When ß-cells fail: lessons from dedifferentiation.

Diabetes is caused by a combination of impaired responsiveness to insulin and reduced production of insulin by the pancreas. Until recently, the decline of insulin production had been ascribed to ß-cell death. But recent research has shown that ß-cells do not die in diabetes, but undergo a silencing process, termed "dedifferentiation." The main implication of this discovery is that ß-cells can be revived by appropriate treatments. We have shown that mitochondrial abnormalities are a key step in the progression of ß-cell dysfunction towards dedifferentiation. In normal ß-cells, mitochondria generate energy required to sustain insulin production and its finely timed release in response to the body's nutritional status. A normal ß-cell can adapt its mitochondrial fuel source based on substrate availability, a concept known as "metabolic flexibility." This capability is the first casualty in the progress of ß-cell failure. ß-Cells lose the ability to select the right fuel for mitochondrial energy production. Mitochondria become overloaded, and accumulate by-products derived from incomplete fuel utilization. Energy production stalls, and insulin production drops, setting the stage for dedifferentiation. The ultimate goal of these investigations is to explore novel treatment paradigms that will benefit people with diabetes.

[Post-transfusion acute lung injury (Trali) after plasma infusion in a patient having a constitutional thrombotic microangiopathy]. / Syndrome d'oedème pulmonaire lésionnel aigu post-transfusionnel (Trali) après perfusion de plasma frais congelé au cours d'une microangiopathie thrombotique congénitale.

INTRODUCTION: Transfusion-related acute lung injury is a post-transfusion interstitial lung injury. CASE REPORT: We reported a post-transfusion acute lung injury in a 23-years old woman having a chronic thrombotic microangiopathy related to an ADAMTS 13 constitutional deficiency receiving monthly plasma infusion for six years. The temporal relationship between the lung injury and the infusion of fresh frozen plasma led to the diagnosis of transfusion-related acute lung injury. The finding in the donor of the transfused plasma of an anti-HLA class II antibody recognizing HLA-DR52 present on leucocytes of the recipient suggests a causal relationship between this antigen-antibody conflict and the triggering of the TRALI. This chronic pathologic state requiring monthly plasma transfusions for thrombotic accident prevention raises the question of the selection of plasma obtained from non-immunized donors. CONCLUSION: The occurrence of a post transfusion pulmonary edema without cardio-vascular overload, must lead to consider a TRALI especially in predisposing clinical situations. In the case reported the role of constitutional ADAMTS 13 deficiency in genesis of TRALI is considered.

[Transfusion-related acute lung injury]. / Oedème pulmonaire lésionnel aigu post-transfusionnel ou Trali.

OBJECTIVES: The transfusion-related acute lung injury frequency was for a long time underestimated since it lacked both a widely accepted clinical definition and a comprehensive etiologic description. Recent clinical and biological data have underlined its frequency and have allowed a better understanding of its mechanisms. CURRENT KNOWLEDGE AND KEY POINTS: Trali is an interstitial lung injury occurring within 6 hours after the beginning of a blood transfusion. This time relationship between blood injection and the occurrence of lung edema is sufficient for a positive diagnosis, if any other cause of interstitial lung edema have been excluded. The clinical definition relies on a desaturation of arterial blood associated to a lack of any cardiac failure or circulation overload. The link between transfusion and lung edema is not univocal and several categories of mechanisms have been discussed. At least 2 of them are well identified; the first one is an immune conflict, and the second one is an activation of neutrophils by injection of biological modifiers such as lipids or CD40 soluble ligand. Evidences exist for the occurrence of Trali only in predisposing condition that mostly consists of a preceding leucostase in lung capillaries. Trali is treated like other lung interstitial edema by oxygen therapy and mechanical ventilation. FUTURE PROJECTS: A better knowledge of Trali offers the opportunity of improving the understanding of the role of blood transfusion in lung edema occurring in complex situations and open the way for a better definition of at risk patient and at risk blood components.

[Combining corticosteroid and aspirin for the prevention of recurrent villitis or intervillositis of unknown etiology]. / Association des corticoïdes à l'aspirine pour la prévention des récidives de villite ou d'intervillite chroniques d'étiologie indéterminée.

