Search PubMed⌕ Search

Biomedical subjects

Alain Fischer

Publications and source records attributed to Alain Fischer.

At least 109 records · Page 6Linked to original sources
PubMed2002

Biochemical and functional characterization of Rab27a mutations occurring in Griscelli syndrome patients.

Rab27a is a member of the Rab family of small GTPase proteins, and thus far is the first member to be associated with a human disease (ie, the Griscelli syndrome type 2). Mutations in the Rab27a gene cause pigment as well as cytotoxic granule transport defects, accounting for the partial albinism and severe immune disorder characteristics of this syndrome. So far, 3 Rab27a missense mutations have been identified. They open a unique opportunity to designate critical structural and functional residues of Rab proteins. We show here that the introduction of a proline residue in the alpha 4 (Ala152Pro) or beta 5 (Leu130Pro) loop, observed in 2 of these spontaneous mutants, dramatically affects both guanosine triphosphate (GTP) and guanosine diphosphate (GDP) nucleotide-binding activity of Rab27a, probably by disrupting protein folding. The third mutant, Trp73Gly, is located within an invariant hydrophobic triad at the switch interface, and was previously shown in active Rab3A to mediate rabphilin3A effector interaction. Trp73Gly is shown to display the same nucleotide-binding and GTPase characteristics as the constitutively active mutant Gln78Leu. However, in contrast to Gln78Leu, Trp73Gly mutant construct neither interacts with the Rab27a effector melanophilin nor modifies melanosome distribution and cytotoxic granule exocytosis. Substitutions introduced at the 73 position, including the leucine residue present in Ras, did not restore Rab27a protein functions. Taken together, our results characterize new critical residues of Rab proteins, and identify the Trp73 residue of Rab27a as a key position for interaction with the specific effectors of Rab27a, both in melanocytes and cytotoxic cells.

Albinism↗
PubMed2002

Mechanisms of CD47-induced caspase-independent cell death in normal and leukemic cells: link between phosphatidylserine exposure and cytoskeleton organization.

Dying cells, apoptotic or necrotic, are swiftly eliminated by professional phagocytes. We previously reported that CD47 engagement by CD47 mAb or thrombospondin induced caspase-independent cell death of chronic lymphocytic leukemic B cells (B-CLL). Here we show that human immature dendritic cells (iDCs) phagocytosed the CD47 mAb-killed leukemic cells in the absence of caspases 3, 7, 8, and 9 activation in the malignant lymphocytes. Yet the dead cells displayed the cytoplasmic features of apoptosis, including cell shrinkage, phosphatidylserine exposure, and decreased mitochondrial transmembrane potential (DeltaPsim). CD47 mAb-induced cell death also occurred in normal resting and activated lymphocytes, with B-CLL cells demonstrating the highest susceptibility. Importantly, iDCs and CD34(+) progenitors were resistant. Structure-function studies in cell lines transfected with various CD47 chimeras demonstrated that killing exclusively required the extracellular and transmembrane domains of the CD47 molecule. Cytochalasin D, an inhibitor of actin polymerization, and antimycin A, an inhibitor of mitochondrial electron transfer, completely suppressed CD47-induced phosphatidylserine exposure. Interestingly, CD47 ligation failed to induce cell death in mononuclear cells isolated from Wiskott-Aldrich syndrome (WAS) patients, suggesting the involvement of Cdc42/WAS protein (WASP) signaling pathway. We propose that CD47-induced caspase-independent cell death be mediated by cytoskeleton reorganization. This form of cell death may be relevant to maintenance of homeostasis and as such might be explored for the development of future therapeutic approaches in lymphoid malignancies.

Antibodies, Monoclonal↗
PubMed2002

Gene therapy of RAG-2-/- mice: sustained correction of the immunodeficiency.

