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Biomedical subjects

J G Sissons

Publications and source records attributed to J G Sissons.

At least 37 records · Page 2Linked to original sources
PubMed1991

Fine specificity of cellular immune responses in humans to human cytomegalovirus immediate-early 1 protein.

Cell-mediated immunity is important in maintaining the virus-host equilibrium in persistent human cytomegalovirus (HCMV) infection. The HCMV 72-kDa major immediate early 1 protein (IE1) is a target for CD8+ cytotoxic T cells in humans, as is the equivalent 89-kDa protein in mouse. Less is known about responses against this protein by CD4+ T cells, which may be important as direct effector cells or helper cells for antibody and CD8+ responses. Proliferative-T-cell responses to HCMV IE1 were studied in normal seropositive subjects. Peripheral blood mononuclear cells from 85% of seropositive subjects proliferated in response to HCMV from infected fibroblasts, and of these, 73% responded to recombinant baculovirus IE1. Responding cells were predominantly CD3+ CD4+. IE1 antigen preparations, including baculovirus recombinant protein, transfected rat cell nuclei, and synthetic peptides, induced IE1-specific T-cell lines which cross-reacted between the preparations. The fine specificity of these IE1-specific T-cell lines was studied by using overlapping synthetic peptides encompassing the entire sequence of the IE1 protein. The regions of the IE1 molecule recognized were identified and these varied between individuals, possibly reflecting differences in major histocompatibility complex (MHC) class II haplotype. In one subject, the peptide specificities of proliferative and MHC class I-restricted cytotoxic determinants on IE1 were spatially distinct. Thus, no single immunodominant T-cell determinant within HCMV IE1 was identified, suggesting that multiple peptides or a region of the 72-kDa IE1 protein would be required to induce specific T-cell responses in humans.

Amino Acid Sequence↗
PubMed1991

Pathogenesis of human cytomegalovirus disease and the kidney.

Uncertainty still surrounds many aspects of the pathogenesis of human cytomegalovirus (HCMV) infection. During asymptomatic infection HCMV is probably present in a small fraction of peripheral blood mononuclear cells (most likely monocytes); in active infection HCMV is detectable in a wider range of cells. In vitro experiments suggest that cellular control of transcription of HCMV immediate early (IE) gene expression is one key determinant of HCMV reactivation. The cytotoxic T cell response to IE and other virus gene products is probably important in subsequently limiting dissemination of HCMV infection. The basis for any association between active HCMV disease and allograft rejection is unclear, although homologies between cellular and viral proteins suggest possible mechanisms. Although HCMV may be associated with tubulointerstitial disease, there is no unequivocally established role for HCMV in the pathogenesis of human glomerular disease.

Animals↗
PubMed1990

Automation of limiting dilution cytotoxicity assays.

Limiting dilution analysis is a valuable technique for the quantitation and clonal analysis of immunocompetent cells. However, manual processing of the large number of samples necessary for satisfactory statistical analysis is laborious, and consequently results in inaccuracies reflected as increased standard errors. We describe the application of an automated robotic liquid handling tool to process samples in limiting dilution cytotoxicity assays. Our studies have shown that automated liquid handling is more accurate than manual methods, and that errors are limited. This results in savings of both time and resources. Furthermore, the process may be adapted for the safe, remote handling of sterile cell cultures, and human pathogens.

Automation↗
PubMed1989

Repression of human cytomegalovirus gene expression associated with a novel immediate early regulatory region binding factor.

A major determinant controlling reactivation of persistent viruses is likely to be the level of cellular factors which regulate virus transcription. Human cytomegalovirus (the largest human herpesvirus) does not replicate in human teratocarcinoma (T2) cells due to a block in transcription of immediate early (IE) gene expression, but these cells become permissive upon retinoic acid induced differentiation. We have analysed changes in DNA binding factors to the major IE promoter/regulatory region of HCMV that occur during differentiation of T2 cells to a permissive phenotype. We show that undifferentiated T2 cells contain a specific nuclear factor that binds to a far upstream region of the major IE regulatory region. Differentiation of T2 cells is associated with a major decrease in this factor and deletion of its specific binding site from IE expression vectors also results in increased levels of expression in undifferentiated cells. Consequently, this novel factor present in undifferentiated cells is a candidate for a differentiation specific negative regulator of HCMV IE gene expression which binds to an element upstream of the major IE enhancer.

Base Sequence↗
PubMed1989

Expression of oncogenic ras in human teratocarcinoma cells induces partial differentiation and permissiveness for human cytomegalovirus infection.

