Pulmonary hypertension in adults completing tuberculosis treatment.

Background: Pulmonary hypertension (PH) after tuberculosis (TB) is typically not included among the chronic lung diseases causing PH (group 3 PH), with few data available to support the inclusion. Objectives: To determine the prevalence of PH in an adult population completing TB treatment. Methods: This single-centre, cross-sectional study only included patients with their first documented episode of TB, and who were in the second half of treatment or had recently completed treatment. PH was assessed using transthoracic echocardiography. Questionnaires were completed, and spirometry and a 6-minute walk test were performed. Results: One hundred patients were enrolled, with a mean age of 37.1 years, of whom 58% were male and 46% HIV positive. The median time since initiation of TB treatment was 22 weeks. The mean (standard deviation) measured right ventricular systolic pressure (RVSP) was 23.6 (6.24) mmHg. One participant had PH (defined as RVSP ≥40 mmHg; 95% confidence interval (CI) 0.0 - 3.0) and a further 3 had possible PH (RVSP ≥35 and <40 mmHg), with a combined PH prevalence of 4% (95% CI 0.2 - 7.8). Airflow obstruction on spirometry was found in 13.3% of 98 patients, while 25.5% had a reduced forced vital capacity. There was no association between RVSP or PH/possible PH and sex, age, HIV status, systemic hypertension, spirometry measurements or 6-minute walking distance. Smoking status was associated with RVSP, but not with the presence of PH/possible PH. Conclusion: There was a significant prevalence of PH in this preliminary study of predominantly young patients completing treatment for a first episode of TB. Larger and more detailed studies are warranted. Study synopsis: What the study adds. Of 100 adult patients with their first episode of tuberculosis (TB) who underwent echocardiograms near the end of treatment completion to determine the prevalence of pulmonary hypertension (PH), 1 (1%) had PH and a further 3 (3%) had possible PH. There was no association between sex, age, HIV status, lung function or 6-minute walking distance and the presence of PH. The study adds to the growing awareness of the association of TB with pulmonary vascular disease. It shows that even in a young population with a first episode of TB treated in an ambulatory setting, there is a significant prevalence of PH on treatment completion.Implications of the findings. Given that 10.6 million people acquire TB annually, the absolute global burden of cases with PH is likely to be high, but is underappreciated to date. Further work is urgently needed in this field.

The RET receptor: function in development and dysfunction in congenital malformation.

Germline mutations in the RET proto-oncogene are responsible for two unrelated neural crest disorders: Hirschsprung disease, a congenital absence of the enteric nervous system in the hindgut, and multiple endocrine neoplasia type 2, a dominantly inherited cancer syndrome. Moreover, somatic rearrangements of RET are causally involved in the genesis of papillary thyroid carcinoma. The receptor tyrosine kinase encoded by the RET gene acts as the subunit of a multimolecular complex that binds four distinct ligands and activates a signalling network crucial for neural and kidney development. Over the past few years, a clearer picture of the mode of RET activation and of its multifaceted role during development has started to emerge. These findings, which provide new clues to the molecular mechanisms underlying RET signalling dysfunction in Hirschsprung disease, are summarized in this review.

Tyrosine phosphorylation of the product of the c-cbl protooncogene is [corrected] induced after integrin stimulation.

Integrin crosslinking on human B cells induces tyrosine phosphorylation of a set of proteins ranging from 105 to 130 kDa, among which is the focal adhesion kinase p125FAK. Here we show that the c-CBL protooncogene product p120c-CBL is a component of these substrates. beta 1 integrin stimulation of p120c-CBL phosphorylation was observed in both transformed and normal human B cells, and was inhibited by prior treatment of cells with cytochalasin B, which disrupts the actin network. In contrast, tyrosine phosphorylation of p120c-CBL following crosslinking of the B cell antigen receptor (BCR) was not affected by cytochalasin B. Integrin stimulation of the promegakaryocytic cell line MO7e also led to a cytoskeleton-dependent tyrosine phosphorylation of p120c-CBL. In MO7e cells, this stimulation was induced by ligation of either beta 1 or beta 2 integrin, whereas only by ligation of beta 1 integrin in B cells. Tyrosine phosphorylation of p120c-CBL links phosphatidylinositol-3 kinase (PI-3K) with the BCR signaling machinery. Although the p85 subunit of PI-3K was increased in p120c-CBL immunoprecipitates from BCR-stimulated B cells, this association was only minimally increased by beta 1 integrin ligation. The function of p120c-CBL remains unknown; however, its interactions in vitro and in vivo with Src homology 2 and 3 (SH2 and SH3) domain-containing proteins suggest that p120c-CBL has a significant function in signal transduction pathways, and therefore may play a role in integrin signaling in lymphoid and hematopoietic cells.

