferation and gamma interferon (IFN-gamma) secretion in response to 28 overlapping 20-mer synthetic peptides covering the complete sequence of the mature (295-amino-acid) 85A component of the major secreted, fibronectin-binding antigen 85 complex from Mycobacterium tuberculosis and Mycobacterium bovis BCG (MTAg85A) was examined by using peripheral blood mononuclear cell (PBMC) cultures from healthy tuberculin- and lepromin-positive volunteers and from patients with tuberculosis and leprosy. Peptide recognition was largely promiscuous, with a variety of human leukocyte antigen haplotypes reacting to the same peptides. PBMC from all tuberculin-positive subjects reacted to Ag85, and the majority proliferated in response to peptide 6 (amino acids 51 to 70), peptides 13, 14, and 15 (amino acids 121 to 160), or peptides 20 and 21 (amino acids 191 to 220). PBMC from tuberculosis patients demonstrated a variable reactivity to Ag85 and its peptides, and the strongest proliferation was observed against peptide 7 (amino acids 61 to 80). MTAg85A peptides were also recognized by PBMC from healthy lepromin-positive volunteers and paucibacillary leprosy patients (again in a promiscuous manner), but despite a 90% homology between the 85A proteins of M. leprae and M. tuberculosis, the peptides recognized were different. PBMC from lepromin-positive healthy contacts reacted against peptide 2 (amino acids 11 to 30), peptide 5 (amino acids 41 to 60), and peptides 25 and 26 (amino acids 241 to 270). PBMC from paucibacillary patients reacted preferentially against peptide 1 (amino acids 1 to 20) and peptide 5. Multibacillary patients were not reactive to Ag85 or the MT85A peptides. IFN-gamma production was generally detected simultaneously with positive lymphoproliferative responses, although peptide 1 mostly stimulated proliferation and peptides 27 and 28 mostly elicited an IFN-gamma response. In conclusion, regions 41 to 80 and 241 to 295 demonstrated powerful and promiscuous T-cell-stimulatory properties, resulting in proliferative responses and IFN-gamma secretion, respectively, in the majority of reactive subjects tested in this study. These results could be of value in the development of a subunit vaccine for tuberculosis and leprosy.

Isodisomy of chromosome 6 in a newborn with methylmalonic acidemia and agenesis of pancreatic beta cells causing diabetes mellitus.

Isodisomy (ID) is a genetic anomaly defined as the inheritance of two copies of the same genetic material from one parent. ID in an offspring is a rare cause of recessive genetic diseases via inheritance of two copies of a mutated gene from one carrier parent. We studied a newborn female with a mut(o) of methylmalonic acidemia and complete absence of insulin-producing beta cells in otherwise normal-appearing pancreatic islets, causing insulin-dependent diabetes mellitus. The patient died 2 wk after birth. Serotyping of the HLA antigens, DNA typing of HLA-B and HLA class II loci, study of polymorphic DNA markers of chromosome 6, and cytogenetic analysis demonstrated paternal ID, involving at least a 25-centiMorgan portion of the chromosome pair that encompasses the MHC. ID probably caused methylmalonic acidemia by duplication of a mutated allele of the corresponding gene on the chromosome 6 inherited from the father. It is also very likely that ID was etiologically related to the agenesis of beta cells and consequent insulin-dependent diabetes mellitus in our patient. We thus speculate on the existence of a gene on chromosome 6 involved in beta cell differentiation.