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Biomedical subjects

J Rusnak

Publications and source records attributed to J Rusnak.

10 recordsLinked to original sources
PubMed1999

Protein kinase C inhibition by UCN-01 induces apoptosis in human glioma cells in a time-dependent fashion.

Recent studies in our laboratory have shown that UCN-01 (7-hydroxystaurosporine), which is a derivative of the non-selective protein kinase inhibitor staurosporine that exhibits relative selectivity for protein kinase C (PKC), is a potent inhibitor of glioma growth in in vitro and in vivo models. This agent exhibits both cytotoxic and cytostatic effects, depending on the time period of drug exposure. In the present study, we examined whether UCN-01-induced cytotoxicity correlated with the induction of apoptosis, and characterized further the time course of this process as a prelude to application of UCN-01 in clinical trials. We first demonstrated that the cytotoxic effects of UCN-01 were associated with the induction of morphological features of apoptosis. Secondly, we identified electrophoretic features of apoptosis semiquantitatively at a series of time points using field inversion gel electrophoresis. These studies showed a peak in the induction of high-molecular-weight DNA fragmentation after 3-6 days of drug treatment. Thirdly, we measured the percentage of cells undergoing apoptosis at various time points using a terminal transferase-catalyzed in situ end-labeling technique, which confirmed a time- and concentration-dependent increase in apoptotic cell numbers. This correlated with a progressive decrease in the percentage of cells that were viable as assessed by trypan blue exclusion. Cell killing peaked within 2-4 days after beginning UCN-01 treatment, but continued at a lower level in the ensuing days. Taken together, these studies demonstrated that extended periods of exposure to UCN-01 are needed for optimal manifestation of cytotoxic effects against glioma cells, a factor that must be taken into consideration in the design of future clinical trials with this agent for malignant gliomas.

Alkaloids↗
PubMed1998

HIV-1 infection induces a selective reduction in STAT5 protein expression.

HIV-1 infection is accompanied by qualitative and quantitative defects in CD4+ T lymphocytes. Loss of immune function in HIV patients is usually associated with a profound dysregulation of cytokine production. To investigate whether cytokine signaling defects occur during HIV infection, PHA blasts from healthy human donors were infected with two strains of HIV-1 and screened for the expression of STAT proteins used in cytokine signaling. A selective decrease in STAT5B was seen 8 days after infection with the BZ167 dual-tropic HIV isolate, but not with the Ba-L, M-tropic strain. Based on these findings, purified T cells from HIV-infected patients in different stages of disease were also tested for STAT expression; decreases in STAT5A, STAT5B, and STAT1alpha were observed in all patients. The reduction in STATs seen in vivo and in vitro after HIV infection may contribute to the loss of T cell function in HIV disease.

Cells, Cultured↗
PubMed1997

In vitro immunologic and virologic effects of interleukin 15 on peripheral blood mononuclear cells from normal donors and human immunodeficiency virus type 1-infected patients.

Interleukin 15 (IL-15) is a cytokine that shares receptor subunits and functional activity, such as T-cell and B-cell stimulation, with IL-2. The effect of IL-2 on immune function and human immunodeficiency virus (HIV) viral load in HIV-infected patients is being actively studied. Thus, we examined how IL-15 compares with IL-2 in several in vitro immunologic and virologic assays in order to explore whether a rationale exists for pursuing initial clinical therapeutic trials with IL-15. The effects of IL-15 on induction of lymphokine-activated killer (LAK) cells, gamma interferon (IFN-gamma) production from HIV-positive peripheral blood mononuclear cells (PBMCs), and HIV production from PBMCs were studied. Induction of LAK cells by IL-15 was found in eight of eight HIV-positive donors. Incubation of PBMCs from some donors with IL-15 (1, 10, 50, and 100 ng/ml) induced production of IFN-gamma. The effect of IL-15 was compared with that of IL-2 on HIV replication in PBMCs from five HIV-positive patients and four HIV-negative donors whose PBMCs were infected in vitro with HIV. Levels of HIV p24 antigen were moderately lower in the presence of 10 ng of IL-15 per ml than with 10 ng of IL-2 per ml, but they were similar for 100 and 500 ng of each cytokine per ml. In summary, IL-15 can induce LAK cell activity in HIV-seropositive patients and can stimulate IFN-gamma production from PBMCs of some donors. IL-15 stimulates levels of HIV production from PBMCs which are similar to or moderately lower than those obtained with IL-2, depending on cytokine concentration.

Cells, Cultured↗
PubMed1996

Molecular analysis of decreased interleukin-12 production in persons infected with human immunodeficiency virus.

Human immunodeficiency virus (HIV) disease is associated with loss of type 1 responses, including interleukin (IL)-12 production. The dramatic drop in p70 production seen at early stages of disease was found not to be associated with a similarly decreased p40 mRNA expression. p35 mRNA expression was more extensively reduced than p40 mRNA expression at these early stages. Monocytes infected in vitro with HIV displayed decreased p35 expression and p70 production, suggesting that such decreased IL-12 expression may contribute to reduced IL-12 production in HIV-positive patients' cells. In addition, treatment of cells with IL-10 increased IL-10 mRNA expression and decreased p40 expression in both HIV-positive and -negative cells, while neutralization of IL-10 increased p40 mRNA levels. These observations, together with the observed hyperproduction of IL-10 in HIV-positive patients, may explain the dysregulation of IL-12 production seen in HIV disease.

