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Biomedical subjects

F Dieli

Publications and source records attributed to F Dieli.

At least 19 recordsLinked to original sources

Novel Munc13-4 mutations in children and young adult patients with haemophagocytic lymphohistiocytosis.

Familial haemophagocytic lymphohistiocytosis (FHL) is a genetically heterogeneous disorder characterised by constitutive defects in cellular cytotoxicity resulting in fever, hepatosplenomegaly and cytopenia, and the outcome is fatal unless treated by chemoimmunotherapy followed by haematopoietic stem-cell transplantation. Since 1999, mutations in the perforin gene giving rise to this disease have been identified; however, these account only for 40% of cases. Lack of a genetic marker hampers the diagnosis, suitability for transplantation, selection of familial donors, identification of carriers, genetic counselling and prenatal diagnosis. Mutations in the Munc13-4 gene have recently been described in patients with FHL. We sequenced the Munc13-4 gene in all patients with haemophagocytic lymphohistiocytosis not due to PRF1 mutations. In 15 of the 30 families studied, 12 novel and 4 known Munc13-4 mutations were found, spread throughout the gene. Among novel mutations, 2650C-->T introduced a stop codon; 441del A, 532del C, 3082del C and 3226ins G caused a frameshift, and seven were mis sense mutations. Median age of diagnosis was 4 months, but six patients developed the disease after 5 years of age and one as a young adult of 18 years. Involvement of central nervous system was present in 9 of 15 patients, activity of natural killer cells was markedly reduced or absent in 13 of 13 tested patients. Chemo-immunotherapy was effective in all patients. Munc13-4 mutations were found in 15 of 30 patients with FHL without PRF1 mutations. Because these patients may develop the disease during adolescence or even later, haematologists should include FHL2 and FHL3 in the differential diagnosis of young adults with fever, cytopenia, splenomegaly and hypercytokinaemia.

Adolescent↗

Time course of mycobacterial infection of dendritic cells in the lungs of intranasally infected mice.

SETTING: Dendritic cells (DC) could regulate between the protective and pathogenic immune responses following tuberculous infection. In this paper we investigated if their early infection in the lungs represents a plausible alternative to cross-priming with mycobacterial antigens acquired from infected macrophages. OBJECTIVE: To determine the extent and time course of infection of lung DCs following intranasal inoculation of BALB/c mice with green fluorescent protein (GFP) tagged Bacillus Calmette-Guerin (BCG). RESULTS: A fraction of GFP-BCG infected lung cells were classified as monocytic DCs with the CD11c+IA+33D1+CD8a- phenotype. These cells represented 5-18% of the total GFP+ cells, the bulk of which were macrophages. The infected DCs could be separated by cell size into two fractions with similar cell surface staining properties during the 2-72 h period after infection. An unexpected difference was observed for the time course of infection between DCs and macrophages: DC infection peaked at 48 h followed by decline at 72 h, while the proportion of infected macrophages remained steady during the same period. CONCLUSION: The presented results are direct evidence that monocytic DCs are recruited to the lungs and take up live bacilli within 48 h of intranasal infection with GFP-BCG. This finding is pertinent for the regulation of pulmonary and systemic immune responses and possibly for the dissemination of mycobacterial infection by DCs.

Administration, Intranasal↗

Cytokine profile, HLA restriction and TCR sequence analysis of human CD4+ T clones specific for an immunodominant epitope of Mycobacterium tuberculosis 16-kDa protein.

The identification of immunodominant and universal mycobacterial peptides could be applied to vaccine design and have an employment as diagnostic reagents. In this paper we have investigated the fine specificity, clonal composition and HLA class II restriction of CD4+ T cell clones specific for an immunodominant epitope spanning amino acids 91-110 of the 16-kDa protein of Mycobacterium tuberculosis. Twenty-one of the tested 28 clones had a Th1 profile, while seven clones had a Th0 profile. None of the clones had a Th2 profile. While the TCR AV gene usage of the clones was heterogeneous, a dominant TCR BV2 gene family was used by 18 of the 28 clones. The CDR3 regions of BV2+ T cell clones showed variation in lengths, but a putative common motif R-L/V-G/S-Y/W-E/D was detected in 13 of the 18 clones. Moreover, the last two to three residues of the putative CDR3 loops, encoded by conserved BJ sequences, could also play a role in peptide recognition. Antibody blockade and fine restriction analysis using HLA-DR homozygous antigen-presenting cells established that 16 of 18 BV2+ peptide-specific clones were DR restricted and two clones were DR-DQ and DR-DP restricted. Additionally, five of the 18 TCRBV2+ clones recognized peptide 91-110 in association with both parental and diverse HLA-DR molecules, indicating their promiscuous recognition pattern. The ability of peptide 91-110 to bind a wide range of HLA-DR molecules, and to stimulate a Th1-type interferon (IFN)-gamma response more readily, encourage the use of this peptide as a subunit vaccine component.

