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Biomedical subjects

L S Kucera

Publications and source records attributed to L S Kucera.

At least 19 recordsLinked to original sources
PubMed1991

Priming of rabbit alveolar macrophages for enhanced oxidative responses by herpes simplex virus type 2 infection.

The effect of herpes simplex virus type 2 (HSV-2) infection on the oxidative response in infant and adult rabbit alveolar macrophages (AM) was studied using either phorbol myristate acetate (0.5 microgram PMA/ml) or latex (250 micrograms/ml) as eliciting agents in a chemiluminescence (CL) assay. Results indicated that uninfected infant AM responded to a latex-elicited but not PMA-elicited CL response. HSV-2 infection (moi = 1.0) of infant AM for 2 hr at 37 degrees C did not alter the PMA or latex-elicited CL responses. In contrast, uninfected adult AM exhibited a markedly increased CL response when elicited with either PMA or latex. HSV-2 infection (moi = 1) of adult AM for 2 hr further increased both PMA- and latex-elicited CL responses. Increasing the moi to 10 inhibited both PMA- and latex-elicited CL responses. Incubation of uninfected control and HSV-2 infected adult AM for 18 hr at 37 degrees C resulted in spontaneous priming of the cells for increased CL responses. In the absence of PMA HSV-2 alone failed to elicit a CL response in adult AM. Infection with heat-inactivated HSV-2 (moi = 1.0 before heat inactivation) did not prime adult AM for enhanced CL responses. AM from BCG immunized adult rabbit produced a considerably higher level CL response that nonimmunized AM; however, HSV-2 infection of these cells did not further enhance the response. In summary, these data indicate that adult AM but not infant AM can be primed by active HSV-2 infection for an increased CL response elicited by either PMA or latex.

Animals↗
PubMed1991

Synthesis and evaluation of novel ether lipid nucleoside conjugates for anti-HIV-1 activity.

Combinations of an amidoalkylphosphocholine, 8, and AZT have been found to cause an apparent synergistic action in suppressing infectious HIV-1 replication. In addition, amidoalkyl, oxyalkyl, and thioalkyl ether lipids have been chemically linked to anti-HIV-1 nucleosides (AZT and DDI) through phosphate and phosphonate linkages. These conjugates have shown promising in vitro anti-HIV-1 activity. Also, the conjugates have a 5-10-fold reduction in cell cytotoxicity compared to AZT alone. The most active compound, an amidoalkyl ether lipid-AZT conjugates, 4A, was found to have a differential selectivity of 1793 in a syncytial plaque assay. In comparison, AZT alone has a value of 1281.

Antiviral Agents↗
PubMed1990

Novel membrane-interactive ether lipid analogs that inhibit infectious HIV-1 production and induce defective virus formation.

A new class of membrane-active ether lipid (EL) analogs of platelet-activating factor were studied for in vitro anti-HIV-1 activity. Human T-cell (CEM-ss) monolayers or suspension cultures were used to determine effects of structural modifications of Type A phosphorus-containing and Type B nonphosphorus EL analogs on (a) the inhibitory concentration50 (IC50) for HIV-1 syncytial plaque formation and cell growth, and, (b) virus budding at the cell plasma membrane. Results indicate that representative Type A and Type B EL inhibit HIV-1 but not herpes simplex virus type 2 plaque formation when added before or up to 2 days after viral infection. Anti-HIV-1 activity does not involve direct inactivation of virus infectivity. Type A EL (IC50 range = 0.2-1.4 microM) with alkyoxy, alkylthio, or alkyamido substitution at glycerol position 1 and ethoxy or methoxy substitution at position 2, and Type B compounds (IC50 range = 0.33-0.63 microM) with an inverse choline or nitrogen heterocyclic substitution at position 3 have selective activity against HIV-1-infected T-cells. EL treatment of HIV-1-infected cells is associated with subsequent release of reverse transcriptase activity, but infectious virus production is inhibited with time after infection. Electron microscopic examination of HIV-1-infected and EL-treated cells revealed absence of detectable budding virus at the plasma membrane but presence of intracytoplasmic vacuolar virus particles. In summary, these data suggest that EL analogs are a novel class of agents that induce defective intracytoplasmic vacuolar HIV-1 formation in T-cells. Being membrane interactive, EL are ideally suited for combination chemotherapy with DNA-interactive anti-HIV nucleoside analogs.