Patients with mutations of either RAG-1 or RAG-2 genes suffer from severe combined immunodeficiency (SCID) characterized by the lack of T and B lymphocytes. The only curative treatment today consists of hematopoietic stem cell (HSC) transplantation, which is only partially successful in the absence of an HLA genoidentical donor, thus justifying research to find an alternative therapeutic approach. To this end, RAG-2-deficient mice were used to test whether retrovirally mediated ex vivo gene transfer into HSCs could provide long-term correction of the immunologic deficiency. Murine RAG-2-/-Sca-1(+) selected bone marrow cells were transduced with a modified Moloney leukemia virus (MLV)-based MND (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted) retroviral vector containing the RAG-2 cDNA and transplanted into RAG-2-/- sublethally irradiated mice (3Gy). Two months later, T- and B-cell development was achieved in all mice. Diverse repertoire of T cells as well as proliferative capacity in the presence of mitogens, allogeneic cells, and keyhole limpet hemocyanin (KLH) were shown. B-cell function as shown by serum Ig levels and antibody response to a challenge by KLH also developed. Lymphoid subsets and function were shown to be stable over a one-year period without evidence of any detectable toxicity. Noteworthy, a selective advantage for transduced lymphoid cells was evidenced by comparative provirus quantification in lymphoid and myeloid lineages. Altogether, this study demonstrates the efficiency of ex vivo RAG-2 gene transfer in HSCs to correct the immune deficiency of RAG-2-/- mice, constituting a significant step toward clinical application.

Animals↗
PubMed2002

Treatment of chronic granulomatous disease with myeloablative conditioning and an unmodified hemopoietic allograft: a survey of the European experience, 1985-2000.

Treatment of chronic granulomatous disease (CGD) with myeloablative bone marrow transplantation is considered risky. This study investigated complications and survival according to different risk factors present at transplantation. The outcomes of 27 transplantations for CGD, from 1985 to 2000, reported to the European Bone Marrow Transplant Registry for primary immunodeficiencies were assessed. Most transplant recipients were children (n = 25), received a myeloablative busulphan-based regimen (n = 23), and had unmodified marrow allografts (n = 23) from human leukocyte antigen (HLA)-identical sibling donors (n = 25). After myeloablative conditioning, all patients fully engrafted with donor cells; after myelosuppressive regimens, 2 of 4 patients fully engrafted. Severe (grade 3 or 4) graft-versus-host disease (GVHD) disease developed in 4 patients: 3 of 9 with pre-existing overt infection, 1 of 2 with acute inflammatory disease. Exacerbation of infection during aplasia was observed in 3 patients; inflammatory flare at the infection site during neutrophil engraftment in 2: all 5 patients belonged to the subgroup of 9 with pre-existing infection. Overall survival was 23 of 27, with 22 of 23 cured of CGD (median follow-up, 2 years). Survival was especially good in patients without infection at the moment of transplantation (18 of 18). Pre-existing infections and inflammatory lesions have cleared in all survivors (except in one with autologous reconstitution). Myeloablative conditioning followed by transplantation of unmodified hemopoietic stem cells, if performed at the first signs of a severe course of the disease, is a valid therapeutic option for children with CGD having an HLA-identical donor.

Adolescent↗
PubMed2002

Immune reconstitution without graft-versus-host disease after haemopoietic stem-cell transplantation: a phase 1/2 study.

BACKGROUND: Allogeneic haemopoietic stem-cell transplantation (HSCT) is the treatment of choice for many haematological malignancies and inherited disorders. When stem cells for transplantation come from a human leucocyte antigen matched unrelated donor, or from a partly mismatched related donor, ex-vivo T-cell depletion of the graft can prevent development of graft-versus-host disease, but lead in turn to a delay in immune reconstitution and a concordant increase in incidence of opportunistic infections and leukaemic relapses. We aimed to infuse T cells selectively depleted in allogeneic T cells that cause graft-versus-host disease using an ex-vivo procedure designed to eliminate alloactivated donor T cells, with an immunotoxin that reacts with a cell surface activation antigen, CD25. METHODS: We did a phase 1/2 study, in which 1-8 x 10(5) allodepleted T cells/kg were infused between days 15 and 47 into 15 paediatric patients who had acquired or congenital haemopoietic disorders and who received HSCT on day 0. Occurrence of graft-versus-host disease and time to immune reconstitution were assessed. No treatment for graft-versus-host disease was given. FINDINGS: Less than 1% residual anti-host alloreactivity was recorded in 12 of 16 procedures. Other immune responses were preserved by the allodepletion procedure in 12 cases. No cases of severe (greater than grade II) graft-versus-host disease arose. Evidence for early T-cell expansion was shown in three patients with continuing viral infections. Specific antiviral responses, such as strong cytolytic activity, were noted. INTERPRETATION: Our results show that ex-vivo selective depletion of T cells that cause graft-versus-host disease is efficient and feasible, even in haploidentical settings.