Retinoic acid (RA)-induced differentiation of human teratocarcinoma (T2) cells results in a change from a normally non-permissive phenotype for human cytomegalovirus (HCMV) infection to cells which are fully permissive. We have used this system to analyse factors associated with differentiation which may regulate HCMV gene expression. Differentiation of T2 cells results in an increase of c-ras expression. Consequently, we have introduced ras expression vectors into T2 cells. We find that, as with RA induction, transfection of T2 cells with oncogenic human Ha-ras results in cells which are permissive for HCMV infection and gene expression. However, unlike RA which induces a cessation of cell proliferation and terminal differentiation, ras transfection only appears to result in changes associated with early events in RA-induced differentiation of T2 cells.

Cell Transformation, Neoplastic↗
PubMed1989

Human cytomegalovirus and monocytes: limited infection and negligible immunosuppression in normal mononuclear cells infected in vitro with mycoplasma-free virus strains.

Human cytomegalovirus (HCMV) infection has previously been associated with the production of immunosuppression. The mechanism by which any such immunosuppressive effect might be mediated is unclear but previous work has implicated an effect of the virus on monocytes. We have attempted to characterize the immunosuppressive activity produced by in vitro infection of normal monocytes with HCMV strain AD169. We first examined the ability of HCMV AD169 and recent clinical isolates to infect normal peripheral blood mononuclear cells in vitro. We have found by immunofluorescence analysis that only a very limited number of peripheral blood mononuclear cells (0.2 to 0.5%) showed evidence of virus infection as demonstrated by expression of the major immediate early protein. We found that the inhibitory activity of supernatants of monocytes exposed to HCMV which suppressed mitogen-driven T cell responsiveness was associated with a protein of about 95K. Experiments to investigate the mechanism of action of this inhibitor suggested the possibility of mycoplasma contamination and we were subsequently able to isolate Mycoplasma hyorhinis from our AD169 virus stock. When a series of low passage clinical isolates of HCMV were examined for their ability to cause immunosuppression, there was a direct correlation between suppression and the presence of contaminating mycoplasmas. Using mycoplasma-free isolates of HCMV we could demonstrate no immunosuppressive effect on mitogen-mediated T cell proliferation of both unseparated human peripheral blood lymphocytes and nylon wool non-adherent T cells; these virus isolates also did not suppress accessory cell function or interleukin 1 production by monocytes infected in vitro. We conclude that the previously reported immunosuppressive effects of HCMV in vitro may be attributable to the presence of mycoplasmas and are unlikely to be due to expression of HCMV in monocytes. We suggest that mycoplasma contamination of isolates of HCMV may be a more extensive problem than is currently recognized.

Antibodies, Bacterial↗
PubMed1988

Human cytomegalovirus-specific cytotoxic T cells. Relative frequency of stage-specific CTL recognizing the 72-kD immediate early protein and glycoprotein B expressed by recombinant vaccinia viruses.

CTL are held to be an important host defense mechanism in persistent herpes-virus infections. We have therefore studied the nature and specificity of human cytomegalovirus (HCMV)-specific CTL in normal persistently infected individuals. This was achieved by using vaccinia recombinants encoding viral genes expressed at different stages of the virus replicative cycle, a structural glycoprotein gB (vac.gB) and the major 72-kD immediate early nonstructural protein (vac.IE) of HCMV, combined with limiting dilution analysis of the CTL response. In two subjects, 43 and 58% of HCMV CTL precursors (CTLp) lysed vac.IE-infected cells, in contrast to less than 6% lysing gB-infected cells. HCMV-specific CTL could also be generated by secondary in vitro stimulation with vac.gB- but not vac.IE-infected autologous fibroblasts. The high frequency of 72-kD IE protein-specific CTL suggests that this is at least a major recognition element for the HCMV-specific CTL response in asymptomatic persistently infected individuals, and CTL with this specificity may be important in maintaining the normal virus/host equilibrium.

Antigens, Viral↗
PubMed1988

Human cytomegalovirus-specific cytotoxic T cells: their precursor frequency and stage specificity.

Human virus-specific cytotoxic T (Tc) cells may be important in maintaining the virus/host equilibrium during persistent herpes virus infections such as that with human cytomegalovirus (HCMV). We have previously shown that HCMV-specific Tc cells are present in peripheral blood in normal asymptomatic seropositive individuals (L. K. Borysiewicz et al., Eur. J. Immunol. 1983. 13: 804). In this study we have used limiting dilution analysis to estimate the precursor frequency of these Tc cells and to further delineate their specificity for viral proteins expressed at different stages of the virus replicative cycle. HCMV-specific Tc precursor cells were present in peripheral blood lymphocytes (PBL) at a frequency of 1/5000 to 20,000 E+ PBL. This frequency was higher than that observed for varicella-zoster virus (VZV)-specific Tc cells (1/30,000 to greater than 500,000) in asymptomatic individuals and was similar to the VZV Tc precursor cell frequencies observed following clinical reactivation (1/30,000). When the stage specificity of clonally derived HCMV-specific Tc cells was analyzed, using target cells treated with phosphonoformate to allow expression of only the nonstructural viral proteins, the majority (60%) of Tc cells lysed these cells. A number of Tc cells lysed only cells which expressed the structural or late HCMV proteins. These results suggest a high precursor frequency of HCMV-specific Tc cells in PBL, and that there are subpopulations of such Tc cells specific for HCMV antigens expressed at different stages of the virus replicative cycle. However, the relative frequencies of these subpopulations suggest that the immunodominant HCMV antigens with respect to the Tc response are expressed at immediate early and/or early times.