Antigens, Bacterial↗

A human leucocyte antigen-DR1 transgene confers susceptibility to experimental allergic encephalomyelitis elicited by an epitope of myelin basic protein.

Much evidence now indicates that human leucocyte antigen (HLA) class I and class II transgenic (Tg) mice can be of value in analysing HLA-restricted presentation of T-cell epitopes relevant to experimental models of autoimmune diseases. One area where this has been applied is the characterization of myelin epitopes presented by HLA class II molecules in experimental model of multiple sclerosis (experimental allergic encephalomyelitis (EAE)). As a first step towards humanized disease models in HLA Tg mice, we have analysed immune response of lymph node cells of HLA-DR1 Tg mice immunized with the human myelin basic protein (MBP) peptides 13-33, 87-106 and 139-154 bound by HLA-DR1. We report here that HLA-DR1 Tg mice display a hierarchy of response in vivo and in vitro to MBP epitopes depending on the binding affinity to DRB*0101 molecule. In fact, the 13-33 epitope induced a strong T helper 1 (Th1) response accompanied by high T-cell precursor frequency and caused mild EAE, while the two other epitopes gave poor (139-154) or no disease (87-106), and these data correlate with in vitro Th1 response. These data could prove a useful tool in understanding the role played by different MBP epitopes in EAE.

Amino Acid Sequence↗

The humoral response in TCR alpha-/- mice. Can gammadelta-T cells support the humoral immune response?

An optimal humoral response requires T-cell help; however, it has been questioned if this help comes exclusively from alphabeta-T cells or whether gammadelta-T cells also contribute. We have attempted to answer this question by studying the humoral response in T-cell receptor alpha-chain knockout (alpha-/-) mice, which lack the alphabetaT cell subset. Two model antigens were used to characterize the response: the thymus-independent (TI) antigen native dextran B512 (Dx), and the thymus-dependent (TD) antigen heat shock protein (HSP65) from Mycobacterium tuberculosis. When challenged with Dx, the alpha-/- mice elicited a strong antibody response and formed rudimentary germinal centres (GCs), a T-cell dependent reaction. In contrast, the humoral response to HSP65 was poor. However, alpha-/- mice became primed when challenged with HSP65, because when supplemented with wild-type thymocytes, the antigen-primed animals were able to mount a stronger response than the nonprimed ones when challenged with HSP65. A crucial step seems to be the collaboration between gammadeltaT cells and antigen presenting cells (APCs), as splenocytes from alpha-/- mice were able to respond to HSP65 in an environment containing primed-APCs. Based on these results, we propose a model for B-cell activation in the alpha-/- mice.

Animals↗

Humoral and cell mediated immune response to cow's milk proteins in Behçet's disease.

OBJECTIVE: To investigate the humoral and cellular immune response against cow's milk proteins in Behçet's disease and to distinguish any behaviour during active or inactive disease. METHODS: Peripheral blood mononuclear cells from 16 patients and from eight normal controls were cultured in the presence of phytohaemagglutinin (PHA), beta-casein, beta-lactoglobulin, or chicken egg albumin. Interferon gamma (IFNgamma) and interleukin 4 (IL4) were measured in the culture supernatants by enzyme linked immunosorbent assay (ELISA). Serum samples from 46 patients with Behçet's disease and from 37 healthy subjects were also studied for antibody detection. Antibodies to beta-casein, beta-lactoglobulin, and chicken egg albumin were determined by ELISA. RESULTS: High IFNgamma but not IL4 levels were found in the supernatants of lymphocytes from patients with active disease cultured in the presence of cow's milk proteins. Levels were comparable with those obtained in cultures stimulated with PHA. A significantly higher level of anti-beta-casein and anti-beta-lactoglobulin IgG and IgA antibodies was found in patients with active Behçet's disease. No relation was found between their occurrence and the age of the patients, the duration of disease, or the presence of gastrointestinal abnormalities. Antibodies to chicken albumin were detected at low levels and with a prevalence similar to that of healthy subjects. CONCLUSION: The results indicate that an active immune response occurs in Behçet's disease. This response involves an increased frequency of antibodies to cow's milk protein and a strong Th1 polarisation after exposure to these antigens. The occurrence of these abnormalities supports a putative role for cow's milk proteins immune response in the pathogenesis of Behçet's disease.

Acute Disease↗

Granulysin-dependent killing of intracellular and extracellular Mycobacterium tuberculosis by Vgamma9/Vdelta2 T lymphocytes.

