Chemokines and chemokine receptors blockers as new drugs for the treatment of chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is characterised by an abnormal inflammatory response of the lung to noxious particles or gases. The cellular inflammatory response in COPD is characterised by an increased number of inflammatory cells in the lungs. Although the molecular and cellular mechanisms responsible for the development of COPD are not well understood; several mediators are assumed to regulate the activation and recruitment of these inflammatory cells into the lung of COPD patients particularly those belonging to the chemokine family. Inhibitors or blockers of chemokine and chemokine receptors are therefore of great interest as potential novel therapies for COPD and many are now in clinical development. A high degree of redundancy exists in the chemokine network and inhibition of a single chemokine or receptor may not be sufficient to block the inflammatory response. Despite this, animal studies suggest a strong rationale for inhibiting the chemokine network in COPD. As such, every leading pharmaceutical company maintains a significant interest in developing agents that regulate leukocyte navigation as potential anti-inflammatory drugs. Drugs and antibodies targeting chemokines and their receptors are generally still in early stages of development and the results of clinical trial are awaited with great interest. These agents may not only provide improved management of COPD but also, importantly, indicate proof-of-concept to further clarify the role of chemokines in the pathophysiology of COPD.

Pharmacological and dietary antioxidant therapies for chronic obstructive pulmonary disease.

The progression and exacerbations of chronic obstructive pulmonary disease (COPD) are intimately associated with tobacco smoke/biomass fuel-induced oxidative and aldehyde/carbonyl stress. Alterations in redox signaling proinflammatory kinases and transcription factors, steroid resistance, unfolded protein response, mucus hypersecretion, extracellular matrix remodeling, autophagy/apoptosis, epigenetic changes, cellular senescence/aging, endothelial dysfunction, autoimmunity, and skeletal muscle dysfunction are some of the pathological hallmarks of COPD. In light of the above it would be prudent to target systemic and local oxidative stress with agents that can modulate the antioxidants/ redox system or by boosting the endogenous levels of antioxidants for the treatment and management of COPD. Identification of various antioxidant agents, such as thiol molecules (glutathione and mucolytic drugs, such as N-acetyl-L-cysteine, N-acystelyn, erdosteine, fudosteine, ergothioneine, and carbocysteine lysine salt), dietary natural product-derived polyphenols and other compounds (curcumin, resveratrol, green tea catechins, quercetin sulforaphane, lycopene, acai, alpha-lipoic acid, tocotrienols, and apocynin) have made it possible to modulate various biochemical aspects of COPD. Various researches and clinical trials have revealed that these antioxidants can detoxify free radicals and oxidants, control expression of redox and glutathione biosynthesis genes, chromatin remodeling, and ultimately inflammatory gene expression. In addition, modulation of cigarette smoke-induced oxidative stress and related cellular changes have also been reported to be effected by synthetic molecules. This includes specific spin traps like α-phenyl-N-tert-butyl nitrone, a catalytic antioxidant (ECSOD mimetic), porphyrins (AEOL 10150 and AEOL 10113), and a superoxide dismutase mimetic M40419, lipid peroxidation and protein carbonylation blockers/inhibitors, such as edaravone and lazaroids/tirilazad, myeloperoxidase inhibitors, as well as specialized pro-resolving mediators/inflammatory resolving lipid mediators, omega-3 fatty acids, vitamin D, and hydrogen sulfide. According to various studies it appears that the administration of multiple antioxidants could be a more effective mode used in the treatment of COPD. In this review, various pharmacological and dietary approaches to enhance lung antioxidant levels and beneficial effects of antioxidant therapeutics in treating or intervening the progression of COPD have been discussed.

A possible overwintering mechanism for bluetongue virus in the absence of the insect vector.

Bluetongue virus (BTV) and several other Orbivirus species are transmitted between mammalian hosts via bites from adults of certain species of Culicoides midges. However, BTV can survive for 9-12 months (typically during the winter), in the absence of adult vectors, with no detectable cases of viraemia, disease or seroconversion in the host. The survival of the virus from one 'vector season' to the next is called 'overwintering' but the mechanism involved is not fully understood. It is demonstrated that BTV can persistently infect ovine gammadelta T-cells in vitro, a process that may also occur during infection and viraemia in mammalian hosts, thus providing a mechanism for virus persistence. Interaction of persistently BTV-infected gammadelta T-cells with antibody to the gammadelta T-cell-specific surface molecule WC-1 resulted in conversion to a lytic infection and increased virus release. Skin fibroblasts induce a similar conversion, indicating that they express a counter ligand for WC-1. Feeding of Culicoides midges induces skin inflammation, which is accompanied by recruitment of large numbers of activated gammadelta T-cells. The interaction of persistently infected gammadelta T-cells with skin fibroblasts would result in increased virus production at 'biting sites', favouring transmission to the insect vector. This suggested mechanism might also involve up-regulation of the WC-1 ligand at inflamed sites. It has been shown previously that cleavage of virus surface proteins by protease enzymes (which may also be associated with inflammation) generates infectious subvirus particles that have enhanced infectivity (100 times) for the insect vector.