Cells, Cultured↗
PubMed1988

Suppressive role of NK cells in pokeweed mitogen-induced immunoglobulin synthesis: effect of depletion/enrichment of Leu 11b+ cells.

Natural killer (NK) cells probably have immunoregulatory effects. However, the evidence to date is mainly based on the suppressive effect of enrichment with relatively impure NK populations (large granular lymphocytes, LGL, Leu 7a+ cells). Here we report on the effect of enrichment and depletion of Leu 7a+ and Leu 11b+ cells (the latter containing virtually all NK activity in freshly prepared lymphocytes) on pokeweed mitogen (PWM)-induced immunoglobulin (Ig) synthesis. Enrichment suppressed Ig synthesis to a degree dependent on the number of cells added, and was not enhanced further by their pretreatment with interferon. Furthermore, depletion of Leu 11b+ cells from peripheral blood lymphocytes (PBL) led to marked enhancement (2-25-fold increase) of Ig synthesis, suggesting these cells may normally exert a suppressive effect. The possible underlying mechanisms were investigated further. Enhanced Ig synthesis by Leu 11b-depleted cultures was associated with an increased number of Ig-secreting cells by plaque assay, but with no change in numbers of CD4+ or CD8+ cells. Treatment of PBL with monoclonal antibodies (anti-Leu 7a/Leu 11b) alone suppressed PWM-induced immunoglobulin synthesis. We conclude that NK cells play a role in the regulation of Ig production, at least in part by an effect on activation/differentiation of B cells, but independent of altered T-cell subpopulations. The effect may be unrelated to their cytotoxic function (being unaffected by interferon, IFN), although the direct effects of anti-Leu 11b and Leu 7a in enhancing the suppressive effect suggest an alternative activation pathway.

Antibodies, Monoclonal↗
PubMed1987

Monoclonal antibodies to restricted and cross-reactive idiotopes on monoclonal rheumatoid factors and their recognition of idiotope-positive cells.

Four human monoclonal rheumatoid factors (MRF) were used to raise a panel of mouse monoclonal antibodies (mAb) which were selected in a solid-phase radioimmunoassay for binding to MRF but not normal IgM. Three mAb, each raised against a different MRF, bound to the majority of MRF and also to most polyclonal RF. Four other mAb bound selectively to the MRF against which they were raised and to no other MRF, and rarely to any polyclonal RF. Competition studies using cold and radiolabeled mAb further indicated that these mAb recognize distinct and different epitopes on MRF. RF activity of MRF was inhibited by 3 of the 4 mAb binding to a single MRF and 2 of the 3 mAb binding to multiple RF. It was thus concluded that of this panel of mAb 3 recognized cross-reactive idiotopes and the remainder demonstrated highly restricted idiotopes on MRF. These mAb identified MRF idiotope-bearing cells in the peripheral blood of 3 of the MRF donors (and a further subject with type II essential cryoglobulinemia), with a frequency ranging from 0.3-10% of all mononuclear cells with the mAb to restricted idiotopes or 1.5-17% with mAb to cross-reactive idiotopes. These anti-idiotopic mAb should thus provide a highly specific means of identifying and monitoring MRF-producing cells in vivo.

Antibodies, Monoclonal↗
PubMed1987

Varicella-zoster virus-specific cytotoxic T lymphocytes (Tc): detection and frequency analysis of HLA class I-restricted Tc in human peripheral blood.