Cell Membrane↗
PubMed1990

Human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus type 2 (HSV-2) can coinfect and simultaneously replicate in the same human CD4+ cell: effect of coinfection on infectious HSV-2 and HIV-1 replication.

Experiments were designed to determine whether HIV-1 and herpes simplex virus type 2 (HSV-2) coinfection leads to simultaneous replication of both viruses in the same human CD4+ cell (MT-4 cell line) and the possible effects of coinfection on infectious virus production. Results from transmission electron microscopy analysis revealed replication of typical HSV-2 nucleocapsids in the nucleus and budding of HIV-1 particles through the plasma membrane and through intracytoplasmic vacuoles containing enveloped HSV-2 particles in the same coinfected cell. Coinfection of HIV-1 persistently infected H9IIIB or promonocytic U1 cells with HSV-2 did not alter total production of infectious HSV-2 or the percentage of HSV-2 infectious centers compared with control H9 and U937 cells infected with HSV-2 alone. However, in coinfected promonocytic U1 cells HSV-2 induced infectious HIV-1 production measured by syncytial plaque assay. In summary, both HIV-1 and HSV-2 can coinfect and simultaneously replicate in the same human CD4+ cell. Interactions between HIV-1 and HSV-2 appear to be unidirectional, resulting in accelerated replication of HIV-1 as reported by Albrecht et al. (J Virol 1989;63:1861-1868), but not HSV-2 as shown by us.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Herpes simplex virus type 2 infection of unstimulated human T-lymphocytes.

Unstimulated human leukemia T-cell lines (MOLT-4, MT-4) were tested for their susceptibility to herpes simplex virus type 2 (HSV-2) infection. Permissive infection of MT-4 cells was demonstrated by growth curve and infectious center assays. In growth curve experiments new progeny virus replication was detected by 24 hrs and maximum titers of HSV-2 replication were measured by 72 hrs after infection of MT-4 cells, whereas, MOLT-4 cells did not produce detectable infectious HSV-2 in growth curve experiments. It may be that a T-cell subset is involved with infectious HSV-2 production, since 5.7% of MT-4 cells were scored as infectious centers after HSV-2 infection compared to only 0.06% of MOLT-4 cells. Furthermore, HSV-2 infected MT-4 (45% of cells) and MOLT-4 cells (30% of cells) expressed viral induced antigen(s) detected by immunofluorescence assays. These data provide the first evidence of infectious HSV-2 replication in T-cells not prestimulated in vitro with mitogens, pharmacologic agents or growth factors. The establishment of T-cell systems that permit rapid and efficient replication of HSV-2 could greatly facilitate studies on interactions between human herpesviruses and AIDS retroviruses since recent published evidence indicates possible synergistic interactions between these virus groups.

Antigens, Viral↗
PubMed1989

Studies of phospholipase and cyclooxygenase activities in herpes simplex virus type 2-transformed rat cells.

In this report we describe results showing that both arachidonic acid (20:4) release and prostaglandin (PG) synthesis in herpes simplex virus type 2 (HSV-2)-transformed tumor-derived rat fibrosarcoma (RFS) cells are inhibited by indomethacin (cyclooxygenase inhibitor). These data suggest that cyclooxygenase (prostaglandin endoperoxide synthetase, PES) and phospholipase are coupled in their regulation in RFS cells. Data obtained using a cell-free assay to measure directly cyclooxygenase specific activity in the absence of phospholipase activity indicate that constitutive cyclooxygenase activity is very low in non-transformed REF and is not induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast, after an initial degradation of constitutive enzyme, cyclooxygenase is synthesized de novo following TPA treatment of HSV-2-transformed RFS clonal cells. At least a 5-fold induction of cyclooxygenase occurs by 6 h post-treatment of HSV-2-transformed RFS clonal cells. This induction is inhibited by pretreatment of cells with cycloheximide (protein synthesis inhibitor) 1 h prior to TPA treatment. Results obtained following delayed addition of cycloheximide to cells after TPA treatment indicate that the newly synthesized cyclooxygenase has a half-life of approximately 5 min. In addition, HSV-2-transformed RFS clonal cells at low passage (less than 10) show over a 3-fold greater increase in cyclooxygenase specific activity after TPA treatment compared to that in TPA-treated high passage (greater than 20) cells, suggesting that induction of high levels of PG synthesis is transient in these cells. These data indicate that HSV-2 transformation of REF cells is associated with alteration of cyclooxygenase regulation.

