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Biomedical subjects

F J Chrest

Publications and source records attributed to F J Chrest.

At least 37 records · Page 2Linked to original sources
PubMed1995

Anti-CD3-induced apoptosis in T-cells from young and old mice.

Light scatter measurements using flow cytometry indicated that T cells from young and old mice undergo apoptosis following activation with immobilized anti-CD3. The percentage of cells in apoptosis after 20 h activation was significantly greater (p < .001) in cultures containing cells from older animals. The mean percentages of apoptotic T cells from young and old mice after 20 h activation were 19.3% and 33.0%, respectively. The proportion of viable cells after 20 h activation was significantly higher (p < .003) in the young (mean = 78.4%) than in the old animals (mean = 65.8%). Simultaneous measurements of light scatter and fluorescence indicated that apoptotic T cells contained both the CD4+ and the CD8+ T-cell phenotypes. The frequency of apoptotic CD8+ T cells was elevated (p < .007) in older animals, where the mean percentage was 15.1%, compared to 5.3% in the young. The most dramatic difference between young and old (p < .0008) was seen in the percentages of viable CD4+ T cells after 20 h activation. The mean viable CD4+ T-cell percentage was 33.7% in- the young and 21.4% in the old. CD4+ cells expressing high levels of CD45RB (CD45RBhi) after activation for 20 h possessed light scatter and bright fluorescence properties characteristic of viable cells, whereas CD4+/CD45RBlo density cells could be identified as apoptotic based on their decreased CD4 fluorescence and scatter characteristics. CD4+ cells from young animals were predominantly CD45RBhi, whereas CD4+ cells from the old had greater levels of CD454RBlo cells. In addition to light scatter changes, measurement of DNA content after 40 h activation revealed the presence of a sub-G1 DNA apoptotic peak and a viable cell cycle distribution. After 40 h of activation, there was an increase in the percentage of apoptotic cells in both young and old mice, with the greatest increase seen in the cells from older animals. Further evidence supporting the process of apoptosis in 40 h-activated cells was confirmed by the appearance of DNA strand breaks detected by in situ nick translation.

Aging↗
PubMed1995

Progression of hormone-dependent adenocarcinoma cells to hormone-independent spindle carcinoma cells in vitro in a clonal spontaneous rat mammary tumor cell line.

A hormone-dependent, clonal carcinoma cell line, designated RM22-F5, was derived from a malignant mammary mixed tumor spontaneously arising in an outbred old female Wistar rat. These cells expressed keratin and desmosomal protein and formed epithelial monolayers in a growth factor and hormone-supplemented medium (LHC-8) containing 10% fetal bovine serum (E-type cells). Cells subcultured for 6 to 8 passages in RPMI 1640 medium containing 10% fetal bovine serum without supplements appeared to be fibroblastic and expressed vimentin (F-type cells). The shift to a fibroblast-like morphology was associated with a more malignant phenotype which included rapid, hormone-independent growth and invasive sarcoma-like character in nude mice. F-type cells were no longer able to express their original epithelial phenotype in LHC-8 medium. Cytogenetic analysis revealed that both E- and F-type cells had essentially the same karyotype. Analysis of PCR-amplified DNA further demonstrated a point mutation of the H-ras-1 gene at codon 12 and loss of the normal H-ras-1 allele in both cell types. Genetic tagging of E-type cells with the neomycin-resistance gene resulted in the generation of F-type cells with neomycin resistance in RPMI 1640 medium, suggesting that F-type cells are a malignant variant of E-type cells arising during in vitro culture. Somatic cell fusion between E- and F-type cells revealed that with most hybrid clones tested, the fibroblast-like phenotype was greatly suppressed. These results suggest that an irreversible phenotypic transition, representative of tumor progression from hormone-dependent adenocarcinoma to more malignant hormone-independent spindle carcinoma cells, is a recessive event and may involve loss of a suppressor function.

Adenocarcinoma↗
PubMed1994

Up-regulation of c-myc induces the gene expression of the murine homologues of p34cdc2 and cyclin-dependent kinase-2 in T lymphocytes.

