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S Berrih-Aknin

Publications and source records attributed to S Berrih-Aknin.

At least 19 recordsLinked to original sources
PubMed2005

Thymic myoid cells protect thymocytes from apoptosis and modulate their differentiation: implication of the ERK and Akt signaling pathways.

Thymic myoid cells correspond to a muscle-like cell population present in the thymic medulla. They are well conserved throughout species evolution, but their biological role is not known. We demonstrated that myoid cells protected thymocytes from apoptosis as evidenced by a strong decrease of annexin-V-FITC positive thymocytes. This effect was (1) specific of myoid cells compared to thymic epithelial cells; (2) dependent on direct cell-to-cell contacts and (3) triggered rapidly after 2 h in cocultures. This protective phenomenon was due to the activation of prosurvival mechanisms. Indeed, myoid cells activated extracellular-regulated kinases (ERK1/2) and Akt in thymocytes. Myoid cells also influenced thymocyte maturation. We observed an increase in CD4(+) and a decrease in CD8(+) single positive (SP) thymocytes when cocultured with myoid cells, independently of a CD8(+)SP increased death or a CD4(+)SP overproliferation. Consequently, thymic myoid cells protect thymocytes from apoptosis and could also modulate their differentiation process.

Annexin A5↗
PubMed2003

Analysis of CD4+CD25+ cell population in the thymus from myasthenia gravis patients.

The present study is aimed at exploring the regulatory CD4(+)CD25(+) T cells in the thymus from myasthenia gravis (MG) patients. In early-onset MG, the thymus is hyperplastic and contains autoreactive activated T cells. Preliminary studies indicate that these CD4(+)CD25(+) cells include activated autoreactive T cells. Studies to characterize the phenotype and suppressive capacity of these cells will be discussed.

CD4 Antigens↗
PubMed2001

Expression of ciliary neurotrophic factor receptor in myasthenia gravis.

In myasthenia gravis, high anti-nicotinic receptor (AChR) antibody titers are not always associated with severity of the disease, suggesting the involvement of other pathological effectors. We showed that ciliary neurotrophic factor receptor (CNTFR) gene expression was higher in muscle biopsy tissue from severely affected MG patients regardless of anti-nAChR antibody titer. This increase was also triggered in vitro by a seric factor from MG patients. CNTFR protein expression was decreased in muscles from seropositive MG patients only.Altogether, our data indicate that the alteration of CNTFR expression in some MG patients could contribute to the severity of the disease in a subgroup of patients.

Adolescent↗
PubMed2001

In vivo and in vitro apoptosis of human thymocytes are associated with nitrotyrosine formation.

Most thymocytes are deleted by thymic selection. The mechanisms of cell death are far from being clear. Peroxynitrite is a powerful oxidant produced in vivo by the reaction of superoxide (O2*-) with nitric oxide (NO*) and is able to mediate apoptosis. The aim of this study was to analyze whether NO and peroxynitrite could play a role in human thymocyte apoptosis. The results indicate that 3-(4-morpholinyl)-sydnonimine (SIN-1, an O2*- and NO* donor) and chemically synthesized peroxynitrite, but not S-nitroso-N-acetyl-D,L-penicillamine (SNAP, an NO* donor), have a strong apoptotic effect on human thymocytes (annexin V staining and TUNEL reaction). This effect was inhibited by exogenous superoxide dismutase (SOD), which interacts with O2*- and inhibits the formation of peroxynitrite. Because peroxynitrite formation requires NO*, thymic stromal cells were investigated to determine if they produced NO*. Inducible NOS was synthesized in cultured thymic epithelial cells in certain conditions of cytokine stimulation, as shown by messenger RNA levels, protein analysis, and nitrite production in the supernatants. SIN-1-treated thymocytes had high levels of tyrosine nitration, abolished by the addition of exogenous SOD. Tyrosine nitration was also detected in thymus extracts and sections, suggesting the presence of peroxynitrite in situ. In thymus sections, clusters of nitrotyrosine-positive cells were found in the cortex and corticomedullary areas colocalized with cells positive in the TUNEL reaction. These data indicate an association between human thymocyte apoptosis and nitrotyrosine formation. Thus, the results support the notion of a physiologic role for peroxynitrite in human thymocyte apoptosis. (Blood. 2001;97:3521-3530)

Annexin A5↗
PubMed2001

Human thymus contains IFN-alpha-producing CD11c(-), myeloid CD11c(+), and mature interdigitating dendritic cells.