Animals↗
PubMed1988

Phospholipid-sensitive, Ca2+-dependent protein kinase activity in rat embryo fibroblasts transformed by herpes simplex virus type 2.

Increased cytosolic phospholipid-sensitive, Ca2+-dependent protein kinase C (PK-C) activity is correlated with the highly tumorigenic potential of rat embryo fibroblasts transformed by herpes simplex virus type 2 (HSV-2). Treatment of the cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) caused a decrease in the cytosolic PK-C with a concomitant increase in PK-C recovered in the membrane fraction. Translocation of the PK-C was dependent upon length of exposure to the phorbol diester. PK-C activity in the cytosolic fraction could be stimulated by TPA without the addition of phosphatidylserine and diacylglycerol. It is tempting to speculate that HSV-2 induction of cellular PK-C activity may be important in phosphorylation of proteins needed for promotion of HSV-2-induced carcinogenesis.

Animals↗
PubMed1987

L-fucose, D-mannose, L-galactose, and their BSA conjugates stimulate macrophage migration.

The effect of selected monosaccharides on the random migration of normal adult rabbit alveolar macrophages (AM) was investigated. It was observed that 10 mM of L-fucose, L-galactose, or D-mannose stimulated AM migration 1.5-2.0 times. In addition, derivatives of L-fucose and D-mannose occupying the carbon-6 position such as L-fucosyl-lactose, D-mannose-6-phosphate, D-mannitol, and mannan enhanced the migration of AM, whereas derivatives of L-fucose and D-mannose in the carbon-1 position produced no migration enhancement. Macrophage migration enhancement activity that was produced spontaneously by spleen cell cultures from normal young rabbits was destroyed by treatment with L-fucosidase. Accordingly, the migration enhancement factor (MEF) found in spleen cell culture supernatants appeared to depend on L-fucose conjugated to some protein carrier because MEF was non-dialyzable. When normal adult AM were treated with L-fucosidase, they lost their responsiveness to migration inhibitory factor (MIF) but retained their responsiveness to MEF. We have interpreted this to mean that the MIF and MEF receptors are distinct. Synthetic MEFs were prepared by conjugating L-fucose, D-mannose, of L-galactose to bovine serum albumin (BSA). It was noted that these sugar-BSA conjugates were about 200 times more effective than the corresponding free sugars in producing migration enhancement. In addition, these sugar-BSA conjugates neutralized MIF activity in a migration inhibition test.

Animals↗
PubMed1987

Induction of cellular functions in spontaneously immortalized rat-2 cells transfected with cloned herpes simplex virus type 2 (HSV-2) DNA.

Experiments were done to determine if cloned transforming sequences from herpes simplex virus type 2 (HSV-2) DNA confer upon transfected Rat-2 cells the capacity to be stimulated in phospholipase and cyclooxygenase activities following 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Tumor-derived Rat-2 cells transformed with sub-fragments (BamHI-E, HindIII/HpaI-ED, or PstI-C) overlapping the right-hand end of the BglII-C transforming region of HSV-2 DNA were stimulated by TPA in both phospholipase activity, measured by deacylation of arachidonic acid, and cyclooxygenase activity, measured by prostaglandin synthesis. Non-transfected Rat-2 control cells showed no increase in these enzyme activities following TPA treatment. To our knowledge, this is the first evidence that cloned HSV-2 DNA has the capacity to induce cellular functions (phospholipase and cyclooxygenase) in transformed mammalian cells.

Animals↗
PubMed1986

Lipid composition of alveolar macrophage plasma membrane during postnatal development.