Case-Control Studies↗
PubMed2002

Chronic infantile neurological cutaneous and articular syndrome is caused by mutations in CIAS1, a gene highly expressed in polymorphonuclear cells and chondrocytes.

Chronic infantile neurological cutaneous and articular (CINCA) syndrome is a severe chronic inflammatory disease of early onset, characterized by cutaneous symptoms, central-nervous-system involvement, and arthropathy. In the present study, we report, in seven unrelated patients with CINCA syndrome, distinct missense mutations within the nucleotide-binding site of CIAS1, a gene encoding cryopyrin and previously shown to cause Muckle-Wells syndrome and familial cold urticaria. Because of the severe cartilage overgrowth observed in some patients with CINCA syndrome and the implications of polymorphonuclear cell infiltration in the cutaneous and neurological manifestations of this syndrome, the tissue-specific expression of CIAS1 was evaluated. A high level of expression of CIAS1 was found to be restricted to polymorphonuclear cells and chondrocytes. These findings demonstrate that CIAS1 missense mutations can result in distinct phenotypes with only a few overlapping symptoms and suggest that this gene may function as a potential inducer of apoptosis.

Amino Acid Sequence↗
PubMed2002

Adenovirally transduced dendritic cells induce bispecific cytotoxic T lymphocyte responses against adenovirus and cytomegalovirus pp65 or against adenovirus and Epstein-Barr virus EBNA3C protein: a novel approach for immunotherapy.

Cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus (Ad) cause significant morbidity and mortality in immunocompromised patients undergoing allogeneic stem cell transplantation. We have established a procedure to generate polyclonal cytotoxic T lymphocyte (CTL) populations with specificity against Ad and CMV or against Ad and EBV. Healthy donor-derived dendritic cells (DCs) were transduced with recombinant adenovirus encoding either CMV pp65 or EBV EBNA3C and used to stimulate autologous T cells. Stimulated T lymphocytes displayed specific simultaneous cytotoxicity against CMV and adenovirus and to a lesser extent against adenovirus and EBV. Recombinant vaccinia virus encoding individual adenovirus proteins showed that the T cell response to the adenovirus was directed mainly against the capsid protein hexon. The frequency of IFN-gamma-secreting T cells was 0.02% for adenovirus alone, and 0.05 and 0.14% for adenoviruses encoding EBNA3C and pp65, respectively. pp65-specific CTLs killed autologous fibroblasts infected with the laboratory strain CMV AD169. The culture conditions were specific as alloreactive T cells were not expanded. Therefore, this approach could be considered in order to generate efficient virus cytolytic T cells to be used as adoptive immunotherapy in transplanted patients.

Adenoviridae↗
PubMed2002

Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy.