The expression and/or up-regulation of several early T cell activation genes is dependent on signals transmitted through the interaction of IL-2 and IL-2R well before entry of the cells into S phase. In these studies, murine G0 T cells activated by immobilized anti-CD3 and subsequently blocked in late G1 expressed normal surface levels and mRNA for IL-2R alpha, IL-2R beta, and transferrin receptor (TfR). However, there was no expression of p34cdc2, and cyclin-dependent kinase (cdk)-2 was not up-regulated even in the presence of exogenous rIL-2. In addition the accumulation of c-myc-specific mRNA and protein was significantly reduced. Pretreatment of G0 T cells with c-myc antisense oligonucleotide effectively reduced the level of specific c-myc protein induced by activation of the cells by immobilized anti-CD3. The presence of antisense c-myc oligonucleotide inhibited the expression of cdc2 and cdk2 without affecting the expression of IL-2R alpha and blocked the activated T cells in the G1 phase. Together these studies demonstrate that c-myc regulates the expression of these cdk and suggest a role for c-myc in the G1/S transition.

Animals↗
PubMed1993

Identification and quantitation of apoptotic cells following anti-CD3 activation of murine G0 T cells.

Multiparameter flow cytometry and cell sorting were used to examine the process of apoptosis after activation of murine resting T cells with immobilized anti-CD3. Activated T cells treated with Hoechst 33342 (HO-33342) and analyzed by flow cytometry showed two major cell populations of high and low fluorescence. These populations were sorted and the DNA extracted and subjected to electrophoresis. Electrophoresis of DNA extracted from T cells showing a low level of HO-33342 fluorescence (HO-Low) resulted in a typical ladder pattern characteristic of internucleosomal DNA degradation associated with apoptosis, whereas the cellular DNA of the cells showing a high level of fluorescence (HO-High) showed a narrow high molecular weight band. Multiparameter analysis further indicated that cells with HO-High characteristics possessed corresponding high-FSC/low-SSC properties, whereas HO-Low cells formed a cluster of low-FSC/high-SSC cells. Analysis of the DNA extracted from cells sorted on the basis of scatter properties alone confirmed that the low-FSC/high-SSC population contained the apoptotic cells and that the high-FSC/low-SSC population was comprised of viable cells. This methodology allowed us to determine the percentage of apoptotic cells following anti-CD3 activation at various time points and to discriminate them from those in cell cycle. We could further quantitate the number of apoptotic versus viable CD4+ and CD8+ cells in the cell cycle.

Animals↗
PubMed1993

Increased production of TGF-beta and Il-6 by aged spleen cells.

Aging is accompanied by a progressive decline in immunity in every species that has been studied. Despite its ubiquity, the causes of immunosenescence are unknown. Transforming growth factor beta (TGF-beta) is a cytokine with potent immunosuppressive properties. Cells from aged mice produce increased levels of TGF-beta in vitro along with similar increases in interleukin 6 (Il-6), a cytokine which is immunosuppressive at elevated concentrations. Il-6 does not upregulate TGF-beta production, but high concentrations of Il-6 increase the percentage of cells expressing the TGF-beta receptor. Increased TGF-beta production and Il-6-induced upregulation of the TGF-beta receptor may be factors contributing to age-associated immunosuppression.

Aging↗
PubMed1992

Expression of the murine homologue of the cell cycle control protein p34cdc2 in T lymphocytes.

The mammalian homologue of the cdc2 gene of the fission yeast Schizosaccharomyces pombe encodes a p34cdc2 cyclin-dependent kinase that regulates the cell cycle of a wide variety of cell types. Resting murine T lymphocytes contained no detectable p34cdc2 protein, histone kinase activity, or specific mRNA for the cdc2 gene. Activation of the T cells by immobilized anti-CD3 resulted in the expression of specific mRNA late in the G1 phase of the cell cycle, and p34cdc2 protein was detectable at or near G1/S. At this point in the cell cycle, the protein was phosphorylated at tyrosine and displayed no H1 histone kinase activity. As the cells progressed through the cycle, the amount of specific mRNA and p34cdc2 increased, and H1 histone kinase activity was detectable when the cells were blocked at G2/M by nocodazole. The activation of T cells by phorbol dibutyrate induced the expression of IL-2R but failed to induce the synthesis of IL-2 or the expression of cdc2-specific mRNA. Under these conditions, the activated cells failed to enter the S phase of the cell cycle. Because the presence of IL-2 added exogenously during activation by phorbol dibutyrate resulted in the expression of cdc2-specific mRNA and progression through the cell cycle, either IL-2 or the interaction with IL-2R may be involved in the expression of cdc2 and regulation of the G1/S transition.

Amino Acid Sequence↗
PubMed1992

Reversible G1 arrest of a human lung epithelial cell line by staurosporine.