We report the cases of two patients who had a favorable outcome with aspirin and corticosteroid therapy during pregnancy for chronic villitis of unknown etiology complicated by labor asphyxia and further intrauterine fetal demise in one gravida 3 patient and for chronic intervillositis of unknown etiology diagnosed after three perinatal deaths in another patient (gravida 4). Chronic villitis of unknown etiology (CVUE) is detected in 7 to 33% of placentas, mainly after intrauterine growth retardation (IUGR), unexplained prematurity, preeclampsia, perinatal asphyxia and intrauterine fetal death (IUFD). The less frequent chronic intervillositis of unknown etiology (CIUE) (0.6 to 0.9/1.000) has been implicated in recurrent severe pregnancy complications, such as spontaneous abortions, IUGR and IUFD. Histopathology and immunohistology are in favor of an immune response against the foreign fetal allograft. The favorable results obtained with corticosteroids and aspirin remain to be confirmed by larger series.

[Autoimmunity induced by low doses of interferon in melanoma stage I]. / Auto-immunité induite par l'interféron à faible dose dans le mélanome stade I.

INTRODUCTION: The principal aim of this work was to determine the prevalence of antinuclear antibodies and antinucleosomes antibodies during a treatment by interferon alpha with low dose for 18 months among patients with a melanoma stage I. The secondary objective consisted to seek the existence or not of a correlation with the clinical relapse, to determine the prevalence of appearance of clinical signs of autoimmune diseases and dysthyroidie. PATIENT AND METHODS: It was an exploratory study. The patients included in the study had a melanoma stage I (French classification), whose excision was realized for 6 weeks maximum, with a Breslow index equal or higher than 1,5 mm. The statistical model of logistic regression was used. RESULTS: Eighty-forth patients were included (38 women and 46 men) old from 21 to 75 years. The prevalence of antinuclear antibodies was 39%. None of the following variables: age, sex, phototype, localisation of melanoma in exposed photo zone, index of Breslow or Clark, were significantly associated with the presence of antinuclear antibodies. As the percentage of patients with anti-nucleosomes was low (5%), no statistical study was carried out. The prevalence of clinical and/or biological dysthyroidie was 37%. 60% of the patients presented at a moment in the evolution antinuclear antibodies or a dysthyroidie. The prevalence of relapses and death different was not correlated significantly with antinuclear antibodies and/or a dysthyroidie. DISCUSSION: Many studies report the appearance of antinuclear antibodies, generally without clinical lesions during the treatment by interferon alpha for cancers (tumours carcinoids, hemopathies) and viral chronic hepatitis. Our study is, to our knowledge, the first evaluating the induction of an autoimmunity during the adjuvant treatment by interferon alpha of melanoma stage I. The induction of autoantibody during the treatment by interferon alpha could constitute a marker of effectiveness of the treatment with improvement of the survival of these patients. In our study, however auto immunity markers do not appear as factors of severity of evolution of the melanoma or predictive factors.

[TRALI: from diagnosis to prevention]. / Le TRALI : du diagnostic à la prévention.

Transfusion-Related Acute Lung Injury (TRALI) is a post-transfusion acute respiratory distress syndrome (ARDS). TRALI is a non-cardiogenic lung oedema occurring within 6 hours following the infusion of a blood component. Its frequency has been estimated from 1 in 5.000 to 1 in 500.000 injected blood products. Its aetiology is still controversial. Antibodies against HLA class I molecules or granulocyte surface molecules recognizing recipient leukocyte antigens were implicated at first. More recently anti HLA class II were involved in some cases. Finally granulocyte activating lipids released from cells during blood storage were claimed to account for cases in which no antibody were detected. Nevertheless, in most cases, none of these triggering factors alone seems sufficient to induce a TRALI. A predisposing condition, associated with leucostasis in pulmonary vessels, is required. Whatever the mechanism, the pulmonary lesion is eventually due to release of neutrophil granule content in contact with endothelial cells of lung micro-capillary vessels. The basement membrane damages leads to fluid and cell extravasations in interstitial and alveolar spaces. Among blood donors multiparous women are the most frequently involved. Up to now there are neither definite guidelines regarding detection of harmful antibodies nor regulation for deferral of potentially dangerous blood donors.

[French training program for medical students in transfusion medicine. Transfusion Medicine Teachers' College]. / Programme en transfusion des étudiants en médecine. Collège des enseignants en transfusion sanguine.

In France, transfusion medicine training program has been updated. A national committee of professors in transfusion medicine propose a series of 13 items which represent the minimum knowledge that general practitioners should possess. This overview of transfusion medicine is far below the level that specialists should reach and they will need an additional specialized training. Several French universities have set up their own training program which is quite similar to the work of the committee of professors. The following recommendations are not strict guidelines but is a common basis which will be improved in 2005 according to new evidence based transfusion medicine.

Disappearance of CD4-lymphocyte circadian cycles in HIV-infected patients: early event during asymptomatic infection.