BACKGROUND: X-linked severe combined immunodeficiency due to a mutation in the gene encoding the common gamma (gamma(c)) chain is a lethal condition that can be cured by allogeneic stem-cell transplantation. We investigated whether infusion of autologous hematopoietic stem cells that had been transduced in vitro with the gamma(c) gene can restore the immune system in patients with severe combined immunodeficiency. METHODS: CD34+ bone marrow cells from five boys with X-linked severe combined immunodeficiency were transduced ex vivo with the use of a defective retroviral vector. Integration and expression of the gamma(c) transgene and development of lymphocyte subgroups and their functions were sequentially analyzed over a period of up to 2.5 years after gene transfer. RESULTS: No adverse effects resulted from the procedure. Transduced T cells and natural killer cells appeared in the blood of four of the five patients within four months. The numbers and phenotypes of T cells, the repertoire of T-cell receptors, and the in vitro proliferative responses of T cells to several antigens after immunization were nearly normal up to two years after treatment. Thymopoiesis was documented by the presence of naive T cells and T-cell antigen-receptor episomes and the development of a normal-sized thymus gland. The frequency of transduced B cells was low, but serum immunoglobulin levels and antibody production after immunization were sufficient to avoid the need for intravenous immunoglobulin. Correction of the immunodeficiency eradicated established infections and allowed patients to have a normal life. CONCLUSIONS: Ex vivo gene therapy with gamma(c) can safely correct the immune deficiency of patients with X-linked severe combined immunodeficiency.

Antibodies↗
PubMed2002

Three novel mutations of the CIITA gene in MHC class II-deficient patients with a severe immunodeficiency.

Four transacting genes, CIITA, RFXANK, RFX5, and RFXAP, control coordinate MHC II expression. In humans, defects in these genes result in the absence of MHC II expression and thus a combined immunodeficiency. CIITA is considered to be a master MHC II regulator and is responsible for the defect in complementation group A. Eight such affected families have been reported. We investigated the molecular basis of the defect in three patients in these families, all presenting a severe immunodeficiency. CIITA transcripts were detected in all three patients but in one at an abnormally low level. Three novel heterozygous mutations of CIITA were found in patients SP and RC. One SP CIITA allele contained a nonsense mutation, G2178A, leading to a premature stop codon and the other allele in SP was found not to be expressed. In patient RC, two in-frame deletions were detected: one of the nucleotides 3003-3084 corresponding to the exon coding from Leu(964)to Asp(991), in the paternal allele, and a CATdel3193-5 of the isoleucine codon at position 1027, in the maternal allele. Transfection of a CIITA-deficient cell line with the recombinant CATdel3193-5-CIITA cDNA revealed a loss of function for this mutant and retention of the protein in the cytoplasm. No mutations were detected in the 4.5-kb cDNA from patient OK but the level of CIITA transcript was found to be profoundly decreased. However, promoters III and IV were not affected. This last case represents the first described CIITA dysfunction due to putative mutation(s) in cis regulatory sequences of CIITA.

Amino Acid Sequence↗
PubMed2002

Differential sensitivity of Jurkat and primary T cells to caspase-independent cell death triggered upon Fas stimulation.

Fas-induced apoptosis, a key event in the regulation of lymphocyte homeostasis, is mainly mediated by the activation of a cascade of caspases. Using caspase- and Fas-associated death domain protein-deficient Jurkat cell lines as well as a pan-caspase inhibitor (Z-VAD-fmk), we observed a second Fas-induced cell death event, independent of any known caspase activation. This pathway is of slow kinetics and displays some features of necrosis. However, this caspase-independent pathway does not seem to play a significant role in the Fas-mediated death of primary activated T cell blasts. Indeed, in this setting, Fas-induced cell death was always substantially inhibited by Z-VAD-fmk, suggesting that caspase activation is an absolute requirement in the Fas-induced death of primary human T lymphocytes.

Apoptosis↗
PubMed2002

Gene therapy of X-linked severe combined immunodeficiency.

Severe combined immunodeficiency (SCID) conditions appear to be the best possible candidates for a gene therapy approach. Transgene expression by lymphocyte precursors should confer to these cells a selective growth advantage that gives rise to long-lived T-lymphocytes. This rationale was used as a basis for a clinical trial of the SCID-X1 disorder caused by common gamma (gamma c) gene mutations. This trial consists of ex vivo retroviral-mediated (MFG-B2 gamma c vector) gammac gene transfer into marrow CD34+ cells in CH-296 fibronectin fragment-coated bags. Up to now, 9 patients with typical SCID-X1 diagnosed within the first year of life and lacking an HLA-identical donor have been enrolled. More than 2 years' assessment of 5 patients and more than 1 year for 7 patients provide evidence for full development of functional, mature T-cells in the absence of any adverse effects. Functional transduced natural killer cells are also detectable, although in low numbers. All but 1 patient with T-cell immunity have also developed immunoglobulin production, which has alleviated the need for intravenous immunoglobulin substitution despite a low detection frequency of transduced B-cells. These 8 patients are doing well and living in a normal environment. This yet successful gene therapy demonstrates that in a setting where transgene expression provides a selective advantage, a clinical benefit can be expected.