Ligation of the WC1 receptor induces gamma delta T cell growth arrest through fumonisin B1-sensitive increases in cellular ceramide.

Ceramide is a powerful regulator of cell fate, inducing either apoptosis or growth arrest. We have previously shown that an Ab to the gammadelta T cell-specific orphan receptor, WC1, is able to induce growth arrest in proliferating IL-2-dependent gammadelta T cells. We now show that this WC1-mediated growth arrest is associated with an increase in cellular ceramide, in the absence of any measurable changes in acidic/neutral sphingomyelinase activity. Moreover, cell-permeable analogues of ceramide also mimicked WC1-induced growth arrest along with an associated decrease in pocket protein expression and phosphorylation status. An important role for ceramide in WC1-induced growth arrest was confirmed by demonstrating that the specific ceramide synthase inhibitor fumonisin B1 blocked WC1-induced growth arrest and the associated molecular effects on the pocket proteins. Finally, we observed constitutive expression of both antiapoptotic factors bcl-2 and bcl-X, the former having increased expression upon WC1 stimulation. It is therefore proposed that ligation of WC1 leads to an accumulation in cellular ceramide through activation of ceramide synthase. This in turn results in a decreased overall expression of the pocket proteins pRb and p107, their hypophosphorylation, and an eventual growth arrest of the gammadelta T cell. To our knowledge, these results demonstrate for the first time that cell surface receptor-mediated ceramide synthase activation can affect cell fate through increases in cellular ceramide and provide further evidence that the orphan receptor WC1 regulates gammadelta T cell biology through a novel signaling pathway.

Transcription factor E2F controls the reversible gamma delta T cell growth arrest mediated through WC1.

IL-2-stimulated expansion of T cells requires continued and sequential passage of the dividing cells through a major cell cycle check point in the G1 phase. We have previously shown that a gamma delta T cell-specific surface receptor, WC1, induces G0/G1 growth arrest, reversible with Con A, in proliferating IL-2-dependent gamma delta T cells. We now show that this reversible WC1-induced cell cycle arrest is correlated with induction of the cyclin kinase inhibitor p27kip1 and an associated down-regulation in cyclins A, D2, and D3 expression, along with dephosphorylation of pocket proteins p107, p130, and pRb. Together with diminished pocket protein phosphorylation, p107 expression levels are significantly down-regulated in response to WC1 stimulation. This coordinated sequence of signaling events is focused on E2F regulation so that, downstream of the pocket proteins, WC1 stimulation results in a diminished DNA binding activity for free E2F as a consequence of reduced E2F1 expression, whereas E2F4 expression is unaffected. Consistent with this interpretation, overexpression of E2F1 overcomes the growth-arresting effects induced by WC1 stimulation. Finally, in accordance with our previous observations at both the cellular and molecular level, subsequent mitogen stimulation can reverse all the above changes induced by WC1. These results, focused on E2F regulation, therefore provide a first insight into the effects of both positive (mitogen) and negative (anti-WC1) stimuli on cell cycle control in IL-2-dependent gamma delta T cells.

Growth arrest of gammadelta T cells induced by monoclonal antibody against WC1 correlates with activation of multiple tyrosine phosphatases and dephosphorylation of MAP kinase erk2.

WC1 is a 215-kDa type 1 transmembrane glycoprotein, the expression of which is restricted to gammadelta T lymphocytes. The binding of an anti-WC1 monoclonal antibody (mAb) (SC-29) induces reversible growth arrest in proliferating interleukin (IL)-2-dependent gammadalta T lymphocytes and this study has examined the relevant biochemical mechanisms. WC1 binding activates multiple protein tyrosine phosphatases causing specific tyrosine dephosphorylation in the absence of calcium mobilization. One of the dephosphorylated proteins was identified as the MAP kinase erk2. Another phosphotyrosine protein of 70 kDa, found to coprecipitate with p85 phosphoinositol (PI)3-kinase was either dephosphorylated or uncoupled from the p85 PI 3-kinase immunoprecipitate after WC1 receptor binding by mAb SC-29. The anti-WC1-induced tyrosine dephosphorylation was reversed by stimulation of gammadelta T cells with concanavalin A or anti-CD3 mAb, demonstrating that at the biochemical level, mitogen stimulation is dominant to the growth-arresting effects of anti-WC1. It is therefore proposed that the activation of tyrosine phosphatases by WC1 binding and the resultant dephosphorylation of certain key signaling protein such as erk2 correlates with and may cause the induction of growth arrest in IL-2-dependent gammadelta T cells, without affecting the cells ability to respond to antigen. Possible mechanisms, which include the inhibition of IL-2 signal transduction pathways, are discussed.