Circadian variations have been observed in peripheral blood lymphocyte counts in normal subjects; they usually reflect variations in absolute CD4-cell count. For this population, nadir occurs around 0800 h (basal value) and the peak value occurs at midnight (1.6 times the 0800-h value). This cycle is thought to be of major importance in the efficacy of the immune response because it expresses the migration of lymphocytes into lymphoid organs.

Follow-up of subjects with isolated and persistent anti-core (anti-p24 or anti-p17) antibodies to HIV.

Systematic screening of blood donations by enzyme-linked immunosorbent assay (ELISA) for HIV antibodies carries a false-positive rate: the sera involved react in Western blot to core antigens (p24 or p17) but reactivity to envelope is absent. We studied 22 subjects with persistent and isolated anti-core reactivities; 75 HIV seropositive patients were controls. The epidemiological data and the follow-up and biological tests performed in these two populations argue that donors with persistent and isolated anti-core antibodies are not seroconverting for HIV. We conclude: (1) that verification of all anti-HIV ELISA-positive sera by Western blot is essential and that the presence of at least once anti-envelope (gp120 or gp41) antibody is indispensable for the diagnosis of HIV infection; (2) that the solitary anti-p24 or anti-p17 bands observed on Western blot are false-positive. There is no evidence that donors with such reactivities are HIV-infected.

High-fold expansion of human cytotoxic T-lymphocytes specific for autologous melanoma cells for use in immunotherapy.

We set up a culture protocol that consistently allows high-fold expansion of tumor-specific T-lymphocytes from most melanoma-invaded biopsies with low doses of recombinant interleukin-2 (rIL-2). Between 2-60 x 10(6) T-lymphocytes could be obtained and cryopreserved from 12 out of 13 patients, by culturing only 50 mm3 tumor tissue with rIL-2. Thawed lymphocytes from 11 of these patients could then be expanded by a median factor of 32,800 by culturing them successively in microplates on irradiated feeder cells with rIL-2 for approximately 2 weeks and then in culture bags or flasks with only rIL-2 for 1-2 additional weeks. Dead feeder cells disappeared during the last phase of the lymphocyte culture with rIL-2. Interestingly, each time they were expanded under these conditions, tumor-infiltrating lymphocytes (TIL) or lymph-node lymphocytes developed a lytic activity apparently restricted to the autologous melanoma line. Tumor-specific lysis, which was maximum at around the end of T-lymphocyte expansion, ranged between 31-63% lysis at an effector:target (E:T) ratio of 20:1. This culture method would thus appear to be suitable for reliable production of over 10(10) T-lymphocytes with good tumor-specific lytic activity from most melanoma-invaded biopsy. It should permit analysis of the immunotherapeutic potential of these populations reinjected into cancer patients.

1,25 Dihydroxyvitamin D3 exerts regional effects in the central nervous system during experimental allergic encephalomyelitis.

1,25-dihydroxyvitamin D3 (1,25-D3) is already known to prevent clinical signs of experimental allergic encephalomyelitis when animals are treated during the immunization phase. In the present work we have evaluated the ability of 1,25-D3 to inhibit chronic relapsing experimental allergic encephalomylitis (EAE) of the Lewis rat, when administered after the beginning of clinical signs. We observed a significant clinical improvement in 1,25-D3-treated rats. This effect was accompanied by a profound inhibition of CD4 antigen expression by central nervous system (CNS) infiltrating monocytes/macrophages and parenchymal microglia. In addition, immunohistochemical analysis performed at the time of the second attack evidenced a region-specific distribution of inflammatory cells. In the same way, some aspects of the effects exerted by 1,25-D3 appeared to vary depending on the region considered, namely spinal cord, brainstem, cerebellum, midbrain or anterior brain. Thus, in 1,25-D3-treated rats, we observed an almost complete inhibition of CD4 antigen expression in the granule cell layer and the adjacent white matter of the cerebellum as well as a marked decrease in the number of OX42-positive cells (macrophages and activated microglia) in anterior brain sections. We conclude that 1,25-D3 can exert immunomodulatory effects inside the CNS during an ongoing immune process and may thus represent a promising therapy for multiple sclerosis.

Improved culture conditions for quantitative evaluation of interleukin 2 production by frozen human lymphocytes.