The cytotoxic T-cell (Tc) response to varicella-zoster virus (VZV) is incompletely characterized. We investigated whether VZV-specific Tc restricted by class I products of the major histocompatibility complex can be generated from the peripheral blood of VZV-immune donors. Cell lines were established from peripheral blood lymphocytes (PBL) of seropositive donors by secondary in vitro restimulation. If cell-free VZV was used as the stimulating antigen, the resulting lines were predominantly CD4+ and did not show class I-restricted cytotoxicity; when autologous infected fibroblasts were used for in vitro stimulation, the resultant lines were usually cytotoxic, although in only 4 of 11 subjects tested was this cytotoxicity HLA restricted and virus specific. PBL were also tested for Tc activity without prior restimulation; VZV-specific Tc activity was only demonstrable in the PBL of a subject convalescent following zoster but not from subjects with recent varicella infection or from normal subjects. VZV-specific Tc precursor frequencies were then determined in six selected subjects by limiting-dilution analysis. A measurable frequency was detectable in four of the six seropositive subjects, ranging from 11/10(6) T cells in an asymptomatic carrier, to 63/10(6) T cells in a subject with recent zoster. We conclude that virus-specific major histocompatibility complex class I-restricted Tc precursors may be present in the peripheral blood of normal individuals seropositive for VZV but at a frequency lower than that for other herpesviruses with nonneuronal sites of latency.

Cell Line↗
PubMed1986

Human natural killer cell lysis of virus-infected cells. Relationship to expression of the transferrin receptor.

Natural killer (NK) cells lyse tumor and virus-infected cells yet the nature of the target structure they recognize is unknown. A normal host cell glycoprotein, the transferrin receptor (TfR), has been proposed as a target structure on tumor cells. We therefore investigated whether changes in the number or physiological recycling of the TfR, consequent on virus infection, were related to the differential susceptibility of virus-infected cells to NK lysis. There was a direct correlation between TfR expression, susceptibility to NK lysis and ability to act as cold target competitors, for human fibroblasts infected with RNA and DNA viruses (cytomegalovirus, herpes simplex, polio, vaccinia and Semliki Forest virus). The NK lysis of human cytomegalovirus-infected fibroblasts was studied in more detail. NK lysis was increased coincident with human cytomegalovirus early antigen expression and this susceptibility to lysis was associated with increased total and recycling TfR but only a slight increase in surface TfR expression. In addition, susceptibility of uninfected human fibroblasts to NK lysis directly correlated with TfR number. However, we were unable to inhibit NK lysis by either excess iron-saturated Tf or affinity-purified TfR. We conclude that there is a direct correlation between total TfR expression and susceptibility to NK lysis of human virus-infected cells; however, the NK target structure on virus-infected cells is probably not the TfR itself.

Cytomegalovirus↗
PubMed1986

Epstein Barr virus-specific immune defects in patients with persistent symptoms following infectious mononucleosis.

In a number of patients recovery from infectious mononucleosis (IM) following primary Epstein Barr virus (EBV) infection, is complicated by the persistence of symptoms for months or years. Normally recovery from infectious mononucleosis is associated with the development of EBV-specific antibodies and memory cytotoxic T-cells, which are present in the peripheral blood of all normal seropositive individuals. We studied four patients who had persistent symptoms for more than two years after infectious mononucleosis to determine if this abnormality was associated with a defect in EBV-specific or non-specific immune responses. All four patients had normal immunoglobulin concentrations, T- and B-cell numbers, T-cell proliferative responses and natural killer cell activity. However three of the four had reduced or absent antibodies to the EBV nuclear antigen (EBNA) although other EBV-specific antibody titres were normal. All four also had reduced EBV-specific cytotoxic T-cell activity as measured by the EBV regression assay. This defect was probably EBV-specific as alloreactive cytotoxic T-cell responses were normal. In addition, three of three patients tested had reduced in vitro antibody synthesis following pokeweed mitogen stimulation. These studies indicate that the syndrome of persistent symptoms following EBV mononucleosis may be associated with a defect in EBV-specific immunity, and thus suggest a possible immunological basis for the syndrome.

Adolescent↗
PubMed1985

Cytotoxic lymphocytes generated in vivo with acute measles virus infection.

We studied the generation of cytotoxic lymphocytes in adults during an outbreak of acute measles virus infection. Nine patients were studied determining in particular whether virus-specific cytotoxic T lymphocytes could be directly detected in peripheral blood during this acute infection. The cytotoxicity of PBL was assayed against measles virus-infected and uninfected phytohemagglutinin-induced blast cells of matched and mismatched HLA, A, B, and C types, in a standard 4-h 51Cr release assay. There was greater cytotoxicity against measles virus-infected than uninfected target cells in at least one sample from every patient. In 4 patients this preferential lysis of virus infected cells was greater (a difference of more than 10% virus-specific lysis) against HLA-matched than mismatched targets. This preference for HLA A and B matched infected target cells was also clearly seen when the effector PBL were depleted of FC receptor bearing cells. The other 5 subjects exhibited no evidence of preferential lysis of HLA-matched measles virus-infected cells. All 9 patients limited the spread of measles virus infection and recovered equally from the acute infection. These studies provide some evidence to suggest that MHC-restricted virus-specific CTL are detectable in human peripheral blood during acute measles virus infection, albeit only with low frequency, but are not necessarily associated with recovery from disease.

Adult↗