Evidence of endogenous volatile organic compounds as biomarkers of diseases in alveolar breath.

The effect of oxygen on markers of oxidative stress has been partially elucidated. Volatile organic compounds (VOCs) are created during the oxidative burst and excreted in the human alveolar breath, which indeed contains biomarkers. A general concept including collection, separation, detection and clinical biomakers validation is presented in this article: (i) a method for the collection and GC-MS of halogenated VOCs in human alveolar breath is described: a transportable apparatus which sampled specifically alveolar breath; the VOCs were captured in a thermal desorption tube, Carbotrap 200® and each sample was thermally desorbed from the trap in an automated GC-MS apparatus; (ii) the inhibitory effects of halogenated alkanes on mitochondria are suspected likely to fight against oxidative stress deleterious reactions; (iii) two-dimensional gas chromatography occurs by the repeated and re-injection of effluent from one chromatographic column into a second column of orthogonal phase. A new commercial GCxGC system is presented; it is accomplished with a dual-stage, quad-jet thermal modulator positioned between the two columns; (iv) the affinity-based sensors usually used in connection with the GCxGC system face a difficulty to take into account different biases coming from different sources of drifting. Compared to other affinity-based sensing modes like electrical ones, gravimetric sensors enable a better decoupling. Nano Electro Mechanical Systems (NEMS)-based resonators are a particular type of gravimetric gas sensors. They are coated with a sensitive layer of polymer where gases of interest present in the atmosphere adsorb, generating an additional mass load which is measured through a frequency shift; (v) examination of exhaled breath has the potential to change the existing routine approaches in human medicine. Breath sampling to identify volatile biomarkers in diseases has been proposed in several respiratory diseases. Several VOCs have been identified in these patients by GC-MS. However, the use of traditional analytical instruments such as GC-MS to detect biomarkers of diseases has not become a routine for clinical applications. Consequently the electronic nose was the logical instrument of choice for disease diagnosis due to the capability of identifying complex mixtures of VOCs (as a whole) within sampled air using pattern-recognition algorithms.

Targeting of c-kit+ haematopoietic progenitor cells prevents hypoxic pulmonary hypertension.

Haematopoietic c-kit+ progenitor cells may contribute to pulmonary vascular remodelling and pulmonary hypertension (PH). Stromal derived factor-1 (SDF-1/CXCL12) and its receptors CXCR4 and CXCR7 have been shown to be critical for homing and mobilisation of haematopoietic c-kit+ progenitor cells in the perivascular niche. We administered AMD3100, a CXCR4 antagonist, and CCX771, a CXCR7 antagonist, to chronic hypoxia exposed mice in order to study the role of c-kit+ progenitor cells in PH. CXCL12, CXCR4 and CXCR7 protein expression, haemodynamic parameters, right ventricular mass, extent of vascular remodelling and perivascular progenitor cell accumulation were studied. Chronic hypoxia-exposed mice showed increased total lung tissue expression of CXCR4, CXCR7 and CXCL12 after development of PH. This was associated with significantly increased right ventricular systolic pressure and evidence of right ventricular hypertrophy, vascular remodelling and perivascular c-kit+/sca-1+ progenitor cell accumulation. CCX771 administration did not abrogate these effects. In contrast, administration of AMD3100, whether alone or combined with CCX771, prevented vascular remodelling, PH and perivascular accumulation of c-kit+/sca-1+ progenitor cells, with a synergistic effect of these agents. This study offers important pathophysiological insights into the role of haematopoietic c-kit+ progenitors in hypoxia-induced vascular remodelling and may have therapeutic implications for PH.

Sustained calcium signalling and caspase-3 activation involve NMDA receptors in thymocytes in contact with dendritic cells.

