Structural and functional features of a cell surface phosphoglycoprotein associated with tumorigenic phenotype in human fibroblast x HeLa cell hybrids

Monoclonal antibodies have been raised against a dimeric cell surface antigen (p75/150) which is specifically associated with the tumorigenic phenotype in human fibroblast X HeLa hybrids. During biosynthesis, a precursor molecule (p70/140), was associated with microsomal membranes in vivo but possessed no detectable cytoplasmic domains. At this stage, each p70 monomer contained 3 "high-mannose" type N-linked glycans which were subsequently processed into endoglycosidase H-insensitive complex oligosaccharides on the mature cell surface forms. Cleavage of this cell surface form with endoglycosidase F yielded non-N-glycosylated polypeptides of Mr = 60,000/120,000. All the monoclonal antibodies identified similar non-N-glycosylated polypeptides in cells grown in the presence of tunicamycin. p75/150 could be weakly labeled with [3H]palmitic or myristic acid. In vivo, p75/150 was found to be phosphorylated on serine residues. Immunoprecipitates of p75/150 from HeLa or tumorigenic hybrid cell lysates exhibited protein kinase activity in vitro, which phosphorylated p75/150 itself, also on serine residues. We were unable to detect this kinase activity in normal fibroblasts and in the nontumorigenic hybrid cells. Furthermore, we were unable to detect p75/150 or its precursors by either cell surface labeling, metabolic labeling, or Western blotting in nontumorigenic cell hybrids; p75/150 thus represents a tumor-specific marker in this system. Tryptic peptides of highly purified p75/150 have been generated, but their amino acid sequences did not reveal any significant homology with previously described proteins.     

Screening for novel human genes associated with CRE pathway activation with cell microarray

In this study, cell microarray technology is used to identify novel human genes associated with CRE pathway activation. By reverse transfection, expression plasmids containing full-length cDNAs were cotransfected with the reporter plasmid pCRE-d2EGFP to monitor the activation of the CRE pathway via enhanced green fluorescence protein (EGFP) expression. Of the 575 predominantly novel genes screened, 22 exhibited relatively higher EGFP fluorescence compared with a negative control. After a functional validation with a dual luciferase reporter system that included both cis- and trans-luciferase assays, 4 of the 22 genes (RNF41, C8orf32, C6orf208, and MEIS3P1) were confirmed as CRE-pathway activators. Western blot analysis revealed that RNF41 can promote CREB phosphorylation. These results demonstrate the successful combination of cell microarray technology with this reporting system and the potential of this tool to characterize functions of novel genes in a highly parallel format.     

An alteration in molecular form associated with activation of human heat shock factor

In higher eucaryotes, heat shock factor (HSF) exists in a cryptic form in unstressed cells. We investigated molecular forms of human HSF before and after activation by sucrose density gradient centrifugation and by gel mobility shift assay using a 32P-labeled heat shock element (HSE). We found that the in vivo or in vitro activated HSF, which is capable of binding to HSE, and its inactive form present in unstressed cells have different sedimentation coefficient; the former is 8 S whereas the latter is 4-5 S. Both the 8 S and 4-5 S forms contain the HSF polypeptide which has the ability to bind to HSE upon activation. The inactive 4-5 S form acquires HSE-binding ability when activated by heat shock or other stimuli. This HSF activity was greatly reduced, however, during recentrifugation in sucrose density gradient and, in addition, the residual activity was not recovered in 8 S fractions. Transformation of the inactive 4-5 S form of HSF to the stable, active 8 S form was achieved when the inactive form was activated and mixed with cytosols of unstressed cells.     

