Changing virus-host interactions in the course of HIV-1 infection.
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Acquired Immunodeficiency Syndrome↗
Biomedical subjects
M R Klein
Publications and source records attributed to M R Klein.
Protection from HIV infection or AIDS?
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AIDS Vaccines↗
Selective in vitro expansion of HLA class I-restricted HIV-1 Gag-specific CD8+ T cells: cytotoxic T-lymphocyte epitopes and precursor frequencies.
OBJECTIVE: To identify HIV-1 Gag cytotoxic T-lymphocyte (CTL) epitopes and HLA restriction of their recognition, and to define precursor frequencies of HIV-1 Gag-specific CTL in the blood of seropositive individuals. METHODS: B-lymphoblastoid cell lines (B-LCL) infected with recombinant vaccinia viruses (rVV) containing a gene coding for HIV-1 Gag (rVV-Gag) were fixed with paraformaldehyde (PFA) and used as antigen-presenting cells (APC) to stimulate peripheral blood mononuclear cells (PBMC) from asymptomatic HIV-seropositive individuals. Specific CTL activity was determined in 51Cr-release assays using B-LCL as targets after infection with rVV-Gag or after pulsing with partially overlapping peptides spanning the Gag sequence. RESULTS: In vitro stimulation resulted in an increased number of CD8+ T cells and CD45R0+ and HLA-DR+ cells. Gag-specific cytotoxicity, mediated predominantly by HLA class I-restricted CD8+ CTL, was observed in all seven individuals studied. Multiple HLA-restricted CTL epitopes were identified with a single culture from one of the individuals. Gag-expressing APC were successfully used as stimulator cells in limiting dilution analysis to determine CTL precursor (CTLp) frequencies. CONCLUSION: PFA-fixed rVV-Gag-infected autologous B-LCL can be used as stimulator cells in bulk PBMC cultures to identify CTL epitopes and to determine CTLp frequencies. This method will facilitate the analysis of HIV-1-specific CTL responses in HIV-infected and vaccinated individuals.
Antigen-Presenting Cells↗
Milk-borne factors affect survival, intestinal growth and development in newborn rats.
Early milk of rats contains high concentrations of many hormones and growth factors. Studies were undertaken to evaluate the physiological effects of early rat milk infranatant, insulin-like growth factors (IGFs) and epidermal growth factor (EGF) upon the growth and development of intestinal tissues in newborn rat pups. In one study, pups were artificially reared for 3 days (d2-d5) then fostered back to the dam. Treatment groups were mother fed controls (MF), artificially reared pups fed rat milk replacer (RMR) or RMR with infranatant from rat milk (r-Infra) or bovine milk (b-Infra). After 8 days, survival in the MF and RMR + r-Infra groups was 100% while survival in the RMR + b-Infra and RMR groups were 50 and 30% respectively. In additional experiments, pups were separated from the dam at birth (no suckling) and sacrificed after 2 days of artificial feeding. Treatments included RMR, RMR + r-Infra, and RMR + long Arg-IGF-I + EGF and were compared to MF control. Histological and autoradiographical evaluation of the intestines from MF controls showed advanced differentiation characterized by proliferative cell populations which were restrictively localized to the basal areas of the mucosa. In contrast, intestinal proliferation in the RMR group was diffusely distributed along both the lower regions of the villi and the crypts. Pups fed RMR + r-Infra exhibited intermediate morphology. Pups artificially fed RMR + long Arg-IGF-I + EGF produced a dramatic establishment of an active crypt-villus axis within two days of feeding.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Expression of the IL-6 gene induces differentiation of a human monoclonal EBV-transformed B cell line.
To study the role of interleukin (IL)-6 as a growth and differentiation factor for Epstein-Barr virus (EBV)-transformed B lymphocytes, we transfected the cDNA coding for human IL-6 in a monoclonal IgG1-secreting EBV B cell line. Two independent clones were selected that constitutively secreted high amounts of IL-6. These clones showed enhanced levels of IL-6 and tumor necrosis factor alpha secretion when compared to non-IL-6 transfected controls. Moreover, they could efficiently be recovered from low cell density cultures in limiting dilutions when plated on a feeder layer of heterologous EBV B cells. IL-6-induced phenotypical changes comprised a significant rise in immunoglobulin secretion levels and enhanced membrane expression of CD25 (the beta chain of the IL-2 receptor) and of the B cell differentiation antigen CD40. IL-6-dependent down modulation of CD38 and of the adhesion structure VLA4 were also observed. Our data support the notion that IL-6 can serve as an growth and differentiation factor for EBV B cells.
Antigens, CD↗
Effects of IL-4, IL-5, and IL-6 on growth and immunoglobulin production of Epstein-Barr virus-infected human B cells.