Staurosporine, a microbial-derived protein kinase inhibitor, reversibly blocked non-synchronized, replicating cultures of the human lung epithelial cell line EKVX in the G1 phase of cell cycle and inhibited DNA synthesis and cell replication. The mechanism of this cell-cycle arrest in EKVX cells by staurosporine was likely due to inhibition of protein kinase C (PKC) because: 1) dose-dependent inhibition of DNA synthesis occurred at levels of staurosporine that inhibit phosphorylation of PKC substrate, 2) inhibition of DNA synthesis was also seen after treatment with another PKC inhibitor H7, but not by the chemically similar HA1004, which has a relative inhibitory specificity for cAMP-dependent protein kinase, and 3) the DNA synthesis was not inhibited by specific tyrosine kinase inhibitors Genistein and Lavendustin A at concentrations that inhibit tyrosine kinase activity. Removal of staurosporine from cell culture media resulted in a rebound in PKC activity and synchronized DNA synthesis in EKVX cultures. The reversibility of the inhibition was noted even after 5 days of treatment with staurosporine, and DNA synthesis remained synchronized for at least two rounds of cell replication after removal of staurosporine. Flow cytometric analysis confirmed that more than 90% of the cell population was blocked in the G1 phase after cells were treated with staurosporine for 24 h. Agents such as staurosporine may be useful for synchronizing cell populations to study cell-cycle specific biochemical events important for the regulation of cell replication in the EKVX cell line.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Comparison of CD3 and CD2 activation pathways in T cells from young and elderly adults.

The ability of purified T cells to be activated by immobilized anti-CD3 and soluble anti-CD2 monoclonal antibodies (mAbs) was compared using cells from young and old donors. Purified T cells from elderly humans activated with immobilized anti-CD3 mAb incorporated less [3H]thymidine (58,780 vs 92,258 cpm; p < 0.02) into cellular DNA, and secreted less IL-2 into the culture supernatants than did T cells from young donors. In contrast, T cells activated with anti-CD2 mAbs displayed no age-related differences in proliferation or IL-2 production. Anti-CD2 stimulation resulted in equal IL-2 synthesis by cells from young and old donors that was comparable to the amount produced by cells from elderly donors stimulated with immobilized anti-CD3. Northern blot analysis of early cell cycle gene expression by anti-CD2 activated T cells demonstrated no age differences in the expression of p55 IL-2R or c-myc specific mRNA, although T cells from elderly individuals activated with immobilized anti-CD3 showed statistically significant decreases in both mRNAs. T cell receptor beta chain mRNA levels did not differ between cells from young or old donors after activation by either anti-CD3 or anti-CD2. The discordance in proliferative ability, IL-2 secretion, and specific mRNA expression between T cells from elderly donors activated through the CD3-TCR complex or by soluble anti-CD2 mAbs provides additional evidence for a multifactorial causation of age-related T cell proliferative defects, and may indicate that the difference in proliferative ability is, in part, attributable to responsiveness to secreted IL-2.

Adult↗
PubMed1990

Increased levels of circulating interleukin 6 in burn patients.

The serum levels of interleukin 6 (IL-6) were determined in a population of burn patients. In all patients, IL-6 levels were increased over a 3-week interval with peak concentrations reached during the first week after injury. Patients receiving intravenous polymyxin B therapy according to a regimen designed to reduce endotoxemia manifested greatly reduced levels of both circulating endotoxins and IL-6. Certain patients not treated with polymyxin B showed extraordinarily large increases in IL-6 which were associated with lethal or life-threatening clinical complications. Increased IL-6 levels were also associated with decreased percentage of circulating T cells and corresponding increases in B cells. However, IL-6 did not produce any direct inhibitory effects in vitro on T cell representation or function.

Biological Factors↗
PubMed1989

Modulation of major histocompatibility complex antigens and inhibition of proliferative activity of YAC lymphoma cells by a natural killer lysis resistance-inducing factor (NK-LRIF).

Concanavalin-A-activated rat spleen cells secrete a natural killer lysis resistance-inducing factor (NK-LRIF) distinct from interleukin-2 and interferon, which induces resistance to NK-cell-mediated lysis in YAC tumor cells. In order for NK-LRIF to have an effect on YAC cells, several hours of incubation is required. When NK-LRIF-treated YAC cells are washed and cultured in the absence of NK-LRIF, normal NK susceptibility is regained. YAC cells treated with NK-LRIF show a significant decrease in the rate of proliferation as judged by changes in cell numbers and rate of thymidine incorporation. Cell cycle studies indicate that the proportion of G0/G1 phase cells increases in YAC preparations treated with NK-LRIF. Major histocompatibility complex (MHC) class I antigen expression is markedly enhanced on YAC cells incubated with NK-LRIF but the expression of MHC class II antigens and Thy-1 antigen remains unchanged. No effect of NK-LRIF treatment on the capacity of YAC cells to bind effector spleen cells could be demonstrated.