Base Sequence↗
PubMed1996

Antigen-stimulated apoptotic T-cell death in HIV infection is selective for CD4+ T cells, modulated by cytokines and effected by lymphotoxin.

OBJECTIVE: To characterize the mechanism of in vitro antigen-induced apoptotic T-cell death in the peripheral blood mononuclear cells (PBMC) of HIV-1-infected individuals. DESIGN AND METHODS: PBMC from HIV-1 infected and uninfected individuals were unstimulated or stimulated with HIV-1 envelope synthetic peptides (Env) or influenza A virus to determine the extent of antigen-stimulated apoptotic T-cell death, whether this death was limited to the CD4+ subset, and the effects of cytokines on T-cell death. Death was assessed by apoptotic nuclear morphology after 7 days of culture by fluorescence microscopy using a DNA-specific dye. Transwell cultures and supernatant transfers were utilized to test whether a soluble factor produced by HIV-positive PBMC induced death of HIV-negative T cells. Exogenous cytokines [interleukin (IL)-12, interferon (IFN)-gamma, IL-4 and IL-10], as well as antibodies against endogenously produced cytokines (IL-4, IL-10, IL-12, and lymphotoxin) were tested for their ability to modulate death. RESULTS: Antigenic stimulation induced death in PBMC from HIV-positive donors, but not in PBMC from HIV-negative donors. Antigen-stimulated death was seen in CD4+ but not CD8+ T-cell subset from the HIV-positive patients. Apoptotic death was blocked by IL-12, IFN-gamma, anti-IL-4, anti-IL-10, and anti-lymphotoxin, but not by anti-IL-12. Transwell and supernatant transfer experiment indicated that antigen-stimulated HIV-positive PBMC produced a factor that killed T-cell blasts. The factor was inhibited by anti-lymphotoxin, but not by anti-IL-10. CONCLUSIONS: Stimulation of HIV-positive PBMC with CD4-dependent antigens results in selective death of CD4+ T cells that is modulated by cytokines. Our results suggest that apoptotic death is not limited to HIV-infected or HIV-specific T cells, but occurs in bystander cells. Lymphotoxin is a mediator of antigen-stimulated T-cell death in this in vitro model.

Antibodies↗
PubMed1994

Treatment of murine cryptosporidiosis with anticryptosporidial immune rat bile.

Persistent cryptosporidiosis was established in nu/nu BALB/c mice by oral inoculation with Cryptosporidium parvum oocysts. The model was used to determine the impact of anticryptosporidial immune rat bile on the resolution of the disease. Presence of C. parvum-specific IgA in the immune rat bile was determined by enzyme-linked immunosorbent assay. Infection of mice was verified by stool analysis for oocytes and by hematoxylin and eosin-stained intestinal sections from control mice (infected but untreated). Efficacy of treatment was determined in control and treated mice by analysis of identical, hematoxylin and eosin-stained sections of the small intestine and cecum. Semi-quantitative comparisons were made by determining the percent of crypts infected with Cryptosporidium organisms. The scores of treated mice were significantly lower then controls. Microscopic analysis of intestinal sections showed less villus atrophy, crypt hyperplasia, and fewer organisms per crypt in the immune bile-treated mice than in controls. These results support a role for humoral immunity in the eradication of cryptosporidiosis.

Animals↗
PubMed1989

Detection of Cryptosporidium oocysts in human fecal specimens by an indirect immunofluorescence assay with monoclonal antibodies.

An indirect fluorescence assay (IFA) with monoclonal antibodies developed for the detection of Cryptosporidium oocysts in fecal smears (Meridian Diagnostics, Inc., Cincinnati, Ohio) was compared with the Zeihl-Neelsen-modified acid-fast stain (MAFS) in 119 human fecal specimens collected between 1984 and 1987. The sensitivity of the IFA was 100%; all 56 specimens positive by MAFS exhibited fluorescence. There were 63 specimens negative for Cryptosporidium sp. by MAFS; of these, 61 were negative by IFA (97% specificity). This discrepancy may reflect an increased sensitivity of the IFA to detect oocysts that were not visualized by MAFS because of faint staining or a paucity of organisms. On average, the IFA required less time than the MAFS (1 versus 5 min, respectively) when only rare or few oocysts were present. Cost comparison of reagents showed the IFA to be three times more expensive. The IFA offers a reasonable alternative to the MAFS because of its high sensitivity and specificity, the simplicity of performing it and interpreting results, and its capability of providing a definitive diagnosis of Cryptosporidium oocysts.

Animals↗
PubMed1989

The current role of sonography in the detection of Down syndrome.

The biparietal diameter (BPD)/femur length ratio, nuchal skin thickening, and measured-to-expected femur length ratio have been suggested as sonographic predictors of Down syndrome. In an effort to validate these parameters, we compared their individual and combined prevalence in 22 fetuses with Down syndrome. Using our normative data, the sensitivity and specificity of the BPD/femur length ratio were 36.4 and 93.4%, respectively. Assuming an incidence of Down syndrome in the general population of one in 1000, the estimated positive predictive value was 0.6%. When the BPD/femur length ratio, nuchal skin thickening, and measured-to-expected femur length ratio were combined, the sensitivity was improved (36.4 versus 45.5%) without significantly altering the specificity (93.4% versus 92.3%). A large prospective study is required to verify the utility of sonographic predictors of Down syndrome before their application can be recommended.

Congenital Abnormalities↗