Animals↗
PubMed2002

Medical perspectives of adults and embryonic stem cells.

In the last 30 years, allogeneic bone marrow transplantation has become the treatment of choice for many hematologic malignancies or inherited disorders and a number of changes have been registered in terms of long-term survival rate of transplanted patients as well as of available sources of hematopoietic stem cell (HSC). In parallel to the publication of better results in HSC transplantation, several recent discoveries have opened a scientific and ethical debate on the therapeutical potential of stem cells isolated from adult or embryonic tissues. One of the major discoveries in this field is the capacity of bone marrow-derived stem cells to treat a genetic liver disease in a mouse model, thus justifying the concept of transdifferentiation of adult stem cell and raising hopes on its possible therapeutical applications. We have tried here to summarise the advances in this field and to discuss the limits of these biological data.

Adult↗
PubMed2002

Gene therapy of severe combined immunodeficiencies.

The concept that the outcome of a devastating disease can be modified by inserting a transgene into abnormal cells is appealing. However, the gene-transfer technologies that are available at present have limited the success of gene therapy so far. Nevertheless, severe combined immunodeficiencies are a useful model, because gene transfer can confer a selective advantage to transduced cells. In this way, a proof of concept for gene therapy has been provided.

Adenosine Deaminase↗
PubMed2002

Functional consequences of perforin gene mutations in 22 patients with familial haemophagocytic lymphohistiocytosis.

Familial haemophagocytic lymphohistiocytosis (FHL), an inherited form of haemophagocytic lymphohistiocytosis (HLH) syndrome, is characterized by the overwhelming activation of T lymphocytes and macrophages invariably leading to death in the absence of treatment. FHL is a heterogeneous autosomal recessive disorder, with one known causative gene which codes for perforin, a cytotoxic effector protein. In this study, we have characterized the genotype and phenotype of 14 unrelated families with perforin deficiency. Four new missense mutations of the perforin gene were identified. In every case, perforin gene mutations led to undetectable intracellular perforin expression within cytotoxic cells, while some residual T-cell cytotoxic activity could be associated with certain missense mutations. Clinical and biological analyses did not differentiate between patients with nonsense or missense mutations, although age at diagnosis, which tended to be similar within members of the same family, was delayed in patients from two families belonging to the second group. In one case, consequences of perforin deficiency, diagnosed at birth, could be assessed prior to onset of clinical manifestations. No evidence for T-cell activation could be shown, suggesting that an exogenous event is required to trigger the disease manifestation. Control assessment of perforin expression and cytotoxic assays by lymphocytes from young children led to the conclusion that perforin content of natural killer cells could be a reliable diagnostic test at any age. Altogether, these data enabled a better characterization of perforin deficiency and its consequences, and defined reliable diagnostic tools.

Codon, Nonsense↗
PubMed2002

Improving immune reconstitution while preventing graft-versus-host disease in allogeneic stem cell transplantation.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the treatment of choice for many hematologic malignancies or inherited disorders. Ex vivo T-cell depletion (TCD) of the graft and post-transplantation immunosuppression efficiently prevent the development of graft-versus-host disease (GVHD). However, the consequence of these nonspecific approaches is a long-lasting immunodeficiency associated with increased disease relapse, graft rejection, and reactivation of viral infections. Donor lymphocyte infusion, to treat leukemic relapse after allogeneic HSCT, can cause severe GVHD. Several strategies are being optimized to specifically inactivate anti-host T cells while preserving antileukemic or antimicrobial immunocompetence, based on ex vivo or in vivo elimination of anti-host T cells or on the modulation of their anti-host activity.

Animals↗