Contribution of Vgamma9/Vdelta2 T lymphocytes to immune protection against Mycobacterium tuberculosis is still a matter of debate. It was reported earlier that Vgamma9/Vdelta2 T lymphocytes kill macrophages harboring live M. tuberculosis through a granule-dependent mechanism that results in killing of intracellular bacilli. This study found that Vgamma9/Vdelta2 T lymphocytes reduce the viability of both extracellular and intracellular M. tuberculosis. Granulysin and perforin, both detected in Vgamma9/Vdelta2 T lymphocytes, play a major role, which indicates that Vgamma9/Vdelta2 T lymphocytes directly contribute to a protective host response against M. tuberculosis infection.

Antigens, Differentiation, T-Lymphocyte↗

Differential activation of human gammadelta cells by nonpeptide phosphoantigens.

Human T cells expressing Vgamma9Ndelta2-encoded TCR recognize several nonpeptide phosphoantigens in the absence of major histocompatibility complex restriction. As these cells respond differentially to increasing concentrations of structurally related phosphoantigens, such ligands constitute agonists of different strengths. By analyzing early cellular events and late effector responses of gammadelta T cells, we compared their patterns of stimulation by weak, medium and strong phosphoantigen agonists. We found that, although the early metabolic activation as assessed by cytosensor microphysiometry directly reflects the intensity of subsequent effector response by gammadelta cells, TCR down-modulation is dissociated from the latter. Weak and mid-range phosphoantigen agonists induce a time- and dose-dependent down-modulation of the gammadelta TCR, whereas strong phosphoantigen agonists induce little or no TCR down-regulation. This indicates that gammadelta TCR down-modulation does not match the extent of TCR signaling as assessed by microphysiometry or conventional effector responses (TNF-alpha production and cytotoxicity). This differential pattern of gammadelta cell activation by phosphoantigens could explain the stronger potencies of some of these agonists.

Acids↗

Biology of gammadelta T cells in tuberculosis and malaria.

Tuberculosis and malaria remain the leading causes of mortality among human infectious diseases in the world. It is estimated that 3 to 5 million people die from tuberculosis and malaria each year. Although it is traditionally believed that CD4 and CD8 alphabeta T lymphocytes are mandatory for protective immune responses against Mycobacterium tuberculosis and Plasmodium falciparum (the ethiologic agents of tuberculosis and the most severe form of malaria, respectively), there is still incomplete understanding of the mechanisms of immune protection and of the causes of its failure in the affected patients. Several studies in humans and animal models have suggested that Vgamma9/Vdelta2 T cells may play an important role in the immune responses against Mycobacterium tuberculosis and Plasmodium falciparum. Vgamma9/Vdelta2 T cells represent about 75% of all circulating gammadelta T cells while they can be greatly expanded during the acute phase of Mycobacterium tuberculosis and Plasmodium falciparum malaria. Vgamma9/Vdelta2 T recognize a new class of antigenic molecules which are nonpeptidic in nature and contain critical phosphate moieties (phosphoantigens). Interestingly, phosphoantigens isolated from Mycobacterium tuberculosis and Plasmodium falciparum share strong structural homology and are probably identical. However, despite a large body of data reported in the literature, it is not yet clear whether Vgamma9/Vdelta2 T cells play a protective or pathogenic role in immune responses against Mycobacterium tuberculosis and Plasmodium falciparum. In this review we summarize our current knowledge of the biology of Vgamma9/Vdelta2 T cells in response to the two pathogens, Mycobacterium tuberculosis and Plasmodium falciparum, and provide evidence suggesting definition of a novel and important protective role through which Vgamma9/Vdelta2 T cells can contribute to the killing of microorganisms residing in intracellular compartments.

Animals↗

Resistance of natural killer T cell-deficient mice to systemic Shwartzman reaction.

The generalized Shwartzman reaction in mice which had been primed and challenged with lipopolysaccharide (LPS) depends on interleukin (IL)-12-induced interferon (IFN)-gamma production at the priming stage. We examined the involvement in the priming mechanism of the unique population of Valpha14 natural killer T (NKT) cells because they promptly produce IFN-gamma after IL-12 stimulation. We report here that LPS- or IL-12-primed NKT cell genetically deficient mice were found to be resistant to LPS-elicited mortality. This outcome can be attributed to the reduction of IFN-gamma production, because injection of recombinant mouse IFN-gamma, but not injection of IL-12, effectively primed the NKT cell-deficient mice. However, priming with high doses of LPS caused mortality of severe combined immunodeficiency, NKT cell-deficient, and CD1-deficient mice, indicating a major contribution of NKT cells to the Shwartzman reaction elicited by low doses of LPS, whereas at higher doses of LPS NK cells play a prominent role. These results suggest that the numerically small NKT cell population of normal mice apparently plays a mandatory role in the priming stage of the generalized Shwartzman reaction.