Three distinct dendritic cell (DC) subsets capable of stimulating allogeneic naive T cells were isolated from human thymus. The most abundant subset was represented by plasmacytoid DCs (pDCs), which secreted high amounts of IFN-alpha upon stimulation with inactivated influenza virus and thus likely correspond to the recently identified peripheral blood natural IFN-alpha/beta-producing cells (IPCs). Like those latter cells, thymic pDCs had distinctive phenotypic features (i.e., Lin(-), HLA-DR(int), IL-3R alpha(hi), CD45RA(hi), CD11c(-), CD13(-), and CD33(lo)) and developed into mature DCs upon culture in IL-3 and CD40L. Of the two other DC subsets, one displayed a phenotype of immature myeloid DCs (imDCs) (HLA-DR(int), CD11c(+), CD13(+), CD33(+)), and the other represented HLA-DR(hi) CD11c(+) mature DCs (mDCs). Since they also expressed DC-LAMP, these mDCs appear to correspond to interdigitating dendritic cells (IDCs). Thymic pDCs, but not myeloid imDCs, strongly expressed lymphoid-specific transcripts such as pre-T alpha, lambda-like, and Spi-B, thereby suggesting a possible lymphoid origin. The detection of Spi-B mRNA, not only upon in vitro maturation of pDCs, but also in freshly purified IDCs, suggests that in vivo pDCs may differentiate into IDCs.

Adolescent↗
PubMed2001

Phenotypic and functional characterization of human thymic stromal cell lines.

To establish new tools for studying human thymic stromal cells, we transfected adherent cells from a human postnatal thymus using a plasmid encoding SV40 large T antigen. Among the cell lines obtained, we characterized four epithelial cell lines (LT-TEC1 to LT-TEC4) and one thymic myoid cell line (MITC). Several morphological, functional and phenotypic differences were observed between these 2 cell types. Epithelial cells were heterogeneous and larger than myoid cells. Untreated LT-TEC lines expressed MHC class I, ICAM-1 and LFA-3 antigens and not MHC class II antigens, similarly to primary thymic epithelial cells (PTEC), while MITC line expressed only class I and LFA-3 antigens. After IFN-gamma treatment, MHC class II and ICAM-1 antigens were markedly upregulated in LT-TEC lines but not in MITC, indicating the absence or a dysfunction of regulatory factors in MITC line. Myoid cells expressed mRNA for all the subunits of the acetylcholine receptor (AChR) while epithelial cells expressed only the alpha, beta and epsilon subunits. Strikingly, LT-TEC produced much more C-C chemokines and IL-6 than MITC cells, while these latter produced higher levels of IL-8 and TNF-alpha. Altogether, these results reveal phenotypic and functional differences between these two stromal cell types, suggesting a potential involvement of myoid cells in the thymic function.

Animals↗
PubMed2000

A specific interferon (IFN)-stimulated response element of the distal HLA-G promoter binds IFN-regulatory factor 1 and mediates enhancement of this nonclassical class I gene by IFN-beta.

Type I interferons display a broad range of immunomodulatory functions. Interferon beta increases gene expression at the transcriptional level through binding of factors to the interferon-stimulated response element (ISRE) within the promoters of interferon-inducible genes, such as HLA class I. Despite mutation of the class I ISRE sequence within the nonclassical HLA-G class I gene promoter, we show that interferon beta enhances both transcription and cell surface expression of HLA-G in trophoblasts and amniotic and thymic epithelial cells that selectively express it in vivo. Deletion and mutagenesis analysis of a putative interferon-regulatory factor (IRF)-1 binding site within the HLA-G promoter show that HLA-G transactivation is mediated through an ISRE sequence 746 base pairs upstream from ATG, which is distinct from the interferon-responsive element described within proximal classical class I gene promoters. Electrophoretic mobility shift analysis and supershift analysis further demonstrate that interferon-responsive transcription factors, including IRF-1, specifically bind to the HLA-G ISRE. Our results provide evidence that IRF-1 binding to a functional ISRE within the HLA-G promoter mediates interferon beta-induced expression of the HLA-G gene. These observations are of general interest considering the implication of HLA-G in mechanisms of immune escape involved in fetal-maternal tolerance and other immune privilege situations.

Amnion↗
PubMed2000

Modulation of HLA-G expression in human thymic and amniotic epithelial cells.