Isolation and partial characterization of a 700 kilodalton human colon carcinoma associated antigen

An antigen of high molecular weight (CA-3) was isolated from the cytosol fraction of GW-39 human colon tumor cells by antibody affinity chromatography. CA-3 was characterized by an acidic pI value of 4.5-4.9, a molecular weight of 700 kilodaltons and a sedimentation coefficient of 13S. It contained all of the commonly occurring amino acids and had an acidic to basic amino acid ratio of 1.4. CA-3 was resistant to dissociation by reducing agents as well as by sodium dodecylsulfate. Quantitation of CA-3 by a radioimmunoassay employing rabbit anti-CA-3 antiserum revealed a marked elevation of CA-3 in the cytosol extracts of human primary colon carcinoma in comparison to normal colon. The molecular properties of CA-3 are compared to those of carcinoembryonic antigen, high molecular weight colon specific antigen CSAp and two other high molecular weight proteins, fibronectin and conglutinin. Colon antigen CA-3 appears to be different from these other molecules in terms of its molecular weight, sedimentation value, isoelectric point and amino acid composition.     

Acquisition of CD80 by human T cells at early stages of activation: functional involvement of CD80 acquisition in T cell to T cell interaction

The interaction between CD28 on T cells and CD80 on APCs intensifies the linkage between TCR and MHC at the site of contact between T cells and APCs. In this study, we demonstrate that during human T cell/human APC interaction, the autologous or allogeneic human CD4(+) T cells become positive for the detection of CD80 at an early stage of activation (24 h). This detection of CD80 is attributable to the acquisition of CD80 from APCs, as opposed to the up-regulation of endogenous CD80, as demonstrated by CD4(+) T cells treated with cyclohexamide. Furthermore, no CD80 mRNA could be detected at 24 h in T cells that had acquired CD80 from APCs. CD80 acquisition by T cells from APCs was enhanced upon TCR engagement. The amount of CD80 acquisition by CD4(+) T cells was shown to be related to the expression of CD80 on APCs. Using soluble fusion proteins (soluble CTLA-4, CD28, and CD80) to block either CD28 on the surface of T cells or CD80 on the surface of APCs, it was demonstrated that CD80 acquisition by T cells is mediated through its receptors, possibly CD28 interaction. Moreover, we demonstrate that T cells that have acquired CD80 have the ability to stimulate other T cells. These data thus suggest that CD80 acquisition by human T cells might play a role in the immunoregulation of T cell responses.     

Mechanisms of thrombin-induced MAPK activation associated with cell proliferation in human cultured tracheal smooth muscle cells

The elevated level of thrombin has been detected in the airway fluids of asthmatic patients. However, the implication of thrombin in the pathogenesis of bronchial hyperreactivity was not completely understood. Therefore, in this study we investigated the effect of thrombin on cell proliferation and p42/p44 mitogen-activated protein kinase (MAPK) activation in human tracheal smooth muscle cells (TSMCs). Thrombin stimulated [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin (PTX) significantly inhibited [3H]thymidine incorporation and phosphorylation of MAPK induced by thrombin. These responses were attenuated by tyrosine kinase inhibitors genistein and herbimycin A, phosphatidyl inositide (PI)-phospholipase C (PLC) inhibitor U73122, protein kinase C (PKC) inhibitor GF109203X, removal of Ca(2+) by addition of BAPTA/AM plus EGTA, and PI 3-kinase inhibitors wortmannin and LY294002. In addition, thrombin-induced [3H]-thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2), indicating that activation of MEK1/2 was required for these responses. Furthermore, overexpression of dominant negative mutants, RasN17 and Raf-301, significantly suppressed p42/p44 MAPK activation induced by thrombin and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. These results conclude that the mitogenic effect of thrombin was mediated through the activation of Ras/Raf/MEK/MAPK pathway. Thrombin-mediated MAPK activation was modulated by PI-PLC, Ca(2+), PKC, tyrosine kinase, and PI 3-kinase associated with cell proliferation in cultured human TSMCs.     