Several culture parameters were studied in order to establish methods for optimal and reproducible production of interleukin 2 (IL2) by thawed lymphocytes. Standard conditions, considered optimal for production by freshly separated lymphocytes (culture medium RPMI 1640 + 1% normal human serum + 10 micrograms/ml PHA), gave low and poorly reproducible results. An increased concentration of human serum (10 and 20%) in the medium improved production but best results were obtained by adding polyethylene glycol (PEG 6000, 0.1 mg/ml) to the culture medium. Furthermore, with the addition of PEG 6000, results became highly reproducible, thus permitting valid comparison of in vitro IL2 production by lymphocytes from normal donors and patients.

Generation of mouse cytolytic T cells against human concanavalin A blasts. Involvement of non-MHC determinants in the antihuman response.

Normal human peripheral blood lymphocytes (PBL) are incapable of eliciting a significant murine cytotoxic T cell (CTL) response either in vivo or in vitro. However, using a primary in vivo and secondary in vitro stimulation with lectin-activated PBL, Thy-l-positive cytotoxic cells were produced. The antigens that these T-cells identified were independent of the serum source employed in the culture medium used for lectin activation. The cells always preferentially lysed cells from the immunizing individual but were also able to lyse target cells from unrelated individuals, regardless of HLA identity or disparity with the immunizing individual, suggesting the presence of both a private (possibly class II antigens) and public specificity. Using the lymphoblasts of different family members as immunogen and targets there was slight preference of the CTL for HLA-identical targets with no apparent difference between the lysis exhibited against semiidentical and nonidentical subjects. Monoclonal antibodies directed against HLA DR or beta 2-microglobulin failed to inhibit the cytotoxicity observed in these experiments. It is suggested that under these circumstances of xenogeneic education, non-MHC-restricted T cells may become cytotoxic, and this model may serve as a useful probe to investigate some of the less-well-defined aspects of the T cell repertoire.

Effects of MHC-encoded TAP1 and TAP2 gene polymorphism and matching on kidney graft rejection.

The products of TAP1 and TAP2 genes, recently mapped within the MHC class II region, are involved in antigen presentation by MHC class I molecules, especially in the transport of endogenous peptides. As for most MHC genes, a polymorphism has been described and the possibility that it could influence the recipient immune response by modulating antigen presentation in kidney transplantation has been tested. The aim of our study was to compare TAP1 and TAP2 gene polymorphism and matching in 53 couples of kidney donors and recipients without any rejection episodes and in 55 other couples who had experienced at least 2 acute cellular rejection episodes; 70 healthy individuals served as controls. Our results showed that allelic variant frequencies of TAP1 alleles (1A to 1C) and TAP2 alleles (2A to 2E), as assessed by amplification refractory mutation system-polymerase chain reaction, were similar among "rejection" and "no rejection" populations. Furthermore, there were no differences of TAP1 and/or TAP2 matching between donors and recipients in the 2 groups. In contrast, we showed that the recipients of the no rejection group were better matched with their corresponding donors for the HLA-DR genes than those of the rejection group. These results suggest that the currently described polymorphism in the limited coding region of TAP1 and TAP2 genes does not influence the incidence of kidney allograft rejection episodes and seems not to be a strong link to the adjacent DR/DQ subregion. Moreover, the observed increase frequency of TAP1B allele in the whole recipient's group as compared with controls (16.2% vs. 7.1% in the healthy individuals; P < 0.02) was not linked to the rejection occurrence but to the presence of glomerulonephritis as initial disease. Our study suggests that, in the clinical conditions tested, neither TAP polymorphism nor TAP matching influences the renal graft outcome.

Treatment of graft-versus-host disease by extracorporeal photochemotherapy: a pilot study.

BACKGROUND: Graft-versus-host disease (GVHD) is a major complication after bone marrow transplantation, which may be refractory to immunosuppressive drugs. As preliminary case reports suggested that extracorporeal photochemotherapy (ECP) using a Therakos device might be beneficial, we conducted a pilot study to assess the efficacy and safety of a new ECP method that does not require administration of 8-methoxypsoralen (8-MOP) to the patient. METHODS: ECP was performed three times a week for 3 weeks and then tapered according to the patient's course. Soluble 8-MOP was added ex vivo to an enriched mononuclear cell suspension obtained by a cell separator. This cellular suspension was then ultraviolet A irradiated and reinfused into the patient. Evaluation was performed using specific objective tests depending on clinical conditions. RESULTS: The two patients in the study with acute GVHD and severe liver dysfunction resistant to steroid pulse showed no improvement with ECP treatment. The five patients with chronic GVHD (c-GVHD) had the following clinical features: three patients had myositis and two patients had severe cutaneous c-GVHD, including one patient with sclerodermoid lesions, one with bronchiolitis obliterans, one with bronchitis, and one with liver involvement. Immunosuppressive drugs were either prohibited or ineffective. The number of procedures for each patient ranged from 13 to 30. Cytapheresis required the use of a double-lumen catheter (4/5) or an arteriovenous fistula (1/5). No side effects were related to 8-MOP or ultraviolet A irradiation. Four of five patients improved after ECP; one patient with bronchiolitis obliterans, a fibrotic condition, remained stable. CONCLUSIONS: ECP treatment may be helpful for the treatment of severe c-GVHD and the avoidance of increased immunosuppression.