This study was undertaken to define the age-related alterations in lipid composition that resident rabbit alveolar macrophages (AM) undergo during postnatal development. The eventual goal is to correlate these changes with the functional maturation of these cells. The number of AM recorded from total lung lavages rose markedly during the first 14 days of life, from 4.9 X 10(5) to 1.1 X 10(7). Adult lungs yielded 1.1 X 10(8) AM. A gradual but significant increase in fluorescence polarization (P) was observed during development when purified AM plasma membranes were tagged with the probe 1,6-diphenyl-1,3,5 hexatriene trimethyl ammonium. The rise ranged from a mean P value of less than or equal to 0.22 to 0.24 (p less than 0.001) for AM plasma membranes from rabbits 1- or 7-day-old to 30- or 150-day-old rabbits, respectively. This finding suggests that the fluidity of the AM plasma membrane decreased during postnatal development. Palmitic, stearic, oleic, and linoleic acids were the most prevalent fatty acids found in the neutral lipid fraction of the AM plasma membrane throughout development. The content of stearic acid rose from 10 to 16%, arachidonic acid rose from 2.8 to 9%, myristic acid decreased from 3.2 to 1.3%, palmitic acid decreased from 42 to 36%, and oleic and linoleic acids changed relatively little during the first 30 days of life. The levels of docosatetraenoic and docosapentaenoic increased gradually during the first 14 days of life, and by 30 days of life the levels declined to that observed at birth. The sum of these changes resulted in an increase in the ratio of unsaturated to saturated fatty acids (1 to 1.15) in the neutral lipid fraction. During the first month of life, the neutral lipid fatty acid pool in the total lipid fraction of AM plasma membrane increased from 12 to 18 mole %, cholesterol increased from 7 to 14 mole %, and total phospholipids decreased from 81 to 67 mole %. These changes resulted in increasing the cholesterol to phospholipid ratio from 0.09 at birth to 0.23 by 150 days of life. The levels of all three major lipid fractions were comparable at 30 days and 150 days of life. Adult levels of choline phosphoglycerides, the predominant phospholipid, were observed by 7 days of life to have decreased from 47 to 34.5 mole %, and the levels of ethanolamine phosphoglycerides and sphingomyelin increased from 17.5 to 25 mole % and from 9 to 13 mole %, respectively. Adult levels of lyso-bis-phosphatidic acid were reached by 30 days of life increasing from 8.2 to 17.8 mole %.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1985

Immunologic characterization of herpes simplex virus type 2 antigens ICP10 and ICSP11/12.

Infected cell protein 10 (ICP10) or antigen 4 (Ag4) and infected cell-specific protein 11/12 (ICSP11/12) have been suggested as specific antigenic markers for cervical carcinoma. Experiments were designed to determine whether ICP10 and ICSP11/12 are distinct antigens and to determine the cellular localization of ICP10. Results indicate that an apparent 160 kdalton (kDa) protein analyzed by 8.5% polyacrylamide gels (= 144 kDa protein analyzed by 7.0% polyacrylamide gels) was detected in HSV-2-infected but not mock-infected extracts. This protein is an early virus-induced protein appearing 2-4 h after HSV-2 infection, and it was synthesized in the presence of successive blocks with cycloheximide and actinomycin D. These properties are characteristic for ICP10 (Ag4), thus establishing the identity of the 160 kDa/144 kDa protein as ICP10. Furthermore, Western blot analyses indicated that ICP10 and ICSP11/12 are distinct antigens recognized by antibodies in sera from immune rabbit or human cervical carcinoma patients. In addition, monoclonal antibodies to the HSV-2-induced ribonucleotide reductase were reactive with ICP10. Antibodies in sera from rabbits immunized against ICP10 and monoclonal antibodies to the HSV-2-induced ribonucleotide reductase were reactive with antigens on the plasma membrane surface of HSV-2-infected cells. Also, the reactivity of monoclonal antibodies with these antigens was blocked by the rabbit antibodies based on immunofluorescence analyses. These data provide evidence that ICP10 is antigenically distinct from ICSP11/12, and that ICP10 is present on the plasma membrane of HSV-2-infected cells. Also, our data confirm and extend the tentative identification of ICP10 with the HSV-2-induced ribonucleotide reductase recently suggested by Bacchetti et al. (J. Virol. 49, 591-593, 1984).

Antigens, Viral↗
PubMed1982

Detection of herpes simplex virus type 2 glycoproteins expressed in virus-transformed rat cells.