CD80 binding polyproline helical peptide inhibits T cell activation

The critical role played by the CD28/CD152-CD80/CD86 costimulatory molecules in mediating T cell activation and suppression provides attractive targets for therapeutic strategies. CD28 and CD152 share a conserved polyproline motif in the ligand-binding region. Similar proline-rich regions in globular domains preferentially adopt a polyproline type II (PP) helical conformation and are involved in transient (II)protein-protein interactions. Interestingly, in the human CD80-CD152 complex, Pro(102) of CD152 restricts the preceding proline to PP(II) helix in the binding orientation in relation to the shallow binding pocket of CD80. Peptide agents derived from binding sites of receptors that mimic the bioactive conformation have been shown to block receptor-ligand interactions. Contact preferences of the interface amino acids at the protein-protein interaction sites and the propensity of each residue to form PP(II) helix were integrated in the design of novel peptide agents referred to as CD80 competitive antagonist peptides. Structural and functional studies suggest potential therapeutic value for select CD80 competitive antagonist peptides.     

2,3,7,8, tetrachlorodibenzo-p-dioxin induces oxygen activation associated with cell respiration

We have investigated the influence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on bioenergetic functions of isolated heart-mitochondria. Electron transfer and energy conservation activities were found to be decreased in the presence of very low amounts of the polychlorinated biphenyl compound (1.5 nmol/mg mitochondrial protein). The effect was greatest when substrates for complex I were used. In this case coupling of oxidative phosphorylation to respiration was drastically diminished, essentially at the expense of state 3 respiration, and P/O values were found around 2 instead of 3. Succinate-related energy conservation remained practically unaffected in the presence of TCDD, suggesting an interference of the toxic compound at coupling site I. SOD plus catalase were found to protect energy-linked respiration from the effect of dioxin indicating the involvement of superoxide radicals and H2O2 in the development of the observed phenomena. The present contribution provides experimental evidence on the formation of these oxygen species in the presence of TCDD. Furthermore, the site of action of TCDD is demonstrated and discussed in relation to the oxygen radical formation observed.     

A 44 kilodalton cell surface homodimer regulates interleukin 2 production by activated human T lymphocytes

We previously described a cell surface antigen, termed Tp44, detected by monoclonal antibody 9.3 on approximately 80% of mature human T lymphocytes. Analysis by SDS-polyacrylamide gel electrophoresis and isoelectric focusing demonstrated that this antigen consists of two identical 44 kilodalton glycopeptides that form a disulfide-linked homodimer. Competitive binding experiments showed that antibody 9.3 and an anti-CD3 antibody (64.1) recognize distinct antigenic determinants; furthermore, the binding of antibody 9.3 was unaffected by prior modulation of CD3. Thus, Tp44 has no detectable cell surface association with CD3. By itself, antibody 9.3 had no detectable effect on either IL 2 receptor expression or IL 2 release, and did not cause T cell proliferation even when monocytes were present and exogenous IL 2 was provided, indicating that binding of antibody 9.3 does not provide a primary signal for T cell activation. However, the proliferative responses of T lymphocytes activated by phytohemagglutinin, concanavalin A, or an anti-CD3 monoclonal antibody were strikingly enhanced in the presence of antibody 9.3, an effect associated with increased IL 2 receptor expression and increased IL 2 secretion. Antibody 9.3 enabled anti-CD3-Sepharose-activated T cells and anti-CD3 antibody-activated Jurkat cells to release IL 2 in the absence of monocytes. Fab fragments of antibody 9.3 had no effect on anti-CD3-induced IL 2 release by Jurkat cells, whereas F(ab')2 fragments had activity comparable to that of unmodified antibody, indicating that bivalent binding of Tp44 molecules is required for IL 2 secretion. Together, these results suggest that TP44 may function as a receptor for accessory signals in the activation of T cells.     