Association of the Cas-like molecule HEF1 with CrkL following integrin and antigen receptor signaling in human B-cells: potential relevance to neoplastic lymphohematopoietic cells.

CrkL, a cellular homologue of the v-crk oncogene, belongs to the family of adaptor proteins, containing SH2 and SH3 domains, but no catalytic domain. Stimulation of normal B-cells and B-cell lines through beta1 integrin or - cell antigen receptor (BCR) promoted the association of CrkL with a set of 105-130 kD tyrosine phosphorylated substrates. The principal substrate is a recently identified molecule known as p105HEF1 (HEF1), which is highly homologous to p130Cas (Cas), the major tyrosine-phosphorylated protein detected in fibroblasts after transformation by v-crk. Immunodepletion studies indicated that all the tyrosine phosphorylated HEF1 or Cas was complexed with CrkL. Furthermore, the guanine nucleotide exchange factor C3G, which is thought to be involved in the regulation of the ras pathway and constitutively binds to the C-terminal SH3 domain of CrkL, could be detected in HEF1 immunoprecipitates. Therefore, CrkL is involved in the formation of a HEF1-CrkL-C3G ternary complex in B-cells, suggesting that it is likely to play an important role, allowing the propagation of the stimulation initiated by both BCR and beta1 integrin ligation.

Human interleukin-6: detection of 10 attomoles by colorimetric sandwich ELISA using immunopurified polyclonal anti-IL-6 antibodies.

A convenient and sensitive sequential sandwich colorimetric ELISA test was established for quantitating IL-6 in culture supernatants or in serum. Immunopurified HRP-labelled rabbit Fab' fragment was used as the tracer and IgG-coated microtiter plate as the capture antibody. The limit of detection was as low as 10 attomoles of analyte (2.5 pg/ml). Unglycosylated recombinant IL-6 and the natural glycosylated cytokine were recognized equally. In addition, IL-6 measurements were unaffected by the presence of various cytokines and assay sensitivity was only slightly reduced in the presence of undiluted serum samples. The technique was applied to the study of in vitro IL-6 production from activated monocytes and to the in vivo determination of IL-6 in various pathological states.

CD3-stimulated Jurkat T cells mediate IL-1 beta production in monocytic THP-1 cells. Role of LFA-1 molecule and participation of CD69 T cell antigen.

In this study we investigated the T cell signals required for monocyte activation. We used an in vitro co-culture system involving two human cell lines: Jurkat T cells and THP-1 monocytes. Monocyte activation was monitored by measuring IL-1 beta production, whereas IL-2 secretion reflected Jurkat activation. We showed that CD-3 -stimulated Jurkat cells delivered an IL-1-inductive signal to THP-1 cells through a cellular contact which was independent of THP-1 Fc receptors cross-linking. Stimulation of IL-1 beta production did not appear to require lymphokine secretion by T cell since a lymphokine defective mutant of Jurkat cell was able to deliver the stimulatory signal. The LFA-1 molecule was clearly shown to participate in the cooperation process, but its role was likely to be restricted to mediating initial adhesive interaction rather than to transducing the IL-1 -inductive signal. Interestingly, the co-culture stimulated by (Fab')2 fragments of CD3 mAb displayed an enhanced IL-1 beta production without any increase of IL-2 secretion. This result indicated that Jurkat cells could stimulate THP-1 cells even when they were only partially activated. The kinetics and conditions of IL-1 beta production called our attention to the early T cell activation antigen CD69. We then showed that CD69 mAb interfered with transmission of the IL-1 inductive signal (40-50% inhibition of IL-1 production). Our results are suggestive of a new role for CD69 molecule intervening in the T lymphocyte-dependent monocyte activation process.

Effects of long duration spaceflight on human T lymphocyte and monocyte activity.

Experiments were performed on blood samples from 5 cosmonauts in order to investigate the effects of long duration spaceflight (26 to 166 days) on immune activity. The experiments were performed on cultured mononuclear cells purified from blood samples collected during the preflight period and 24 h after landing. The production of interleukin 2, which is the major cytokine involved in T lymphocyte proliferation, was found to be enhanced after flight in some individuals, whereas the ability of mitogen-stimulated cells to express interleukin 2 receptor was impaired 24 h after flight for two cosmonauts out of five. Normal interleukin 2 receptor expression was obtained in all cases when lymphocytes were directly activated by a protein kinase C activating phorbol ester. On the other hand, no significant changes were observed in interleukin 1 production by cultured peripheral blood mononuclear cells. Lastly, the distribution of T lymphocytes subsets was examined in peripheral blood sampled 24 h after landing and was found to be within normal values.