L-glutamate, the major excitatory neurotransmitter, also has a role in non-neuronal tissues and modulates immune responses. Whether NMDA receptor (NMDAR) signalling is involved in T-cell development is unknown. In this study, we show that mouse thymocytes expressed an array of glutamate receptors, including NMDARs subunits. Sustained calcium (Ca(2+)) signals and caspase-3 activation in thymocytes were induced by interaction with antigen-pulsed dendritic cells (DCs) and were inhibited by NMDAR antagonists MK801 and memantine. NMDARs were transiently activated, triggered the sustained Ca(2+) signal and were corecruited with the PDZ-domain adaptor postsynaptic density (PSD)-95 to thymocyte-DC contact zones. Although T-cell receptor (TCR) activation was sufficient for relocalization of NMDAR and PSD-95 at the contact zone, NMDAR could be activated only in a synaptic context. In these T-DC contacts, thymocyte activation occurred in the absence of exogenous glutamate, indicating that DCs could be a physiological source of glutamate. DCs expressed glutamate, glutamate-specific vesicular glutamate transporters and were capable of fast glutamate release through a Ca(2+)-dependent mechanism. We suggest that glutamate released by DCs could elicit focal responses through NMDAR-signalling in T cells undergoing apoptosis. Thus, synapses between T and DCs could provide a functional platform for coupling TCR activation and NMDAR signalling, which might reflect on T-cell development and modulation of the immune response.

Immunoregulation by Vbeta specific antibodies in myasthenia gravis: mining physiological T cell homeostasis for TCR specific therapy.

Besides regulatory T cells, also comprising T cell receptor (CR)-specific T cells, it is increasingly evident that natural autoantibodies, among which anti-TCR antibodies represent additional immunomodulators in the immune system. We took advantage of myasthenia gravis (MG), a well-characterized antibody-mediated autoimmune disease, to demonstrate that without prior vaccination against TCR determinants, patients with MG present increased circulating anti-TCR antibodies directed to the dominant TCR used by pathogenic T cells. These findings, pointing to a regulatory protective role of anti-TCR antibodies, are discussed in the context of the mechanisms of action and the physiological role of anti-TCR antibodies in T cell homeostasis, and of the puzzling world of regulatory T cells. Natural anti-TCR antibodies are found in the serum of all individuals, with prevalence in physiological and pathological situations such as ageing, pregnancy, allograft transplantation, retroviral infection, and autoimmune diseases, including MG. The common link is the mounting of immune responses against alloantigens, pathogens or autoantigens, conferring on anti-TCR antibodies a broader role in controlling responses to any antigen (self or non-self) and more generally in T cell homeostasis. This homeostasis mechanism may well be exploited in therapeutic strategies based on TCR peptide vaccination in autoimmune diseases.

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.

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.

Chromatin immunoselection defines a TAL-1 target gene.

Despite the major functions of the basic helix-loop-helix transcription factor TAL-1 in hematopoiesis and T-cell leukemogenesis, no TAL-1 target gene has been identified. Using immunoprecipitation of genomic fragments bound to TAL-1 in the chromatin of murine erythro-leukemia (MEL) cells, we found that 10% of the immunoselected fragments contained a CAGATG or a CAGGTG E-box, followed by a GATA site. We studied one of these fragments containing two E-boxes, CAGATG and CAGGTC, followed by a GATA motif, and showed that TAL-1 binds to the CAGGTG E-box with an affinity modulated by the CAGATG or the GATA site, and that the CAGGTG-GATA motif exhibits positive transcriptional activity in MEL but not in HeLa cells. This immunoselected sequence is located within an intron of a new gene co-expressed with TAL-1 in endothelial and erythroid cells, but not expressed in fibroblasts or adult liver where no TAL-1 mRNA was detected. Finally, in vitro differentiation of embryonic stem cells towards the erythro/megakaryocytic pathways showed that the TAL-1 target gene expression followed TAL-1 and GATA-1 expression. These results establish that TAL-1 is likely to activate its target genes through a complex that binds an E-box-GATA motif and define the first gene regulated by TAL-1.

p45 NF-E2 regulates expression of thromboxane synthase in megakaryocytes.