Animals↗
PubMed1988

Decreased proliferation, interleukin 2 synthesis, and interleukin 2 receptor expression are accompanied by decreased mRNA expression in phytohemagglutinin-stimulated cells from elderly donors.

Using cDNA probes to human interleukin 2 (IL2) and interleukin 2 receptor (IL2R), the amount of IL2 and IL2R mRNA produced by PHA stimulated peripheral blood mononuclear cells from young (less than 40 yr) and old (greater than 60 yr) donors was quantitated. Stimulated cell cultures from each individual were also examined for proliferative ability, expression of membrane IL2R, membrane IL2R density, and for the amount of IL2R shed into the culture supernatant. Induction of IL2 and IL2R mRNAs were decreased in cells from elderly individuals, as were the levels of IL2 secretion, the percentage of IL2R+ T cells and the density of membrane IL2R per cell. The results suggest that decreased expression of both IL2 and IL2R mRNA contributes to the low synthesis of IL2 and membrane IL2R, respectively, and is partially responsible for the diminished proliferative activity observed in lymphocytes from the elderly.

Aging↗
PubMed1987

Regulation of interleukin 2 and interleukin 2 receptor gene expression in human T cells: I. Effect of Ca2+-ionophore on phorbol myristate acetate co-stimulated cells.

Human peripheral blood T lymphocytes were treated with phorbol myristate acetate (PMA), an activator of protein kinase C (PKC) activity, and with the calcium ionophore A23187. The resulting accumulation of specific mRNA for interleukin 2 (IL-2) and interleukin 2 receptor (IL-2R), as well as IL-2 secretion and membrane IL-2R expression were examined. At low concentrations (0.1 microM), A23187 synergized maximally with PMA to induce proliferation, to increase IL-2R mRNA levels and the expression of membrane IL-2R, and to produce a low but sufficient accumulation of IL-2 mRNA and IL-2 secretion. A high concentration of A23187 (1.0 microM) did not show any synergism for the accumulation of IL-2R mRNA, membrane IL-2R expression and inhibited the proliferation of PMA co-stimulated T cells. It did, however, induce maximum accumulation of IL-2 mRNA and IL-2 secretion.

Calcimycin↗
PubMed1987

Impaired phorbol ester and calcium ionophore induced proliferation of T cells from old humans.

Current models of T cell activation implicate increases in intracellular free Ca2+ concentration and activation of the Ca2+ and phospholipid dependent enzyme protein kinase C (PKC) as important early events leading to interleukin 2 (IL-2) production, interleukin 2 receptor (IL-2R) expression, and subsequent cell proliferation. The present study examined the age-related defect in T cell proliferation to determine if signals that activate PKC and increase intracytosolic free Ca2+ concentration might be defective. Using phorbol myristate acetate (PMA), which directly activates PKC, and Ca2+ ionophore A23187, which increases intracellular cytoplasmic free Ca2+ concentration, the induction of IL-2 secretion, IL-2R expression and cell proliferation were studied. The results demonstrate that following stimulation with PMA and A23187, purified T cells from elderly subjects demonstrate low levels of IL-2 production, IL-2R expression and cell proliferation. Exogenous purified human IL-2 did not fully correct the low proliferative responses of T cells from old donors, however, did markedly boost the response. While it appears that the inability of T cells to express IL-2R and respond to IL-2, along with a lower endogenous IL-2 production are limiting factors in cell proliferation, the inability of PMA and A23187 to correct this defect suggests that the early phases of signal transduction per se are probably not a primary cause of the immunodeficiency seen in ageing.

Adult↗
PubMed1986

Phenotypic expression of natural killer cell associated membrane antigens and cytolytic function of peripheral blood cells from different aged humans.

The function and phenotypes of peripheral blood NK cells from different aged adults were examined using a 4 hr 51Cr release NK assay and laser flow cytometry utilizing the Leu-7 and Leu-11 monoclonal antibodies. It was found that there was no difference between aged and young adults in the expression of Leu-7+, Leu-11+, Leu-7-11+ or Leu-7+11+ cells. A significant decrease, however, was seen in both percent representation and absolute number of Leu-7+11- cells in older subjects. When examined by age and gender, women aged 17-59 yr had a significantly lower percent representation and absolute number of Leu-11+, Leu-7-11+ and Leu-7+11+ cells and baseline NK activity than did men of similar age. No differences were seen between men and women aged 60-84 yr. No differences were seen in the degree of augmentation by interleukin-2 of NK function in any group. Our data indicate that NK function is correlated with cells bearing the Leu-11 surface antigen and that the gender of the cell donor is a significant factor in the representation of NK cells and function in persons under 60 yr of age. Studies on age differences in NK number and function must therefore take into account the percent of women in the populations studied.