Animals↗

Vgamma9/Vdelta2 T lymphocytes reduce the viability of intracellular Mycobacterium tuberculosis.

An effective immune response against the intracellular pathogen Mycobacterium tuberculosis is strictly dependent on T cell activation. Although this protective response mainly depends on local release of pro-inflammatory cytokines by Th1 CD4(+) T cells, contribution of Vgamma9 / Vdelta2 T lymphocytes to immune protection against this pathogen is suggested by the antimycobacterial reactivity of this subset and its ability to produce large amounts of Th1 cytokines. Here we show that Vgamma9 / Vdelta2 T lymphocytes kill macrophages harboring live M. tuberculosis. The cytotoxic activity of Vgamma9 / Vdelta2 T lymphocytes was not MHC class I or class II restricted but was blocked by anti-TCR monoclonal antibodies, thus indicating that it involved specific interaction between the TCR and the target cell. The cytotoxicity of Vgamma9 / Vdelta2 T lymphocytes was not mediated by TNF-alpha or Fas-Fas ligand, but was shown to occur through a granule-dependent mechanism that resulted in reduction of the viability of intracellular bacilli. Perforin was shown to play an important role in killing of both infected macrophages and intracellular mycobacteria. These data strongly suggest that Vgamma9 / Vdelta2 T lymphocytes contribute to the host defense against M. tuberculosis infection.

Cells, Cultured↗

Whole body irradiation induces IFN-gamma production in BALB/c mice by preventing the appearance of a V alpha 14(+)NK T downregulatory population.

Lymph node cells from TNCB-immune BALB/c mice fail to produce IFN-gamma when exposed to antigen in vitro. Conversely, lymph node cells of irradiated (550 rads) BALB/c mice produce IFN-gamma. Transfer experiments show that normal BALB/c mice contain cells which suppress IFN-gamma production. These downregulatory cells are CD4(+)alpha beta(+)and rearrange the invariant V alpha 14-J alpha 281 T cell receptor alpha chain, thus belonging to the NK T cell subset. Downregulatory cells probably act by producing IL-4 as their effect is blocked by mAb to IL-4.

Animals↗

Evidence of alloreactive T lymphocytes in fetal liver: implications for fetal hematopoietic stem cell transplantation.

The use of hematopoietic stem cells for in utero transplantation to create permanent hematochimerism represents a new concept in fetal therapy, although this approach has provided heterogeneous results. In this paper we have undertaken molecular, phenotypic and functional studies aimed at identifying the presence of fully competent T lymphocytes in samples of fetal livers and cord blood. We found mature VDJ TCR beta chain transcripts in fetal liver cells taken from 7 to 16 weeks of gestation and a similar pattern was detected in cord blood cells sampled from 13.5 to 20.5 weeks of gestation. A Vbeta8 gene sequence comparable to that detected in adult PBMC was found in fetal liver samples at 9 or 17 weeks gestation. PreTalpha message was detected in all samples and its expression decreased in fetal blood samples with increasing gestational age while Calpha message appeared at 9.4 weeks and its expression increased during gestational age. T cell clones obtained from fetal liver cells showed a mature TCR alphabeta+, CD8+ phenotype and displayed strong alloreactivity against allo-MHC class I molecules. The presence of alloreactive T lymphocytes may explain the failure to engraft in fetuses older than 13 to 16 weeks and may provide insights into fetal liver transplantation. Bone Marrow Transplantation (2000) 25, 135-141.

CD8 Antigens↗

Change of Th0 to Th1 cell-cytokine profile following tuberculosis chemotherapy.

T cells mediate protection against tuberculosis, but little is known about their role during chemotherapy of patients with active disease. Here we examined the cytokine profile of CD4 T cells before and after four months of chemotherapy in six initial skin test anergic cases. Purified protein derivative (PPD) and 16-kDa antigen-reactive CD4 T-cell clones prior to therapy resided mostly in disease-associated body fluids and were of the Th0 (interferon (IFN)-gamma + interleukin (IL)-4) secreting profile. In contrast, the majority of postchemotherapy CD4 T-cell clones originated from blood and were of the IFN-gamma secreting Th1 type. However, the recognition of several peptides derived from the 16-kDa antigen was not significantly different between the Th1 and Th0 clones. We conclude that chemotherapy shifts CD4 T cells from the affected body fluids to the blood circulation, accompanied by a change from Th0 to Th1 cytokine profile.

CD4-Positive T-Lymphocytes↗