Transcription factor p45 NF-E2 is highly expressed in the erythroid and megakaryocytic lineages. Although p45 recognizes regulatory regions of several erythroid genes, mice deficient for this protein display only mild dyserythropoiesis but have abnormal megakaryocytes and lack circulating platelets. A number of megakaryocytic marker genes have been extensively studied, but none of them is regulated by NF-E2. To find target genes for p45 NF-E2 in megakaryopoiesis, we used an in vivo immunoselection assay: genomic fragments bound to p45 NF-E2 in the chromatin of a megakaryocytic cell line were immunoprecipitated with an anti-p45 antiserum and cloned. One of these fragments belongs to the second intron of the thromboxane synthase gene (TXS). We demonstrate that the TXS gene, which is mainly expressed in megakaryocytes, is indeed directly regulated by p45 NF-E2. First, its promoter contains a functional NF-E2 binding site; second, the intronic NF-E2 binding site is located within a chromatin-dependent enhancer element; third, p45-null murine megakaryocytes do not express detectable TXS mRNA, although TXS expression can be detected in other cells. These data, and the structure of the TXS promoter and enhancer, suggest that TXS belongs to a distinct subgroup of genes involved in platelet formation and function.

Altered intrathymic T-cell repertoire in human myasthenia gravis.

In myasthenia gravis, the thymus is thought to be the primary site of autosensitization. We investigated the V beta T-cell repertoire at different intrathymic differentiation stages in 17 patients with myasthenia gravis and 8 age-matched control subjects by tricolor immunofluorescence, using a panel of six anti-V beta antibodies. We observed an increased expression of V beta 5.1 and V beta 8 subfamilies in the patients compared to the control subjects. These increases were observed not only in mature cells but also in the latest thymic precursors of mature cells (double-positive CD3 high), while there was no change in intermediate precursors (double-positive CD3 low), pointing to biased selection during intrathymic differentiation. In addition, there was a strong correlation between the percentage of V beta 5.1+ and V beta 8+ cells among both the CD4 and CD8 subsets in the patients, but not in control subjects, suggesting that thymic events relevant to the disease lead to these selected populations. Finally, location studies of V beta 5.1+ cells on thymic sections indicated that these cells were overrepresented both in the core of germinal centers and in perifollicular areas of hyperplastic thymuses, suggesting a role in the autoimmune response. Taken together, these findings are compatible with the hypothesis of a biased intrathymic selection in myasthenia gravis.

Expression of acetylcholine receptor genes in human thymic epithelial cells: implications for myasthenia gravis.

The intrathymic presence of the muscle acetylcholine receptor (AChR) is controversial, and the nature of the cell(s) expressing it is unclear. We thus analyzed the molecular expression of muscle AChR in human thymi. mRNA studies indicated that the two isoforms (P3A+ and P3A-) of the alpha-subunit were present in thymic extracts and in cultured thymic epithelial cells (TEC), while expression in thymocytes was low and not consistently detectable. The amount of mRNA coding for the alpha-subunit, evaluated by means of quantitative PCR, was about 20 times less in TEC than in muscle, and was similar in TEC from normal subjects and from patients with myasthenia gravis (MG). The beta- and epsilon-subunits present in adult AChR were also expressed in TEC (but not in thymocytes), while the embryonic subunit (gamma) was absent. In TEC cultures, the AChR alpha- and epsilon-subunit mRNA levels were down-regulated by forskolin, as also observed in the TE671 rhabdomyosarcoma cell line, suggesting similar regulation of AChR subunits in thymus and muscle. Protein expression was evidenced on TEC (but not on thymocytes), by Western blotting as well as by immunofluorescence, thus demonstrating AChR expression on human thymic epithelial cells. There was no difference in the expression of AChR between TEC from MG patients and controls, meaning that the expression of AChR subunits alone is not sufficient to explain the onset of MG.

In vivo preferential usage of TCR V beta 8 in Torpedo acetylcholine receptor immune response in the murine experimental model of myasthenia gravis.

The aim of our study was to determine the T cell receptor (TCR) V beta gene usage involved in the T cell response to Torpedo AChR in C57BL/6 mice. The specific proliferation towards AChR was found to be blocked by anti-V beta 8.1,2,3 and to a lesser extent by anti-V beta 5 mAbs, but not by the other antibodies used (anti-V beta 2, V beta 6, V beta 9). In addition, a significant expansion of CD4+ V beta 8+ cells was observed when lymph node cells from these primed mice were stimulated in vitro with purified AChR. Involvement of V beta 8 subfamilies was also explored in vivo. After 7 days of treatment, there was a striking inhibition of the proliferative response of cells from anti-V beta 8.1,2,3-treated mice and a moderate inhibition when using anti-V beta 8.1,2 and anti-V beta 8.2 antibodies. Thus our in vitro and in vivo analysis indicate that in C57Bl/6 mice, T cell response to AChR is restricted to few V beta TCR, mostly belonging to the V beta 8 sub-families.