HLA antigens and factor VIII antibody in classic hemophilia. European study group of factor VIII antibody.

Possible interrelations between the immune response factor VIII and the major histocompatibility system were investigated in 57 multi-transfused hemophilic brothers belonging to 26 families. Linkage appears very unlikely although formal proof of independence cannot be offered. The HLA system, therefore, does not provide markers predictive for the development of antibodies to factor VIII in severe hemophilia A.

Crucial role of the third and fourth hypervariable regions of HLA-DPB1 allelic sequences in the mixed lymphocyte reaction.

Since HLA-DP mismatches are known to induce proliferative response in MLR I, we investigated the real impact of the different DP alleles and the possible role of one or several hypervariable regions of the DPB allelic sequences. Accordingly, we performed MLR I between HLA-A, B, DR, DQ, and Dw identical individuals DP oligotyped after DNA amplification. A total of 23 one-DP-mismatched healthy stimulator and responder cells displaying nine different DP specificities were thus evaluated in 52 MLRs I. This allowed us to analyze the impact of amino acid composition of each of the six hypervariable regions independently of the amino acid matching or mismatching in the five others. We show here that DP combinations sharing the same amino acid sequence in the third (C) and fourth (D) hypervariable regions are associated with a low proliferative response in vitro (p less than 0.01). These data imply that a perfect HLA-DP matching may not be requisite in selecting bone marrow donors. Indeed, the choice of donors may rely on determination of these particular mismatched HVRs between the DP alleles involved especially in GvHD direction. This policy including prospective DP oligotyping should be of great interest, especially when MLRs I are false negative or nonevaluable. It will enable a better definition of which DP mismatches are acceptable in BMT.

Susceptibility to alloimmunization to platelet HPA-1a antigen involves TAP1 polymorphism.

The alloimmunization against platelet HPA-1a antigen in mothers of thrombocytopenic neonates is strongly associated with HLA class II structures (DR3 and DR13) and especially with HLA-DR52a antigen (98% of the cases reported here). Because new genes have recently been mapped within the MHC class II region, we typed TAP1 and TAP2 gene polymorphisms by ARMS-PCR in order to characterize more effectively MHC genes involved in this alloimmunization. Our results showed that TAP1*0102 allele was significantly associated with NAIT only in the population of HLA-DR 13-DR52a-immunized women (50%) versus HLA-DR 13-DR52a controls (20%) (p < 0.05), and not in HLA-DR3-DR52a-immunized women versus HLA-DR3-DR52a controls. There is no linkage disequilibrium between TAP1*0102 and DRB1*13 alleles (delta = -0.0063) that could account for this result. The higher frequency of TAP1*0102 allele among HLA-DR 13-DR52a-immunized women suggests that HPA-1a antigen presentation and recognition may be influenced by nonclassic HLA class II gene polymorphisms, or that other linked but yet unknown genes could interfere.

Impact of the MHC-encoded HLA-DMA, DMB, and LMP2 gene polymorphisms on kidney graft outcome.

We previously studied the relationship between TAP1 and TAP2 gene polymorphism and compatibility in kidney graft outcome and reported that the currently described TAP1 and TAP2 gene polymorphisms did not influence the incidence of acute rejection episodes. In this study, we report on the effect of polymorphism and matching of HLA-DMA, -DMB, and LMP2 genes on kidney graft outcome. This study was performed on 102 selected kidney recipients who experienced two or more acute rejection episodes (rejection group) during follow up and who were compared to a group of 150 patients who never had rejection (non rejection group). Although a significant effect of HLA-DR matching was observed between these two groups, our data suggest that matching for all the new genes located in the HLA class II region (TAP1, TAP2, LMP2, HLA-DMA and -DMB) does not influence the kidney graft outcome. However, a significant increase (pc < 0.05) of DMA*0102 allele was observed in the recipients of the rejection group as compared to those of the non rejection group. This effect was not due to a linkage disequilibrium between DMA and HLA-DR loci and suggests that this specific HLA-DMA allele could play a role in the indirect pathway of class II presentation of donor antigens.