A gamma delta T cell specific surface receptor (WC1) signaling G0/G1 cell cycle arrest.

Three monoclonal antibodies (mAb; SC-6, SC-12, and SC-29) reactive with the gammadelta T cell-restricted antigen WC1 were obtained immunizing mice with an ovine interleukin (IL)-2-dependent gammadelta T cell line. These mAb strongly inhibited DNA synthesis in IL-2-dependent gammadelta T cell lines with cell cycle arrest in G0/G1 phase, but did not induce apoptosis. The mAb-induced growth arrest was reversible, either by removing the mAb or by co-culture with mitogen or anti-CD3 in the presence of IL-2. In contrast, addition of phorbol ester, ionomycin and IL-2 had no effect on the mAb-induced growth arrest. The observations define a biologically important role for the cell surface molecule WC1 in the regulation of gammadelta T cell proliferation and also provide a suitable system to study the relevant signal transduction events.

Preparation of monoclonal anti-porcine CD3 antibodies and preliminary characterization of porcine T lymphocytes.

The CD3-T-cell receptor complex is the clonotypic surface structure by which T lymphocytes recognize foreign antigens and are subsequently activated. Because of the low immunogenicity of the CD3 molecules, anti-CD3 monoclonal antibodies (mAb) are difficult to prepare and have not been available in several species. Following isolation of porcine CD3, 14 anti-porcine CD3 mAb were prepared, which define six groups of CD3-epsilon epitopes, coprecipitate two types of TCR and reveal considerable heterogeneity of CD3 expression amongst lymphocyte subpopulations. Thus, both CD3 positive and negative subpopulations of CD2 or CD8 positive cells were found in the blood. The density of CD3 on CD2+ or CD8+ cells was relatively low and heterogeneous, whereas the CD2-, CD8- or MAC320+ T cells expressed CD3 at a higher and more homogeneous level. Finally, in the thymus, staining with anti-CD3 resolved large thymocytes into two subsets: one expressing a high level of CD3 and the other being negative. In contrast, small thymocytes expressed CD3 at a low and more homogeneous level. Immunohistological studies confirmed the presence of clearly detectable CD3 in thymus medulla and the T-cell regions of peripheral lymphoid tissues. Most of the mAb were mitogenic, when presented to peripheral blood mononuclear cells in immobilized form. The anti-CD3 mAb also induced redirected cytotoxicity which was shown to be Fc receptor dependent.

Porcine CD3 epsilon: its characterization, expression and involvement in activation of porcine T lymphocytes.

The cloning, characterization and expression of porcine CD3 epsilon and establishment of its role in T-cell activation using an anti-porcine CD3 epsilon monoclonal antibody, as described here, provides a first step towards a greater understanding of the porcine immune response. Porcine CD3 epsilon was cloned from a porcine T-cell cDNA library by polymerase chain reaction and found to have up to 72% identity with other CD3 epsilon chains, retaining all the necessary primary structural motifs for correct functioning of porcine CD3 epsilon. When expressed in COS7 cells porcine CD3 epsilon was an intracellularly localized, monomeric 23,000 MW protein exhibiting no evidence of N-glycosylation. A monoclonal antibody, PPT3, recognized expressed porcine CD3 epsilon and activated porcine T cells as demonstrated by stimulation of calcium mobilization, an increase in protein tyrosine phosphorylation and proliferation. These results further reaffirm and identify CD3 epsilon as an important cell surface protein involved in signal transduction of activation signals in porcine T cells.

Murine B-cell activation via CD38 and protein tyrosine phosphorylation.

CD38 has been implicated in the regulation of both proliferation and rescue from apoptosis of B cells. The signalling events associated with CD38-mediated activation of murine B cells are, as yet, not well defined but it is clear that ligation of CD38 by a mitogenic antibody, NIMR-5, induces a calcium influx in resting B cells. Interestingly, however, cross-linking of CD38 does not mobilize intracellular stores of calcium. We now provide a rationale for these findings by demonstrating that CD38 is not coupled to the generation of inositol phosphates in resting B cells. We do, however, show that CD38 ligation stimulates one, or more, protein tyrosine kinase activities which may play a central role in the transduction of CD38-mediated signals leading to B-cell activation.