In the present study we investigated whether interleukin-4 (IL-4), IL-5, and IL-6 could enhance the efficiency of Epstein-Barr virus (EBV) transformation for the generation of specific human monoclonal antibody (HuMAb)-producing B-cell lines directed against erythrocyte Rhesus(D) antigen. In newly EBV-infected B cells, IL-4 and IL-6 caused a comparable enhancement of proliferation and of total IgG and IgA production. IL-6 showed a much stronger effect than IL-4 on IgM production, whereas IL-4 was unique in inducing IgE production. No stimulatory effects of IL-5 on either growth or Ig production were observed. Although addition of IL-6 resulted during the early phase after EBV infection in high numbers of Ag-specific antibody-producing wells, this did not result in an increased number of stable HuMAb-secreting cell lines. When the effects of cytokines were tested on established polyclonal EBV B cells, in a high cell density culture system, only IL-6 was able to enhance Ig secretion, while no effect could be demonstrated on proliferation. These studies substantiate that IL-6 is an important regulator of proliferation and Ig production, and that it acts at distinct stages after EBV infection, but does not increase the final overall recovery of Ag-specific EBV B-cell lines.
Antibody Formation↗
Protein kinase C regulates secretion of lymphotoxin (LT/TNF beta) and TNF alpha by human EBV-transformed B cells.
Epstein-Barr virus (EBV)-transformed cell lines constitutively secrete lymphotoxin (LT/TNF beta) and not tumor necrosis factor-alpha (TNF alpha). To analyze the cellular processes that regulate LT and TNF alpha secretion by lymphoblastoid cell lines, we studied the role of two signal transduction pathways leading to either protein kinase C (PK-C) or PK-A activation. We demonstrate that PK-C activation, either after cross-linking of surface Ig or by direct activation with phorbolester, leads to increased production of both LT and TNF alpha, whereas no prominent role for PK-A was found. Interleukin (Il)-4 was found to synergize with PK-C activation in raising levels of secreted LT and TNF alpha. Increased levels of LT and TNF alpha did not correlate with augmented levels of immunoglobulin secreted by the cell lines nor with improved proliferation. These observations demonstrate that EBV B cells respond to B cell activation signals leading to PK-C activation with increased production of both LT and TNF alpha. It is, however, unlikely that these molecules serve as autostimulatory factors for EBV B cells, but in contrast might play a role in downregulation of biological functions in these cells.
B-Lymphocytes↗
Heterogeneity in both cytokine production and responsiveness of a panel of monoclonal human Epstein-Barr virus-transformed B-cell lines.
To optimize growth and Ig production of in vitro-cultured Epstein-Barr virus (EBV)-transformed B cells, a panel of six monoclonal EBV B-cell lines was analyzed for autocrine growth factor production and responsiveness to various cytokines. Three cell lines produced Il-I and four produced Il-6, although differences concerning the amount of lymphokines produced were observed. Interestingly a considerable tumor necrosis factor beta (lymphotoxin) activity was found in supernatants of all cell lines. One IgM-producing cell line that did not secrete either Il-1 or Il-6 was exceptional in its ability to respond to the addition of rIl-6 with a 5- to 10-times elevated IgM production. In contrast, cell lines in the panel capable of Il-6 production showed only a minimal elevation of Ig production on addition of exogenous Il-6. Ig production was slightly less in some cell lines when Il-6 was neutralized. Antibodies against lymphotoxin or Il-6 did not influence growth rate of the cell lines significantly, implying that neither Il-6 nor lymphotoxin had an autostimulatory effect on the analyzed cell lines. This study demonstrates a heterogeneity regarding the amount and type of lymphokines produced by long-term monoclonal EBV cell lines, which may account for the diverse responses exhibited by these cells towards exogenously added lymphokines.
B-Lymphocytes↗
Riboflavin deficiency in cultured rat hepatoma cells: a model for studying the hepatic effects of riboflavin deficiency.
The acyl-CoA dehydrogenases are a family of mitochondrial flavoenzymes required for fatty acid beta-oxidation and branched-chain amino acid degradation. The hepatic activity of these enzymes, particularly the short-chain acyl-coenzyme A (CoA) dehydrogenase, is markedly decreased in riboflavin deficient rats. We now report that the in vivo effects of riboflavin deficiency on the beta-oxidation enzymes of this group are reproduced in FAO rat hepatoma cells cultured in riboflavin-deficient medium. Although it has been long known that hepatic short-chain acyl-CoA dehydrogenase activity is the most severely affected of the straight-chain specific enzymes in riboflavin deficiency, the mechanism by which its activity is decreased has not been reported. We have used this new cell culture system to characterize further this mechanism. Whole cell extracts from riboflavin-deficient and control cells were subjected to analysis by denaturing polyacrylamide gel electrophoresis. The contents of the gels were then electroblotted onto nitrocellulose filters and probed with short-chain acyl-CoA dehydrogenase-specific antiserum. The relative abundance of enzyme antigen was estimated autoradiographically. Our findings indicate that short-chain acyl-CoA dehydrogenase activity changes in parallel with its antigen, suggesting that riboflavin deprivation does not affect the activity of individual enzyme molecules. Further, no evidence of extramitochondrial enzyme precursor was found on the blots, making unlikely a significant block in the mitochondrial uptake process. These findings suggest that changes in short-chain acyl-CoA dehydrogenase activity in riboflavin deficiency result from either increased synthesis or decreased degradation of the enzyme.
Acyl-CoA Dehydrogenase↗