Streptococcus group B--association with Aerobic vaginitis and ability to human cell lines activation

The aim of this study was to estimate: the frequency of aerobic vaginitis, susceptibility of the GBS isolated from vagina of non-pregnant women with and without cervicitis to selected antibiotics and chemotherapeutics and the proinflammatory cytokines production by HeLa, THP-I, U - 937 cells after stimulation by vaginal GBS. Our results indicated low frequency of the aerobic vaginitis -4.5% among non-pregnant young women and ability of the vaginal GBS to release proinflammatory cytokines by human cell lines in vitro.     

Changes in cytokine levels and NK cell activation associated with influenza

Several studies have highlighted the important role played by murine natural killer (NK) cells in the control of influenza infection. However, human NK cell responses in acute influenza infection, including infection with the 2009 pandemic H1N1 influenza virus, are poorly documented. Here, we examined changes in NK cell phenotype and function and plasma cytokine levels associated with influenza infection and vaccination. We show that absolute numbers of peripheral blood NK cells, and particularly those of CD56(bright) NK cells, decreased upon acute influenza infection while this NK cell subset expanded following intramuscular influenza vaccination. NK cells exposed to influenza antigens were activated, with higher proportions of NK cells expressing CD69 in study subjects infected with seasonal influenza strains. Vaccination led to increased levels of CD25+ NK cells, and notably CD56(bright) CD25+ NK cells, whereas decreased amounts of this subset were present in the peripheral blood of influenza infected individuals, and predominantly in study subjects infected with the 2009 pandemic H1N1 influenza virus. Finally, acute influenza infection was associated with low plasma concentrations of inflammatory cytokines, including IFN-γ, MIP-1β, IL-2 and IL-15, and high levels of the anti-inflammatory cytokines IL-10 and IL-1ra. Altogether, these data suggest a role for the CD56(bright) NK cell subset in the response to influenza, potentially involving their recruitment to infected tissues and a local production and/or uptake of inflammatory cytokines.     

Preterm labor and chorioamnionitis are associated with neonatal T cell activation

Background

Preterm parturition is characterized by innate immune activation and increased proinflammatory cytokine levels. This well established association leads us to hypothesize that preterm delivery is also associated with neonatal T lymphocyte activation and maturation.

Methodology/Principal Findings

Cord blood samples were obtained following term, preterm, and deliveries complicated by clinical chorioamnionitis. Activation marker expression was quantitated by flow cytometric analysis. Infants born following preterm delivery demonstrated enhanced CD4⁺ T lymphocyte activation, as determined by CD25 (Term 9.72% vs. Preterm 17.67%, p = 0.0001), HLA-DR (Term 0.91% vs. Preterm 1.92%, p = 0.0012), and CD69 expression (Term 0.38% vs. Preterm 1.20%, p = 0.0003). Neonates delivered following clinical chorioamnionitis also demonstrated increased T cell activation. Preterm neonates had an increased frequency of CD45RO⁺ T cells.

Conclusion/Significance

Preterm parturition is associated with neonatal CD4⁺ T cell activation, and an increased frequency of CD45RO⁺ T cells. These findings support the concept that activation of the fetal adaptive immune system in utero is closely associated with preterm labor.     

Membrane dynamic alterations associated with activation of human platelets by thrombin

Two fluorescent probes, N-carboxymethylisatoic anhydride, which binds to membrane proteins, and 1,6-diphenyl-1,3,5-hexatriene, a lipophilic label, have been used to follow membrane microenvironmental changes. Activation of human platelets by thrombin resulted in a simultaneous increase in values of fluorescence polarization (P) of both probes during the stages of shape change and secretion, which further increased during platelet aggregation. The similar pattern of changes in P for both probes indicates the interdependence of lipids and proteins in the activated platelet membrane.     

Mechanisms of human cell protection associated with genetic polymorphism

Systems ensuring protection of human cells against endogenous and exogenous mutagenic factors are considered in terms of genetic polymorphism. Some protection mechanisms are described, including those connected with capturing free radicals, biotransformation of xenobiotics, excision repair of DNA damage (excision of nitrous bases, nucleotides, mismatch repair). A special section is devoted to some issues of using antimutagens in context of genetic polymorphism. The problem of adaptive response is discussed, providing evidence for independence (in some cases) of DNA repair systems and the formation of adaptive response. Some results of the author obtained many years ago but still relevant are presented.     