Adolescent↗
PubMed1985

G0 B cells activated by anti-mu acquire the ability to proliferate in response to B cell-activating factors independently from entry into G1 phase.

In the present studies we investigated the early steps in B cell activation and determined that activation could be separated from entry into cell cycle. Purified B cells from BALB/c nu/nu mice, activated under controlled conditions by affinity-purified rabbit IgG anti-mu or F(ab')2 fragments prepared from the same antibodies, were sorted according to size by flow cytometry. Approximately 80% of the B cells were small and were shown to be in Go state by quantitative RNA analysis and by [3H]uridine and [3H]thymidine incorporation studies. The sorted Go B cells, when again incubated in serum-free medium with the appropriate anti-mu preparations, remained in Go. However, Go B cells in the presence of anti-mu have undergone significant change(s), in as much as they were now able to proliferate in response to soluble mediators. The ability to develop a proliferative response to B cell-activating factor(s), acquired independently from entry into cell cycle, characterizes another important step in early B cell activation and also indicates that Go B cells characterized on the basis of cell size, density, and/or RNA content may be in an activated state.

Animals↗
PubMed1984

Analysis of immune status of hemodialyzed adults: association with prior transfusions.

Peripheral blood leukocytes of 29 hemodialyzed adults, 19 transfused and 10 nontransfused, were studied using immunofluorescent staining with monoclonal antibodies and in vitro measurement of natural killer (NK) cell activity. When compared with control subjects, the absolute number of leukocytes in transfused hemodialyzed patients was significantly reduced (P less than 0.01), as were the absolute numbers of OKT11+ cells (P less than 0.01), and OKT4+ cells (P less than 0.0001). The percent representation of OKT11+ and OKT4+ cells was also significantly lower among transfused hemodialyzed patients (P less than 0.01 and 0.001, respectively), and this loss of OKT4+ cells resulted in a decrease in the ratio of OKT4+/OKT8+ cells (P less than 0.01). The absolute number of Leu-7+ cells was also decreased in the transfused group (P less than 0.05). A decrease in in vitro NK cell activity was present in both transfused and nontransfused hemodialyzed subjects. Whether these differences in peripheral blood lymphocytes were induced by the erythrocyte transfusions could not be determined; however, if they reflect changes in central lymphoid tissues, then these results may help explain the prolonged survival of renal allografts in transfused individuals.

Adult↗
PubMed1983

Monoclonal antibody analysis of T-lymphocyte subsets in young and aged adults.

Eleven monoclonal antibodies identifying surface antigens present on human T lymphocytes were utilized to investigate the effects of advanced age on peripheral blood lymphocyte subsets. Both the proportion and number of lymphocytes recognized by five antibodies reactive with 'pan' T cell antigens (OKT3, OKT11, Leu4, T101 and Lyt3) decreased with age. The percentage of helper/inducer (OKT4+, Leu3a+) cells remained constant; however the proportion of Leu2a+, suppressor/cytotoxic cells declined. There was no change with age in the percent representation of OKT9+ or OKT10+ cells, nor in the ratio of helper/inducer to suppressor/cytotoxic cells (OKT4+/OKT8+ or Leu3a+/Leu2a+). Absolute numbers of helper/inducer (OKT4+, Leu3a+), suppressor/cytotoxic (OKT8+, Leu2a+), OKT9+ and OKT10+ cells were lower in elderly individuals as the result of lymphocytes constituting a lower percentage of the peripheral blood white cell population in this age group. While only small differences existed between the lymphocyte populations of young and aged men; aged women, compared to young women, had more dramatic shifts in their T cell populations. Comparison of antibodies putatively identifying similar (the same) functional groups of T cells demonstrated excellent correlation between the percentage of cells enumerated with the antibodies OKT3+: Leu4+ (r = .951), OKT4+: Leu3a+ (r = .914), OKT8+: Leu2a+ (r = .896), and in the ratio of helper/inducer to cytotoxic/suppressor OKT4+/OKT8+: Leu3a+/Leu2a+ (r = .926) cells.

Adult↗