Rat embryo fibroblasts transformed by herpes simplex virus type 2 (HSV-2) were assayed for the expression of certain virus-specific glycoproteins on the surface membranes. Monospecific antisera to HSV-2-specific glycoproteins, designated gAgB, gC, and gX, were used in membrane immunofluorescence studies with HSV-2-transformed cell lines tREF-G-1, tREF-G-2, and a tumor-derived rat fibrosarcoma cells line produced in syngeneic rats inoculated with tREF-G-1 cells. Analysis of the three HSV-2-transformed cell lines showed that antisera to the gAgB and gX glycoproteins were reactive with these cells. In contrast, no significant reactivity was observed when anti-gC serum was reacted with the HSV-2-transformed cell lines. All three antiglycoprotein sera reacted positively with rat cells productively infected with HSV-2. Additionally, the HSV-2-transformed and tumor-derived cell lines showed positive internal immunofluorescence after reaction with antiserum to an early, nonstructural viral protein designated VP143 (molecular weight, 143,000). Infectivity of HSV-2 in standard plaque assays was neutralized by hyperimmune rat antisera to tREF-G-2 or rat fibrosarcoma cells and to HSV-2 virions and by sera from rats bearing the fibrosarcoma. Adsorption of rat-anti-HSV-2 serum with tREF-G-2 or rat fibrosarcoma cells reduced neutralizing activity to 10 and 12%, respectively, compared with 90% neutralization by antiserum adsorbed with nontransformed rat embryo fibroblast cells and 100% neutralization with unadsorbed antiserum. In summary, HSV-2-transformed rat cells retained and expressed genetic information necessary for the production of HSV-2 glycoproteins and a nonstructural protein after high passage in tissue culture or in the syngeneic host.

Animals↗
PubMed1981

Phospholipid synthesis in human embryo fibroblasts infected with herpes simplex virus type 2.

The effect of herpes simplex virus type 2 infection on the synthesis of phospholipids in human embryo fibroblasts was determined at temperatures permissive (35 C) or nonpermissive (42 C) for virus replication. Incorporation of [32P]i was decreased by herpes simplex virus type 2 infection after 6 hr, which corresponds to the time of initiation of progeny virus production. No differences were observed in the relative incorporation of [32P]i phospholipid classes. In another series of experiments cells were labeled with [3H]ethanolamine before infection and with [14C]ethanolamine after infection. The incorporation of [14C]ethanolamine was also decreased after 6 hr of infection. When choline was substituted for ethanolamine, a similar, although less pronounced, decrease in incorporation was seen in infected cells compared to mock-infected cells. During abortive infections at 42 C, incorporation of [3H]thymidine into cellular DNA was stimulated, but the incorporation of phospholipid precursors was decreased. Total phospholipid composition and phospholipid acyl group composition were not changed appreciably during abortive or productive infection, regardless of whether the cells were labeled before or after infection. In conclusion, these data indicated that, during herpes simplex virus type 2 infection, the incorporation of lipid precursors into phospholipid was decreased. The stimulation of cellular DNA synthesis previously observed during abortive infection at 42 C was not paralleled by a detectable stimulation of total phospholipid synthesis. Neither productive nor abortive infection resulted in significant phospholipid compositional changes in the host cell; however, both resulted in a marked inhibition of phospholipid synthesis.

Cells, Cultured↗
PubMed1981

Aminoacyl fucosides as possible biochemical markers at tumorigenic and metastatic potential in herpes simplex virus type 2-transformed rat cells.

Two classes of aminoacyl fucosides termed FL3 and FL4 were studied as possible markers of tumorigenic and metastatic potential in herpes simplex virus type 2 transformed rat cells. In the present study, clonal cell lines of transformed highly tumorigenic and metastatic (t-REF-G-1.1), weakly tumorigenic and nonmetastatic (t-REF-G-2.1), nontumorigenic (t-REF-G-2.0), and secondary nontransformed rat embryo fibroblast cells were labeled with [3H]fucose, and cell extracts were analyzed for ratio of radioactivity incorporated into FL3 and FL4. Results indicated that, in extracts from t-REF-G-2.0 and nontransformed rat embryo fibroblast cells, the ratios of FL4/FL3 were 5.78 and 5.71, respectively. In contrast, t-REF-G-2.1 cells exhibited a FL4/FL3 ratio of 1.45, while t-REF-G-1.1 cells exhibited a FL4/FL3 ratio of 0.74. In subclonal cell lines isolated from TPA-treated and mock-treated t-REF-G-2.1 cells, the FL4/FL3 ratios correlated with the tumorigenic and metastatic potential of these subclones in newborn syngeneic White Buffalo rats. These data suggested that alterations in fucose-labeled components can be used to predict the tumorigenic and metastatic potential of herpes simplex virus type 2-transformed rat cells.

Aminoglycosides↗