Expression of the nonclassical HLA class I antigen, HLA-G, is tightly regulated. HLA-G physiologic expression is mostly restricted to some placental and thymic cell types. Only few established cell lines express HLA-G in vitro. Cytokine-induced expression of HLA-G is hardly observed and also depends on the cell lineage. We assessed expression and cytokine regulation of HLA-G in primary cultures derived from human thymus and amnion epithelial cells, which also express HLA-G in vivo. We show that HLA-G cell surface expression is maintained, but decreases gradually, in primary cultures derived from human thymus and amnion epithelial cells. We also show that IFN-gamma re-induces HLA-G cell surface expression and upregulates classical class I gene expression in both primary cultures and in a thymus derived cell line. We further show that IFN-gamma also upregulates levels of HLA-G transcripts in TEC primary cultures. This study provides evidence that IFN-gamma induction of HLA-G expression occurs in the human amnion and the thymus, and is mediated at the transcriptional level in these tissues. These results also suggest a role for the microenvironment in regulating HLA-G in vivo gene expression in the thymus and amnion membrane.

Amnion↗
PubMed2000

T cell deletion and unresponsiveness induced by intrathymic injection of staphylococcal enterotoxin B.

Intrathymic injection of alloantigens appears to be the most efficient route to induce alterations of T cell reactivity. In the present study, we explored the modifications of Vbeta8.1, 8.2 T cell population and T cell reactivity in the thymus and in the spleen induced by intrathymic injection of staphylococcal enterotoxin B to adult mice. Vbeta8 antigen expression was investigated by flow cytometry analysis. T Cell reactivity was studied in vitro by the proliferative response to SEB. SEB induced a significant reduction in the percentage of mature Vbeta8+ T cells in the thymus (days 7-14), and in the spleen (days 7-28). Interestingly, this depletion occurs in the CD4- CD8+ cells in the thymus whereas in the CD4+ CD8- cells in the spleen. In parallel, the proliferative response to SEB but not to SEA was significantly decreased in the thymus on days 7 and 14, and in the spleen from day 7 to day 28. Moreover, this unresponsiveness was more pronounced in the spleen than in the thymus. Anergy was SEB-specific and fully reversed by exogenous IL-2. SEB injected intrathymically induced significantly more pronounced and more durable T cell alterations than intraperitoneal and subcutaneous injections. This may be related to the observation that after i.t. injection, SEB was detected both at a higher amount and for a longer period in the central and peripheral compartments. Our results clearly demonstrate that the intrathymic route is definitely the most efficient to induce not only thymic but also peripheral pivotal immune alterations in our model.

Animals↗
PubMed2000

Kinetic analysis of microchimerism induced by intrathymic injection of allogeneic splenocytes in mice.

Allograft survival facilitated by intrathymic (i.t.) injection of allogeneic cells have shown that modifications of T-cell development induce specific tolerance. One hypothesis is that the resulting microchimerism may play a role in preparing the host immune system for the allograft. To investigate whether the deliberate introduction of allogeneic splenocytes into the thymus of adult mice allows the establishment of a lasting donor/recipient microchimerism, a full allogeneic mouse system (H-2 and Mls) with additional sex mismatch was used. Male cells injected into female mice were detected using an optimized nested-polymerase chain reaction which specifically amplifies the SRY gene with a sensitivity of 1/10(4). After i.t. injection, donor cells were observed early both in the lymph nodes and spleen (75 and 25% of mice, respectively). They were still present on day 6, although preferentially in the thymus (100% of mice) than in the lymph nodes (50% of mice) or in the spleen (22% of mice). After intraperitoneal (i.p.) or subcutaneous (s.c.) injection, donor cells were early (2 h) but transiently detected in the thymus, since on day 6 they were detected in 0 and 17% of mice after i.p. and s.c. injection, respectively. Kinetics of donor-cell detection was similar both in the spleen and lymph nodes with a clear decrease in the percentage of mice with donor-cell detection between day 2 and day 6 (20 and 17% of positive mice for the spleen after i.p. and s.c. injections, respectively--20 and 33% of positive mice for the lymph nodes after i.p. and s.c. injections, respectively). Our results clearly show that i.t. injection of allogeneic splenocytes induces a microchimerism which is both more lasting and detected in a higher percentage of mice than by the i.p. and s.c. routes, both at the central (thymus) and peripheral (spleen) levels.

Animals↗
PubMed2000

[Modulation of experimental myasthenia gravis by IVIg].

Myasthenia Gravis (MG) is an autoimmune disease mediated by antibodies directed against the acetylcholine receptor (AChR). Treatment by IVIg is effective in acute forms of myasthenia gravis. In order to determine the in vivo effects of the various fractions of human immunoglobulins, we used an experimental model of myasthenia gravis in SCID mice. To this end, thymic cells from MG patients are transferred to these mice according to a well defined protocol. When establishing of the model, we noticed the appearance of anti-AChR antibodies and the loss of AChR expression at the muscle level. After treatment with IVIgG or IVIgM, the mice displayed a lower anti-AChR antibody titer compared to control mice (albumin treated) and the loss of the AChR number at the muscle was significantly reduced. These results obtained from one MG patient indicate that the human immunoglobulin preparations induce significant effects on pathogenic parameters in the SCID mouse model. Therefore this model is interesting to approach the mechanisms of action of human immunoglobulins and deserves further investigation.