LAMTOR1 depletion induces p53-dependent apoptosis via aberrant lysosomal activation.

Lysosomal regulation is a poorly understood mechanism that is central to degradation and recycling processes. Here we report that LAMTOR1 (late endosomal/lysosomal adaptor, MAPK and mTOR activator 1) downregulation affects lysosomal activation, through mechanisms that are not solely due to mTORC1 inhibition. LAMTOR1 depletion strongly increases lysosomal structures that display a scattered intracellular positioning. Despite their altered positioning, those dispersed structures remain overall functional: (i) the trafficking and maturation of the lysosomal enzyme cathepsin B is not altered; (ii) the autophagic flux, ending up in the degradation of autophagic substrate inside lysosomes, is stimulated. Consequently, LAMTOR1-depleted cells face an aberrant lysosomal catabolism that produces excessive reactive oxygen species (ROS). ROS accumulation in turn triggers p53-dependent cell cycle arrest and apoptosis. Both mTORC1 activity and the stimulated autophagy are not necessary to this lysosomal cell death pathway. Thus, LAMTOR1 expression affects the tuning of lysosomal activation that can lead to p53-dependent apoptosis through excessive catabolism.

Measles virus assembly within membrane rafts.

During measles virus (MV) replication, approximately half of the internal M and N proteins, together with envelope H and F glycoproteins, are selectively enriched in microdomains rich in cholesterol and sphingolipids called membrane rafts. Rafts isolated from MV-infected cells after cold Triton X-100 solubilization and flotation in a sucrose gradient contain all MV components and are infectious. Furthermore, the H and F glycoproteins from released virus are also partly in membrane rafts (S. N. Manié et al., J. Virol. 74:305-311, 2000). When expressed alone, the M but not N protein shows a low partitioning (around 10%) into rafts; this distribution is unchanged when all of the internal proteins, M, N, P, and L, are coexpressed. After infection with MGV, a chimeric MV where both H and F proteins have been replaced by vesicular stomatitis virus G protein, both the M and N proteins were found enriched in membrane rafts, whereas the G protein was not. These data suggest that assembly of internal MV proteins into rafts requires the presence of the MV genome. The F but not H glycoprotein has the intrinsic ability to be localized in rafts. When coexpressed with F, the H glycoprotein is dragged into the rafts. This is not observed following coexpression of either the M or N protein. We propose a model for MV assembly into membrane rafts where the virus envelope and the ribonucleoparticle colocalize and associate.

High expression of the antigen recognized by the monoclonal antibody GB24 on human breast carcinomas: a preventive mechanism of malignant tumor cells against complement attack?

GB24 is a mouse monoclonal antibody raised against a common trophoblast-lymphocyte cross-reactive antigen. GB24 detects the membrane cofactor protein (MCP, CD46), a member of the complement regulatory protein family, which serves as a cofactor for factor 1 mediated cleavage of C3b. This study investigated the reactivity of GB24 on 38 breast carcinomas and 34 normal/benign breast tissues by immunochemistry as well as the reactivity of F2B7-2, an antibody specific to the decay accelerating factor (DAF, CD55) of the complement. GB24 staining was present on both normal tissue and benign lesions, but very strong diffuse reactivity was observed on carcinomas. This reactivity increased with the tumor grade. By contrast, malignant tumor cells lacked DAF expression. F2B7-2 antibody reacted weakly with benign epithelial cells. Results were studied by computer assisted image analysis to accurately define the mean optical densities. The densitometric analysis of MCP positive carcinomas showed a high intensity of the staining. Expression of MCP and DAF on MCF-7 cell lines was analyzed by flow cytometry. MCF-7 cell lines were strongly stained by mAb GB24 only. These data suggest that selectively enhanced expression of the antigen recognized by GB24 is associated with malignant breast disorders. This high expression, which may reflect a protective mechanism by which tumor cells survive complement activation, may prove useful as a marker of malignant transformation.

Suppressive effect of T cell proliferation via the CD29 molecule. The CD29 mAb 1 "K20" decreases diacylglycerol and phosphatidic acid levels in activated T cells.

