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.

Electronic properties of the interface between p-CuI and anatase-phase n-TiO2 single crystal and nanoparticulate surfaces: a photoemission study.

We present a study of the growth of the p-type inorganic semiconductor CuI on n-type TiO2 anatase single crystal (101) surfaces and on nanoparticulate anatase surfaces using synchrotron radiation photoemission spectroscopy. Core level photoemission data obtained using synchrotron radiation reveal that both the substrate (TiO2) and the overlayer (CuI) core levels shift to a lower binding energy to different degrees following the growth of CuI on TiO2. Valence band photoemission data show that the valence band maximum of the clean substrate differs from that of the dosed surface which may be interpreted qualitatively as due to the introduction of a new density of states within the band gap of TiO2 as a result of the growth of CuI. The valence band offset for the heterojunction n-TiO2p-CuI has been measured using photoemission for both nanoparticulate and single crystal TiO2 surfaces, and the band energy alignment for these heterojunction interfaces is presented. With the information obtained here, it is suggested that the interface between p-CuI and single crystal anatase-phase n-TiO2 is a type-II heterojunction interface, with significant band bending. The measured total band bending matches the work function change at the interface, i.e., there is no interface dipole. In the case of the nanoparticulate interface, an interface dipole is found, but band bending within the anatase nanoparticles remains quite significant. We show that the corresponding depletion layer may be accommodated within the dimension of the nanoparticles. The results are discussed in the context of the functional properties of dye-sensitized solid state solar cells.

Oxidative stress and macrophage function: a failure to resolve the inflammatory response.

The suppression of pro-inflammatory gene expression along with the clearance of apoptotic cells by phagocytosis can play an important role in resolving the inflammatory response. Any impairment of these processes can therefore lead to a chronic inflammatory state. Oxidative stress can have both direct and indirect effects on macrophage function. This mini-review highlights a mechanism through which oxidative stress via the production of reactive carbonyls alters the ECM (extracellular matrix) environment of macrophages, thereby altering their behaviour. Carbonyl modification of ECM proteins causes increased macrophage adhesion and activation through receptors that are also involved in phagocytosis. Moreover, interaction of macrophages with these carbonyl-modified ECM proteins leads to decreased phagocytic activity towards apoptotic cells. At a more direct level, both oxidative and carbonyl stress inhibits activity of the transcriptional co-repressor HDAC-2 (histone deacetylase 2), which under normoxic conditions helps to suppress pro-inflammatory gene expression. Consequently, macrophages activated under conditions of oxidative or carbonyl stress can lead to a more enhanced inflammatory response. Coupled with an impairment of the phagocytic response, this can lead to ineffective clearance of apoptotic cells and secondary necrosis, with the result being failure to resolve the inflammatory response and the establishment of a chronic inflammatory state.

Mapping of SOMU1 and M1 epitopes on the apomucin encoded by the 5' end of the MUC5AC gene.

We have developed 11 monoclonal antibodies (MAbs) against human gastric mucin, (1-13M1, 2-11M1, 2-12M1, 9-13M1, 58M1, 19M1, 21M1, 45M1, 463M, 589M, 62M1), which specifically stained by immunohistochemisty both the human gastric surface mucosa and colon adenoma. Among them, five (19M1, 21M1, 463M, 589M, 62M1) immunoreacted with the peptide encoded by the 3' region of the MUC5AC gene (Nollet et al: Int J Cancer 2002;99:336-343). In this study, we identified in the 5' region of this gene the nucleotide fragments encoding peptides immunoreacting with three other anti-M1 MAbs (1-13M1, 2-11M1 and 9-13M1), as well as the SOMU1 MAb (Sotozono et al: J Immunol Methods 1996;192:187-196). 1-13M1 MAb immunoreacts with peptides, including the Cys 2 and Cys 4 domains. The SOMU1 MAb recognized the Cys 5 domain, and the MAbs 2-11M1 and 9-13M1 the globular D1/D2 and D3 domains, respectively. Using serial sections of the mucosae adjacent to colon adenocarcinomas and colon adenomas, we observed that the anti-M1 and anti-SOMU1 MAbs displayed the same immunostaining patterns. The three anti-M1 MAbs (2-12M1, 58M1, and 45M1) did not react with the products of the MUC5AC gene tested until now. The MUC5AC apomucin is now well characterized by MAbs immunoreacting against seven different epitopes belonging to the different main cystein globular domains of this macromolecule. Such antibodies are useful tools for studying the biosynthesis, polymerization, and degradation of mucin.

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.

IgM heavy chain complementarity-determining region 3 diversity is constrained by genetic and somatic mechanisms until two months after birth.

Due to the greater range of lengths available to the third complementarity determining region of the heavy chain (HCDR3), the Ab repertoire of normal adults includes larger Ag binding site structures than those seen in first and second trimester fetal tissues. Transition to a steady state range of HCDR3 lengths is not complete until the infant reaches 2 mo of age. Fetal constraints on length begin with a genetic predilection for use of short DH (D7-27 or DQ52) gene segments and against use of long DH (e.g., D3 or DXP) and JH (JH6) gene segments in both fetal liver and fetal bone marrow. Further control of length is achieved through DH-specific limitations in N addition, with D7-27 DJ joins including extensive N addition and D3-containing DJ joins showing a paucity of N addition. DH-specific constraints on N addition are no longer apparent in adult bone marrow. Superimposed upon these genetic mechanisms to control length is a process of somatic selection that appears to ensure expression of a restricted range of HCDR3 lengths in both fetus and adult. B cells that express Abs of an "inappropriate" length appear to be eliminated when they first display IgM on their cell surface. Control of N addition appears aberrant in X-linked agammaglobulinemia, which may exacerbate the block in B cell development seen in this disease. Restriction of the fetal repertoire appears to be an active process, forcing limits on the diversity, and hence range of Ab specificities, available to the young.