Acetylcholine↗
PubMed2000

Modulation of acetylcholine receptor expression in seronegative myasthenia gravis.

In a previous study, we demonstrated a compensatory mechanism for regulating acetylcholine receptor (AChR) gene expression in muscle biopsies from seropositive and seronegative (SN) myasthenia gravis (MG) patients. To further characterize the AChR regulation mechanisms involved in SNMG disease, we investigated the effects of MG sera on nicotinic AChR expression (at the protein and messenger RNA [mRNA] levels) in cultured human muscle cells. Sera from SNMG patients induced an in vitro increase in the level of nicotinic AChR beta-subunit mRNA but did not cause a decrease in AChR protein level. This apparent discrepancy was not due to a higher level of AChR synthesis as demonstrated by analysis of AChR turnover. In SN patients, the increase in beta-subunit mRNA level was followed after 48 hours by a slight increase in the amount of AChR surface protein. This regulation of nicotinic receptor expression was due to the purified IgG-containing fraction. Thus, sera from SNMG patients contain an immunoglobulin that induces an increase in AChR mRNA without causing a decrease in AChR protein level, suggesting an indirect regulatory mechanism involving another surface molecule. This model is therefore useful for defining the targets involved in the pathogenesis of SNMG disease.

Adolescent↗
PubMed1999

Functional Fas expression in human thymic epithelial cells.

Fas, a cell surface receptor, can induce apoptosis after cross-linking with its ligand. We report that Fas antigen is constitutively expressed in medullary epithelial cells of the human thymus. Expression is decreased in cultured thymic epithelial cells (TEC), similarly to HLA-DR antigen. TEC are resistant to anti-Fas-induced apoptosis after 4 days of primary culture, and this resistance is reversed by concomitant addition of cycloheximide. Cycloheximide also downregulated the expression of Fas-associated phosphatase-1, which has been found to inhibit Fas-induced apoptosis. This phosphatase could be involved in the resistance to Fas-induced apoptosis observed on day 4 of TEC culture. When TEC were subcultured after 10 to 13 days of primary culture, exposure to interleukin-1-beta, tumor necrosis factor-alpha, and interferon-gamma, alone or together, reinduced Fas mRNA and protein expression. In coculture with activated thymocytes, TEC also upregulated Fas protein expression. Cytokine-activated TEC became sensitive to apoptosis induced by an agonistic anti-Fas antibody. This apoptosis was inhibited by Z-VAD-fmk but not by Z-DEVD-fmk and DEVDase activity was slightly increased in Fas-stimulated TEC, suggesting that DEVDase activity is not sufficient to induce TEC apoptosis. Taken together, these data show that the Fas receptor is expressed in medullary epithelial cells of the human thymus and is able to induce apoptosis.

Apoptosis↗
PubMed1999

Functional expression of receptors for calcitonin gene-related peptide, calcitonin, and vasoactive intestinal peptide in the human thymus and thymomas from myasthenia gravis patients.

The molecular and functional expression of serpentine membrane receptors for vasoactive intestinal peptide (VIP), calcitonin gene-related peptide (CGRP), and calcitonin (CT) were characterized in human thymus and thymomas from myasthenia gravis (MG) patients and thymic epithelial cells either in primary culture (PTEC) or transformed by the simian virus 40 large T (SV40LT) oncogene (LT-TEC). Using RT-PCR combined with Southern analysis, we identified the PCR products corresponding to the receptor (-R) transcripts for VIP, CGRP, and CT in thymus from control subjects and MG patients with either hyperplasia or thymoma. Similar expressions of the VIP- and CGRP-R transcripts were observed in PTEC, whereas the CT-R message was not detected. In LT-TEC, the signals for VIP-R, CGRP-R, and CT-R transcripts were seen with a lower intensity than those in control and MG thymus. In agreement with our molecular analysis, 1) VIP was the most potent peptide among VIP-related peptides (VIP > PACAP > PHM > PHV) to stimulate cAMP production through specific type 1 VIP receptors in both PTEC and LT-TEC; 2) cAMP generation was induced by CGRP in PTEC and by CT in LT-TEC; 3) in frozen thymic sections and by flow cytometry, type 1 VIP-R, CGRP-R, and CT-R were localized in epithelial cells; and 4) in parallel, the transcription of the acetylcholine receptor alpha subunit (the main autoantigen in MG) was induced by CGRP and CT in PTEC and LT-TEC, respectively. Our data suggest that the neuroendocrine peptides VIP, CGRP, and CT may exert functional roles during MG and malignant transformation of the human thymus.