We had previously reported that the CD29 mAb "K20," presented in a soluble form, blocks peripheral T cell proliferation/activation induced by a CD3 mAb. To better characterize the negative signal delivered by soluble K20, we have investigated its effects on the phospholipid metabolism, both in Jurkat and CD4+ T cells. In CD3-activated T cells, K20 inhibited the increase of diacylglycerol (DAG) and phosphatidic acid levels, but did not modify phosphatidylinositol 4,5-bisphosphate levels, cytosolic Ca2+ raise, and inositolphosphates formation, indicating that K20 did not inhibit phosphatidylinositol 4,5-bisphosphate hydrolysis by phospholipase C-gamma. Moreover, in these conditions, K20 increased phosphatidylethanolamine levels, without variation of phosphatidylcholine, phosphatidylserine, and phosphatidylinositol, suggesting that K20 specifically increased the phosphatidylethanolamine biosynthesis from DAG. Thus, the effects of K20 on DAG and phosphatidic acid levels resulted from an accelerated catabolism rather than from a defect of synthesis. That K20 acts solely at an early step of T cell activation, namely before the binding of IL-2 to its receptor, is supported by the observation that adding exogenous rIL-2 increased proliferation in spite of K20. These results suggest that the beta 1 integrin molecules interact with the membrane phospholipid metabolism and they appear to be the hallmark of a peculiar negative pathway of T cell activation, likely to play an important regulatory role mediated via the T cell integrin molecules.

Inhibition of phorbol ester-induced cell activation in microgravity.

T lymphocytes and monocytes were exposed to microgravity and activated to produce interleukin 2 and interleukin 1, respectively. When Jurkat T cells were triggered with monoclonal antibodies directed against the CD3/T cell receptor complex in the presence of THP-1 monocytes used as accessory cells, cell-to-cell contacts took place in microgravity leading to normal production of interleukin 2 and interleukin 1, as compared to ground controls. In contrast, when cells were individually stimulated by soluble substances including a protein kinase C activating phorbol ester, the production of interleukin 1 and interleukin 2 was dramatically inhibited during microgravity exposure. This result indicates that microgravity may affect the cellular target of phorbol ester.

Disruption of microtubule network in human monocytes induces expression of interleukin-1 but not that of interleukin-6 nor tumor necrosis factor-alpha. Involvement of protein kinase A stimulation.

We have reported recently that colchicine and other microtubule-disrupting agents stimulated interleukin-1 (IL-1) alpha and beta synthesis in human monocytes. In this study we found that unexpectedly colchicine failed to stimulate the expression of two other potent immune mediators, namely tumor necrosis factor-alpha and interleukin-6. Remarkably, taxol which induces stable microtubule bundles, antagonized the colchicine but not the LPS-induced IL-1 synthesis. These results suggested that the colchicine-mediated IL-1 induction was generated by microtubule disassembly. We next demonstrated that microtubule disruption triggered an elevation of intracellular levels of cAMP and a subsequent stimulation of protein kinase A. The use of different protein kinase inhibitors supported a role of the PKA, but not the PKC, in the colchicine-induced IL-1 production. Furthermore, elevation of intracellular cAMP levels by 8-bromo-cAMP, forskolin, or cholera toxin potentiated the effect of suboptimal concentration of colchicine on IL-1 synthesis. However, these agents alone were unable to induce IL-1 synthesis. Therefore, our data indicate that the cAMP/protein kinase A-signaling pathway is necessary but not sufficient to generate IL-1 synthesis by microtubule disruption. Thus, microtubule-disrupting drugs appear as useful tools to further characterize the molecular events which regulate IL-1 production in human monocytes.

[VLA and alpha6, beta4 integrins. Expression in normal and neoplastic human tissues]. / Les integrines VLA et alpha6beta4 expression sur les tissus humains normaux et neoplasiques.

Among the cellular adhesion molecules, the integrin family, more particularly the VLA (Very late antigen) integrins, is currently the subject of numerous investigations in pathology. These integrins are involved in cell-cell contact or cell-matrix adhesions. During neoplastic diseases, cellular expression of integrins changes and a study of the modifications could allow a new etiopathogenic approach carcinogenesis and metastatic phenomena. New prognostic factors may be defined in tumor pathology. We describe the general structure of integrins and the mechanisms of their binding with matricial ligands and with cytoskeleton. The expression of VLA integrins and the alpha6beta4 heterodimer on normal and neoplastic human tissues is then described. Finally, we describe the involvement of these proteins in tumor progression and tissue invasion.

Measles virus structural components are enriched into lipid raft microdomains: a potential cellular location for virus assembly.