Towards the design of an antibody that recognises a given protein epitope.

We have explored the possibility of designing repertoires of antibodies complementary to a given protein epitope, specifically the face of the ribonuclease inhibitor barstar that binds to the enzyme barnase. An antibody repertoire was created by mutation of ten residues in the hypervariable loops of a synthetic antibody fragment and displayed on filamentous bacteriophage. The positions of three of the ten residues of the antibody (VL 32, 50 and 94) were chosen to match a triangle of three negative charges on the face of barstar and mutated to favour residues of opposite charge or those with hydrogen-bonding potential. The other seven residues, chosen to allow for variation in the surface of interaction, were mutated at random. One of the antibody fragments isolated after selection of the repertoire (10(8) clones per library) was shown to bind to barstar with an affinity of 1.0x10(-7) M and the binding was competed by barnase. Furthermore, the binding of the antibody to barstar was highly sensitive to mutation of any of five residues of barstar known to contact barnase. This indicates that it may be possible, by a combination of design and selection, to build antibodies to a given epitope.

Tie-over bolster dressings may not be necessary to secure small full thickness skin grafts.

BACKGROUND: The accepted method for securing full thickness skin grafts (FTSG) is with a tie-over bolster dressing, with or without basting sutures. We question the need for tie-over bolster dressings for small FTSGs. OBJECTIVE: We describe our method of FTSG reconstruction and the outcome in a consecutive series. METHODS: Thirty patients with surgical defects following tumour excision from the face24, scalp1, ear2, and finger3 ranging in diameter from 8 to 45 mm (mean 20 mm) were reconstructed with a FTSG. Interrupted monofilament nylon perimeter sutures only were used, with antibiotic ointment at the wound edge with either a light dressing or no dressing. RESULTS: A series of 30 FTSG secured without a tie-over bolster dressing or basting sutures is described, all with good to excellent long term results. In two cases there was early superficial necrosis of the graft, but ultimately 100% graft take. CONCLUSION: We have found tie-over bolster dressings and basting sutures to be unnecessary in our series of small FTSG. This technique saves time and material and minimizes handling of the graft.

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.

Analyses of monoclonal antibodies reacting with porcine CD3: results from the Second International Swine CD Workshop.

Among the 57 monoclonal antibodies (mAb) analyzed within the T-cell group from the Second Swine CD Workshop, six mAb fell within clusters T10 and T11 (No. 088, STH164; No. 148, FY1A3; No. 149, FY2C1; No. 150, FY1H2; No. 151, FY2A11; No. 169, BB23-8E6). The mAb within these two groups gave a similar appearance on flow cytometry and stained all peripheral blood T-cells as defined by CD4 and wCD8 staining. All six mAb precipitated a 24 kDa protein. On the basis of inhibition analyses performed as part of the workshop and from published data, the mAb define at least three epitopes. There is only minimal stimulation of resting peripheral lymphocytes, but four of the mAb produce strong stimulation in the presence of PMA. With the exception of STH164, all have been shown to react with CD3 epsilon-transfected COS cells. The new mAb, therefore, react with three epitopes on porcine CD3 epsilon designated CD3a (BB23-8E6, FY2A11), CD3b (FY1A3, FY2C1), and CD3c (FY1H2). mAb STH164 appears to be reactive with another epitope, however, since its reactivity with CD3 has not been confirmed it is designated as wCD3.

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.

Biophysical methods for the determination of antibody-antigen affinities.

Binding specificities against virtually any antigen can be isolated from antibody libraries displayed on filamentous phage. The determination of antibody-antigen affinity constants and binding kinetics is an important part of antibody characterization, and may be predictive of antibody performance in biotechnological applications. This article, intended as a guideline for the scientist who isolates a novel antibody and wishes to characterize its binding properties, presents the authors' view on widely used methodologies for the quantitative determination of antibody-antigen interactions.

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.

Comparison of two reflectance photometers in the assessment of neonatal hypoglycaemia.

To assess the accuracy and reliability of reflectance photometers in estimating blood glucose concentrations, two were assessed: the Ames Glucometer 3 (Bayer Diagnostics) with Glucofilm Test strips; and the Reflolux S (Boehringer Mannheim) with BM Test Glycemia 20-800 strips. These were compared with laboratory blood glucose estimations in 100 assays (50 comparisons for each machine, measuring the difference (d) between the glucose reading and the mean of the reflectance photometer and the laboratory value). The Ames Glucometer 3 (mean d = + 0.7 mmol/l, (SD 1.1) mmol/l) was less accurate than the Boehringer Reflolux S (mean d = 0.2 mmol/l, (SD (0.7) mmol/l). The range of error of both machines is wide (Ames 2 SD range + 2.9 mmol/l to -1.5 mmol/l true readings; Boehringer + 1.8 mmol/l to -1.2 mmol/l of true readings). Because of this, any reflectance photometer readings that are even slightly low should be checked with laboratory estimations. The clinical value of such machines is limited in infants with low blood glucose concentrations.