A B lymphocyte surface molecule mediating activation and protection from apoptosis via calcium channels.

A rat mAb (NIM-R5) was prepared against a 42-kD B cell activation Ag (p42). The expression of p42 is increased upon activation. NIM-R5 induces an increase of intracellular Ca2+, due to influx from the exterior milieu via calcium channels. This stimulation does not prejudice further stimulation with anti-Ig, and thus p42 constitutes an activation signal independent of membrane Ig. The antibody induces increased expression of class II molecules on resting B lymphocytes and prepares the cells for "spreading" when interacted with immobilized anti-class II antibody. The antibody alone is weakly mitogenic and comitogenic with IL-4 on resting B cells. Of particular interest, NIM-R5 induces proliferation and rescue from apoptosis in B cells activated in vitro. In conclusion, NIM-R5 induces an Ig-independent activation and proliferation of resting and activated B cells. This antibody does not recognize other known B cell activation Ag such as CD23, CD40, or CD72. We therefore propose that the p42 Ag is a glycoprotein with an important role in the regulation of B lymphocyte activation and survival.

Identification and characterization of the murine homologue of CD22, a B lymphocyte-restricted adhesion molecule.

The human B lymphocyte-specific Ag, CD22, is a cell adhesion molecule expressed on the surface during a narrow window of B cell development, coincident with surface IgD. A ligand for CD22 has recently been identified on human T cells as the low molecular mass isoform of the leukocyte common Ag, CD45RO. CD22 has been reported to function in the regulation of both T and B cell activation in vitro. In this study, we report the isolation and expression of a molecular cDNA clone encoding the murine homologue of CD22, mCD22. Within their predicted protein sequences, murine and human sequences overall have 62% identity, which includes 18 of 20 extracellular cysteines and six of six cytoplasmic tyrosines. BHK cells transfected with mCD22 cDNA specifically adhere to resting and activated T lymphocytes and in addition bound activated, but not resting, B cells. Five Th clones were analyzed for their ability to adhere to mCD22; two Th0 clones and one Th1 clone bound CD22+ BHK transfectants, but not all T cell clones bound CD22+ cells: another Th1 clone and a Th2 clone did not. mCD22+ BHK transfectants were also specifically bound by the B cell-specific mAb, NIM-R6, demonstrating that this mAb is specific for murine CD22. Human cell lines expressing the counter-receptors for human CD22 were also examined for adhesion to the murine CD22 homologue; the epitope responsible for B cell adhesion to CD22 is conserved, whereas the T cell epitope binding to CD22 is not. The cDNA and mAb to murine CD22 will be useful for defining the in vivo function of CD22.

The detection of an early advanced glycation product which co-elutes with the Amadori product on aminophenylboronate affinity chromatography.

The production of an antiserum recognizing an early advanced glycation product of glycated human serum albumin (HSA) is reported. The antiserum was produced with the intention of recognizing the Amadori product, i.e. the monofructosamine derivative, of any glycated protein. In retrospect, however, the immunogen appears to have been transformed in vivo which led to the production of antibodies to an early advanced glycation product. Two-site immunometric and competitive ELISAs showed that the affinity-purified antibodies recognized glycated HSA only after it had been stored for several months. This recognition, by the antibody, was more specific for the transformed product than for the original hapten (1-amino-1-deoxy-D-fructose-6-aminohexanoic acid) used for immunization by a factor of more than 1,000. These antibodies also detected immunoreactive material present in the elution fraction after in vivo glycated HSA had been chromatographed on an aminophenylboronate affinity column, indicating that an early advanced glycation product can co-elute with the Amadori product of glycated HSA on aminophenylboronate affinity chromatography. This suggests that the antiserum recognized an early advanced glycation product that also contained cis-diols as does the Amadori product, and may prove useful in the early detection of clinical complications in diabetic patients.

[Real-time automatic analysis of sleep-waking behavior in the rat recorded by telemetry (author's transl)]. / Analyse automatique en temps réel du comportement veille-sommeil chez le rat enregistré par radiotélémesure.

The authors present a method of real-time automatic analysis of the sleep-waking cycle in the rat recorded by a miniature telemetry system. This method detects seven behavioural phases second by second. The correspondance computer-corrector is 88 p. 100 for the three principal phases: waking, slow sleep and paradoxical sleep.