Macrophage subpopulations in the thymic hyperplasia of patients with myasthenia gravis.

Macrophages were studied in sections of the thymus from patients with Myasthenia Gravis, using a panel of monoclonal antibodies against the monocyte/macrophage lineage. In normal areas, CD14 and CD35 were preferentially expressed in the medulla while RM3/1 and 25F9 markers stained essentially cortical macrophages. CD11c-labelled cells were densely located throughout the thymus. The antibody 27E10 recognized clusters of cells located around or in the perivascular areas, that could be migrating monocytes. In the germinal centers, cells were stained with CD14, CD35, CD11c and 25F9 but not with RM3/1 and 27E10 markers. The numbers of CD14- and CD35-positive cells were significantly increased in Myasthenia Gravis thymic hyperplasia compared to control thymus (p < 0.025 and p < 0.01, respectively). This increase was clearly due to the higher number of positive cells in the germinal centers and in the areas surrounding the lymphoid follicles. The potential role of these cells in accessory cell function and antigen presentation could be relevant in terms of intrathymic sensitization and autoantibody production that have been demonstrated in thymic hyperplasia from Myasthenia Gravis patients.

Identification by genomic typing of non-DR3 HLA class II genes associated with myasthenia gravis.

HLA association with myasthenia gravis (MG) has been studied in a series of 114 patients using class I and class II genotyping after PCR amplification. Positive association was found with DR3, particularly in women (RR = 2.6) and in early MG onset (RR = 3.4). DRB1, DRB3, DQB1, DQA1 and B (B8 and B18) genotyping revealed that the association was predominantly with the B8 DRB1*03 DRB3*0101 DQB1*0201 DQA1*0501 ancestral haplotype. This haplotype frequency was also increased in patients with thymic hyperplasia (RR = 3.5) and was greatly reduced in patients with thymoma (RR = 0.35). Sixteen out of 48 patients carrying this 8.1 ancestral haplotype showed absence of B8 (n = 4) or of DR3 (n = 12). HLA class II genotyping further revealed the existence of two other significant associations. MG was positively associated with the DQB1*0604 allele (RR = 3.4), particularly in patients with thymoma (RR = 5.7). Furthermore, the disease was negatively associated with DR1 in females (RR = 0.32). These data suggest that MG is placed under the control of at least three distinct genes: (1) a class II predisposing gene in the 8.1 ancestral haplotype; (2) a thymoma-associated class II allele on the DQB1*0604 haplotype; and (3) a protective allele DR1.

IL-1 mRNA expression detected by in situ hybridization and polymerase chain reaction in human thymus and thymoma. Correlation with late T-cell maturation in normal versus neoplastic thymus.

Interleukin-1 has long been suggested to participate in the development of T-cells in the thymus. To determine whether IL-1 was produced in the human thymus, we used the technique of in situ hybridization and PCR amplification of mRNA. High levels of IL-1 mRNA were detected in cells at the cortico-medullary border, which separates immature from mature T-cells, suggesting a role for IL-1 in a late stage in T-cell maturation. IL-1 transcripts were detectable by a single step PCR reaction in these samples. We examined a series thymomas (human thymic epithelial tumors) that induce partial T-cell maturation: In this group of thymomas T-cells develop to the cortical stage but not beyond. We failed to detect high levels of IL-1 mRNA expression in these tumors by in-situ hybridization, IL-1 mRNA was detected only in one of these samples by a single step PCR amplification. However, all of the samples were positive, when analyzed with a more sensitive, two step amplification of IL-1 mRNA by the use of nested primers. These results are consistent with the hypothesis that higher level of IL-1 expression is either the consequence of, or required for late T-cell maturation.