The process of measles virus (MV) assembly and subsequent budding is thought to occur in localized regions of the plasma membrane, to favor specific incorporation of viral components, and to facilitate the exclusion of host proteins. We demonstrate that during the course of virus replication, a significant proportion of MV structural proteins were selectively enriched in the detergent-resistant glycosphingolipids and cholesterol-rich membranes (rafts). Isolated rafts could infect the cell through a membrane fusion step and thus contained all of the components required to create a functional virion. However, they could be distinguished from the mature virions with regards to density and Triton X-100 resistance behavior. We further show that raft localization of the viral internal nucleoprotein and matrix protein was independent of the envelope glycoproteins, indicating that raft membranes could provide a platform for MV assembly. Finally, at least part of the raft MV components were included in the viral particle during the budding process. Taken together, these results strongly suggest a role for raft membranes in the processes of MV assembly and budding.

CD46 (membrane cofactor protein) associates with multiple beta1 integrins and tetraspans.

The tetraspans associate with a large number of surface molecules, including a subset of beta1 integrins and, indirectly through CD19, with the complement receptor CD21. To further characterize the tetraspan complexes we have raised and selected monoclonal antibodies (mAb) for their ability to immunoprecipitate a molecule associated with CD9. A unique mAb was identified which recognizes the complement regulator CD46 (membrane cofactor protein). CD46 associated in part with several tetranspans and with all beta1 integrins that were tested (CD29/CD49a, CD29/CD49b, CD29/CD49c, CD29/CD49e, CD29/CD49f) but not with beta4 integrins. These data, together with cross-linking experiments showing the existence in living cells of CD46/integrin complexes, suggest that CD46 associates directly with beta1 integrins and indirectly with tetraspans. CD46 also acts as a receptor for measles virus; however, mAb to various integrins and tetraspans did not modify the virus fusion entry step.

Nonstructural C protein is required for efficient measles virus replication in human peripheral blood cells.

The P gene of measles virus (MV) encodes the phosphoprotein, a component of the virus ribonucleoprotein complex, and two nonstructural proteins, C and V, with unknown functions. Growth of recombinant MV, defective in C or V expression, was explored in human peripheral blood mononuclear cells (PBMC). The production of infectious recombinant MV V- was comparable to that of parental MV tag in simian Vero fibroblasts and in PBMC. In contrast, MV C- progeny was strongly reduced in PBMC but not in Vero cells. Consistently, the expression of both hemagglutinin and fusion proteins, as well as that of nucleoprotein mRNA, was lower in MV C--infected PBMC. Thus, efficient replication of MV in natural host cells requires the expression of the nonstructural C protein. The immunosuppression that accompanies MV infection is associated with a decrease in the in vitro lymphoproliferative response to mitogens. MV C- was as potent as MV tag or MV V- in inhibiting the phytohemagglutinin-induced proliferation of PBMC, indicating that neither the C protein nor the V protein is directly involved in this effect.

Inefficient measles virus budding in murine L.CD46 fibroblasts.

Infection of mouse L.CD46 fibroblasts with measles virus resulted in a poor virus yield, although no defects in the steps of virus binding, entry or fusion, were detected. Two days post-infection, the level of expression of the viral F protein was found to be similar on the surface of infected L.CD46 and HeLa cells using a virus multiplicity enabling an equal number of cells to be infected. After immunofluorescence labelling and confocal microscopy, L.CD46 cells also displayed a significant increase in the co-localisation of the N protein with the cell surface H and F proteins. Immunogold labelling and transmission electron microscopy demonstrated the accumulation of numerous nucleocapsids near the plasma membrane of L. CD46 cells with little virus budding, in contrast to infected HeLa cells which displayed fewer cortical nucleocapsids and more enveloped viral particles. Purified virus particles from infected L. CD46 contained a reduced amount of H, F and M protein. Altogether, these data indicate that, in L.CD46 cells, the late stage of measles virus assembly is defective. This cellular model will be helpful for the identification of cellular factors controlling measles virus maturation.

Stimulation of human interleukin 1 production and specific mRNA expression by microtubule-disrupting drugs.

The production of interleukin 1 (IL1), a pleiotropic monocyte-derived interleukin, can be induced in vitro by various stimuli. The present study shows that cytochalasins which inhibit actin filament polymerization in various cell types have no significant effect on IL1 production from human monocytic cells. On the contrary, microtubule disrupters such as colchicine, vinblastine, and vincristine dramatically potentiate (15- to 35-fold), in a dose-dependent fashion, cell-associated IL1 and to a lesser extent (2.5- to 7-fold) released IL1 in the myelomonocytic THP1 cell line and in adherent peripheral blood mononuclear cells. The enhancing effect of the drugs was blocked by actinomycin D and by cycloheximide and was accompanied by an increase of specific IL1 beta mRNA expression as measured by Northern blot analysis, thus indicating that these drugs act at a transcriptional or post-transcriptional IL1 gene expression level.