Mechanisms of human cell protection associated with genetic polymorphism

Systems ensuring protection of human cells against endogenous and exogenous mutagenic factors are considered in terms of genetic polymorphism. Some protection mechanisms are described, including those connected with capturing free radicals, biotransformation of xenobiotics, excision repair of DNA damage (excision of nitrous bases, nucleotides, mismatch repair). A special section is devoted to some issues of using antimutagens in context of genetic polymorphism. The problem of adaptive response is discussed, providing evidence for independence (in some cases) of DNA repair systems and the formation of adaptive response. Some results of the author obtained many years ago but still relevant are presented.__________Translated from Genetika, Vol. 41, No. 4, 2005, pp. 520–535.Original Russian Text Copyright © 2005 by Zasukhina.     

Low-level CD4+ T cell activation is associated with low susceptibility to HIV-1 infection

Different features have been associated with low susceptibility to HIV type 1 (HIV-1) infection in exposed seronegative individuals. These include genetic make-up such as homozygosity for the CCR5-Delta32 allele and the presence of HIV-specific CTLs. We studied immune activation and immune responsiveness in relation to HIV-1 susceptibility in 42 high-risk seronegative (HRSN) participants of the Amsterdam Cohort Studies and 54 men from the same cohort who were seronegative at the moment of analysis but later became HIV seropositive. HRSN had higher naive (CD45RO CD27) CD4 and CD8 T cell numbers and lower percentages of activated (HLADR CD38, CD70) CD4 and proliferating (Ki67) CD4 and CD8 T cells, irrespective of previous episodes of sexually transmittable infections. Furthermore, whole blood cultures from HRSN showed lower lymphoproliferative responses than healthy laboratory controls. These data suggest that low levels of immune activation and low T cell responsiveness may contribute to low HIV susceptibility.     

Antibodies against the CD44 p80, lymphocyte homing receptor molecule augment human peripheral blood T cell activation

The CD44 inhibitor Lutheran [In(Lu)]-related p80 molecule has recently been shown to be identical to the Hermes-1 lymphocyte homing receptor and to the human Pgp-1 molecule. We have determined the effect of addition of CD44 antibodies to in vitro activation assays of PBMC. CD44 antibodies did not induce PBMC proliferation alone, but markedly enhanced PBMC proliferation induced by a mitogenic CD2 antibody pair or by CD3 antibody. CD44 antibody addition had no effect upon PBMC activation induced by PHA or tetanus toxoid. CD44 antibody enhancement of CD2 antibody-induced T cell activation was specific for mature T cells as thymocytes could not be activated in the presence of combinations of CD2 and CD44 antibodies. CD44 antibody enhancement of CD2-mediated T cell triggering occurred if CD44 antibody was placed either on monocytes or on T cells. In experiments with purified monocyte and T cell suspensions, CD44 antibodies A3D8 and A1G3 augmented CD2-mediated T cell activation by three mechanisms. First, CD44 antibody binding to monocytes induced monocyte IL-1 release, second, CD44 antibodies enhanced the adhesion of T cells and monocytes in CD2 antibody-stimulated cultures, and third, CD44 antibodies augmented T cell IL-2 production in response to CD2 antibodies. Thus, ligand binding to CD44 molecules on T cells and monocytes may regulate numerous events on both cell types that are important for T cell activation. Given that recent data suggest that the CD44 molecule may bind to specific ligands on endothelial cells (vascular addressin) and within the extracellular matrix (collagen, fibronectin), these data raise the possibility that binding of T cells to endothelial cells or extracellular matrix proteins may induce or up-regulate T cell activation in inflammatory sites.