Adolescent↗
PubMed1999

Normal human immunoglobulin suppresses experimental myasthenia gravis in SCID mice.

Serum IgM has been shown to participate in the control of IgG autoreactivity in healthy subjects. We have recently shown that an immunoglobulin preparation of pooled normal human IgM (IVIgM) contains anti-idiotypic antibodies against disease-associated IgG autoantibodies in autoimmune patients and protects rats from experimental autoimmunity. The aim of the present study was to asses the in vitro and in vivo immunomodulatory effects of IVIgM in comparison with IgG, in SCID mice reconstituted with thymic cells from a myasthenia gravis patient. Non-leaky SCID mice were injected i.p. with 60 x 10(6) thymic cells from a patient with myasthenia gravis and 1 day later boosted with 10(6) irradiated acetylcholine receptor (AchR)-expressing TE671 cells. On days 14, 21 and 28, mice were treated with IVIgM or with equimolar amounts of human serum albumin. The level of anti-AchR antibodies in the sera of three out of four IgM-treated animals was less than 1 nM. Further, there was a significant decrease in the loss of endplate AchR on the diaphragms of IgM-treated SCID mice. These findings indicate that pooled normal IgM exerts an immunoregulatory role in experimental myasthenia gravis, and suggests that IgM may be considered as an alternative approach in the therapy of autommune diseases.

Animals↗
PubMed1999

Prevention of autoimmune attack by targeting specific T-cell receptors in a severe combined immunodeficiency mouse model of myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disease targeting the skeletal muscle acetylcholine receptor. We have previously demonstrated a selection bias of CD4+ T cells expressing the Vbeta5.1 T-cell receptor gene in the thymus of HLA-DR3 patients with MG. To evaluate the pathogenicity of these cells, severe combined immunodeficiency mice engrafted with MG thymic lymphocytes were treated with anti-Vbeta5.1 antibody. Signs of pathogenicity (eg, acetylcholine receptor loss and complement deposits at the muscle end plates of chimeric mice) were prevented in anti-Vbeta5.1-treated severe combined immunodeficiency chimeras. Pathogenicity was mediated by autoantibodies against acetylcholine receptor. Thymic cells depleted of Vbeta5.1-positive cells in vitro before cell transfer were nonpathogenic, indicating that Vbeta5.1-positive cells are involved in the production of pathogenic autoantibodies. Acetylcholine receptor loss was prevented by Vbeta5.1 targeting in HLA-DR3 patients only, demonstrating specificity for HLA-DR3-peptide complexes. The action of the anti-Vbeta5.1 antibody involved both the in vivo depletion of Vbeta5.1-expressing cells and an increase in the interferon-gamma/interleukin-4 ratio, pointing to an immune deviation-based mechanism. This demonstration that a selective and specific T-helper cell population is involved in controlling pathogenic autoantibodies in MG holds promise for the treatment of MG.

Adolescent↗
PubMed1999

Establishment of a human thymic myoid cell line. Phenotypic and functional characteristics.

The subset of myoid cells is a normal component of the thymic stroma. To characterize these cells, we immortalized stromal cells from human thymus by using a plasmid vector encoding the SV40 T oncogene. Among the eight cell lines obtained, one had myoid characteristics including desmin and troponin antigens. This new line was designated MITC (myoid immortalized thymic cells). These cells expressed both the fetal and adult forms of muscle acetylcholine receptor (AChR) at the mRNA level, as well as the myogenic transcription factor MyoD1. alpha-Subunit AChR protein expression was detected by flow cytometry and the AChR was functional in patch-clamp studies. In addition, AChR expression was down-modulated by myasthenia gravis sera or by monoclonal antibody anti-AChR on MITC line similarly to TE671 rhabdomyosarcoma cells, making the MITC line an interesting tool for AChR antigenic modulation experiments. Finally, the MITC line expressed LFA-3, produced several cytokines able to act on T cells, and protected total thymocytes from spontaneous apoptosis in vitro. These results are compatible with a role of thymic myoid cells in some steps of thymocyte development. Therefore MITC line appears to be a useful tool to investigate the physiological role of thymic myoid cells.

Antigenic Modulation↗