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.

Cellular aspects of myasthenia gravis.

Several cellular aspects were investigated in a large series of patients with MG. First, non-Ag-specific proliferation was tested by measuring the response to r-IL2. Thymocytes from most MG patients showed hyperactivity to r-IL2. Peripheral blood lymphocytes (PBL) from some patients also showed a high response to r-IL2. These responding patients were generally those tested before thymectomy, presenting a high anti-AChR Ab titer and a severe form of the disease. Second, Ag-specific proliferation of MG PBL was assayed using 8 synthetic peptides corresponding to selected domains of torpedo or human AChR. Only 2 peptides gave a positive response in a significant number of patients, essentially in those presenting high anti-AChR Ab titer. The first is located near the alpha-bungarotoxin binding site and the second is in a cytoplasmic domain, according to models predicting the AChR transmembrane orientation. The positive results were essentially obtained with the human peptides; the corresponding torpedo peptides were positive in very few patients. Both human and torpedo peptides which include a part of the alpha-bungarotoxin binding site were negative. Finally, although morphological abnormalities were clearly visible in thymic hyperplasia, no correlation could be established between the thymus type and the cellular proliferation either to r-IL2, or to the peptides. Overall, our data indicate that cell-dependent mechanisms participate in the pathogenesis of MG, but the level of their involvement deserves further investigation.

Antibodies to thymic epithelial cells in myasthenia gravis.

The presence of anti-thymus antibodies was investigated in the serum of 36 patients with myasthenia gravis (MG). Using an immunofluorescence technique on frozen thymic sections, we found 45% of patients sera reacting with normal or MG thymuses. Staining was confined to subcapsular and medullary keratin-positive epithelial cells. Thirty-five out of 36 sera from healthy controls and all 15 sera from patients presenting another autoimmune disorder were negative. Antibodies to thymic epithelial cells were almost exclusively detected in patients presenting thymic hyperplasia and did not disappear after thymectomy. They were not clearly associated with antiacetylcholine receptor antibody titer, nor with disease severity. Their strong association to thymic abnormalities highlights the role of the thymus in pathogenesis of MG. The reasons for the appearance of these antibodies, the structure they recognize on thymic epithelial cells and their possible etiological role are discussed.

Evidence of enhanced recombinant interleukin-2 sensitivity in thymic lymphocytes from patients with myasthenia gravis: possible role in autoimmune pathogenesis.

We evaluated the activation state of thymic lymphocytes in patients with myasthenia gravis (MG) by cytofluorographic analysis of CD25 expression and by testing their sensitivity to recombinant interleukin-2 (rIL-2) in the absence of any known previous stimulation. We detected no phenotypic signs of activation in fresh MG thymic lymphocyte suspensions, while functional signs of activation were reflected in a significantly higher sensitivity to rIL-2 in MG patients than in controls. The responses to rIL-2 were time- and dose-dependent, were inhibited by a blocking anti-IL-2 receptor antibody, and were associated with an increase in CD25+ T cells in both patients and controls. The T cells with functional signs of previous activation may represent autoreactive cells involved in the autoimmune process and confirm thymus gland hyperactivity in MG. These cells could result from primary autosensitization against the thymic acetylcholine receptor (AChR)-like molecule or from altered migration of peripheral activated cells into an abnormal thymic environment. Our results also provide a clue for understanding the effect of thymectomy in myasthenia gravis.

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.

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.

In situ production of interleukins in hyperplastic thymus from myasthenia gravis patients.

We analyzed by in situ hybridization the expression of four interleukin genes (interleukin-beta [IL-1 beta], IL-6, IL-2, and interferon-gamma) in seven thymuses displaying a follicular hyperplasia. The seven thymuses were obtained from patients with myasthenia gravis. Interleukin-1 beta- and IL-6-producing cells were detected in similar amounts and with similar distributions: mainly in perifollicular areas and in the connective structures emerging from the septae at the site of cortex disruption. The comparison of in situ hybridization and immunohistochemical results suggested that thymic epithelial cells and/or perifollicular macrophages were responsible for this production. Interleukin-2-producing cells were detected in perifollicular areas and, to a lesser extent, inside follicles. They were clearly outnumbered by CD25-positive cells which were similarly distributed. Despite the expression of these molecular and immunohistochemical markers of T-cell activation, interferon-gamma-producing cells were extremely rare in myasthenic thymuses. The pattern of interleukin production (which was virtually absent in normal control thymuses) in myasthenic thymuses was different from that in benign hyperplastic lymph nodes. This interleukin production may play a role in the development of follicular hyperplasia in myasthenic thymuses, a phenomenon which is associated with the in situ production of autoantibodies.

Interleukin-6 overproduction by cultured thymic epithelial cells from patients with myasthenia gravis is potentially involved in thymic hyperplasia.

Most patients with Myasthenia Gravis (MG) present a thymic hyperplasia characterized by the presence of lymphoid follicles. The acetylcholine receptor autoantigen, as well as autoantigen specific activated T and B cells found in the thymus, strongly suggest that auto-sensitization could take place in this organ. Since IL-6 is involved in T and B cell growth and differentiation, we thought that abnormal IL-6 expression by thymic epithelial cells (TEC) could be related to thymic hyperplasia in MG. In this paper, IL-6 protein and gene expression by cultured TEC from patients with MG were examined. TEC from patients presented a dramatic IL-6 hyperproduction phenotype as compared to controls when stimulated by exogenous signals such as LPS and cytokines (IL-1 beta, TNF-alpha) alone or in combination. Moreover, we observed a similar effect with a physiological signal such as the syngeneic lympho-epithelial cell contact. Autologous thymocytes stimulated normal and MG TEC IL-6 production in a time- and dose- dependent way, and with a higher magnitude in MG TEC compared to controls. In all stimulation conditions, induction of IL-6 production required protein synthesis and was associated with increased IL-6 mRNA level expression as assessed by computer-aided quantification after in situ mRNA hybridization. In addition, recombinant IL-6 induced in vitro growth of TEC, demonstrating that IL-6 is a possible autocrine growth factor for these cells. This deregulated IL-6 production as well as the ability of TEC to use it as a growth factor may be of pathophysiological relevance in MG. It provides an explanation for morphological changes of the thymus and may have a key role in initiation, exacerbation and ongoing of the autoimmune response in MG. Therefore this study extends our current understanding of the molecular pathophysiology of MG.

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.