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Biomedical subjects

G A Cabral

Publications and source records attributed to G A Cabral.

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

Differential inhibition of RAW264.7 macrophage tumoricidal activity by delta 9tetrahydrocannabinol.

delta 9tetrahydrocannabinol (THC), the major psychoactive component of marijuana, has been shown to inhibit macrophage cell contact-dependent cytolysis of tumor cells. The purpose of this study was to determine whether THC inhibited macrophage cytolytic function by targeting selectively tumor necrosis factor (TNF)-dependent pathways versus L-arginine-dependent reactive nitrogen intermediates. An in vitro system employing RAW264.7 macrophage-like cells as effectors and TNF-sensitive mouse L929 fibroblasts or nitric oxide (NO.)-sensitive P815 mastocytoma cells as targets, was employed to assess the effect of THC on cytolysis. Macrophages were pretreated with THC or vehicle for 48 hr, subjected to multistep activation with 10 U/ml recombinant mouse gamma-interferon (IFN-gamma) plus 100 ng/ml LPS or to direct activation with 1 microgram/ml LPS, and co-cultured with tumor cells in the presence or absence of THC. THC inhibited TNF-dependent killing by macrophages subjected to either multistep or direct activation. Decreased amounts of TNF-alpha were detected in medium of macrophage cultures treated with THC. In contrast, THC inhibited NO.-dependent cell contact killing only for macrophages subjected to direct activation. Decreased levels of NO2-, a stable degradation product of the short-lived and highly toxic effector molecule NO., were produced by these macrophages. In addition, the effect of the enantiomeric pairs (-)CP55,940/(+)CP56,667 or (-)HU-210/(+)HU-211 on macrophage cell contact-dependent killing was assessed. Inhibition of macrophage tumoricidal activity against TNF-sensitive L929 cells was effected by both isomers of THC analogs. In contrast, both of the enantiomeric pairs had an effect on killing of NO.-sensitive P815 mastocytoma cells only for macrophages subjected to direct activation. These data suggest that cannabinoids inhibit macrophage cell contact-dependent killing of tumor cells by a noncannabinoid receptor-mediated mechanism. However, specific cytolytic pathways are inhibited differentially by cannabinoids depending on the activation stimuli to which macrophages are exposed.

Animals↗
PubMed1995

delta 9-Tetrahydrocannabinol modulates antigen processing by macrophages.

delta 9-Tetrahydrocannabinol (THC) exposure suppresses multiple immunological functions of macrophages. The ability of macrophages exposed to THC to process and present soluble protein antigens was investigated by the stimulation of antigen-specific helper T cell hybridomas to secrete interleukin-2. The T cell response to hen egg lysozyme was dramatically reduced after a 24-hr pretreatment of a macrophage hybridoma with THC. In contrast, THC exposure did not alter the capacity of the macrophage hybridoma to process chicken ovalbumin and augmented their presenting cell function for a pigeon cytochrome c response. These findings could not be attributed to differential effects of THC on either cell viability or expression of the antigen receptor-associated CD3 complex by the T cells. The level of T cell activation with peptides of lysozyme and cytochrome c, which do not require processing, was inhibited only at the highest concentrations of THC, suggesting that THC mainly affects antigen processing. Peritoneal macrophages exposed to THC during an antigen pulse and fixed with paraformaldehyde showed similar effects on the subsequent T cell responses to lysozyme and cytochrome c in the absence of THC, arguing against a possible influence of THC on the T cells. Therefore, THC differentially modulates the capacity of macrophages to process antigens that is necessary for the activation of CD4+ T cells.

Animals↗
PubMed1994

Expression of a cannabinoid receptor in baculovirus-infected insect cells.

A cannabinoid receptor recombinant baculovirus (AcNPV-THCR) has been constructed and employed to express rat neural cannabinoid receptors. Northern analysis of total RNA from Spodoptera frugiperda (Sf9) insect cells infected with AcNPV-THCR revealed novel hyper-production of a 3.3 kb transcript when probed with nick-translated rat cannabinoid receptor cDNA. Optimal viral protein expression was observed in 35S-metabolically labeled AcNPV-THCR-infected Sf9 cells at a multiplicity of infection of 2.5. Transmission electron microscopy of AcNPV-THCR-infected Sf9 cells showed extensive membrane perturbation and electron-dense cytoplasmic perinuclear accumulation, indicative of receptor glycoprotein expression. Immunofluorescence staining using antiserum produced to a fusion protein consisting of the external domain of the cannabinoid receptor and hepatitis B core antigen revealed cannabinoid receptor expression in AcNPV-THCR-infected Sf9 cells. Scatchard-Rosenthal analysis of [3H]CP55,940 receptor binding indicated a Kd of 3.4 nM and a Bmax equal to 3.17 pmol/mg protein. Western immunoblotting performed on AcNPV-THCR-infected Sf9 cell lysates revealed immunoreactive bands with relative molecular weights ranging from 45 to 79 kDa. The predominant species (55 kDa) exhibited a relative molecular weight consistent with that predicted for the translational product obtained from the cannabinoid receptor cDNA coding sequence. In vitro translation using AcNPV-THCR mRNA also yielded a 55 kDa immunoreactive species. These data indicate that the baculovirus expression system is a viable means of expressing relatively large quantities of cannabinoid receptor recombinant protein.

Animals↗
PubMed1994

Inhibition of macrophage inducible protein expression by delta-9-tetrahydrocannabinol.

Macrophages have been shown to undergo a sequential process to full activation in response to priming and triggering signals such as gamma interferon (IFN gamma) and bacterial lipopolysaccharide (LPS). These cells also may be driven directly to full activation by exposure to relatively high concentrations of LPS. Each of the stages to activation is associated with differential protein expression suggesting that newly synthesized proteins are associated with the functional activities attributable to that activation state. These observations indicate that protein profiles may serve as a barometer of the macrophage activation state. Delta-9-tetrahydrocannabinol (THC), the major psychoactive component in marijuana, was shown to inhibit inducible protein expression in response to the priming agents Concanavalin A (Con A) supernatant and IFN gamma. THC also suppressed protein expression in response to LPS. P388D1 and RAW264.7 macrophage-like cells, treated with Con A supernatant or IFN gamma, exhibited restructuring of protein profiles based on iso-Dalt two-dimensional gel electrophoresis. Protein profile restructuring, distinctive from that elicited in response to priming agents, was seen for macrophages treated with LPS. Treatment of macrophages with Con A supernatant, IFN gamma, or LPS in concert with THC (10(-7) M to 10(-5) M), resulted in the generation of protein profiles whose patterns reverted approximately to those of unprimed or unactivated macrophages. THC was shown to alter the expression of select proteins whose induction is associated with macrophage priming or activation. The expression of P388D1 macrophage class II Ia molecules of the major histocompatibility complex (MHC), in response to Con A supernatant and IFN gamma, was inhibited. THC also altered the expression of tumor necrosis factor alpha (TNF alpha) elicited by RAW264.7 cells in response to LPS. These results suggest that THC alters macrophage functional activities, at least in part, by suppressing their capacity to express effector molecules elicited in response to priming and activating signals.

Animals↗
PubMed1993

delta-9-Tetrahydrocannabinol inhibits cell contact-dependent cytotoxicity of Bacillus Calmétte-Guérin-activated macrophages.

The effect of delta-9-tetrahydrocannabinol (delta-9-THC), the major psychoactive component of marijuana, on the capacity of Bacillus Calmétte-Guérin (BCG)-activated macrophages to lyse L929 tumor cells, Naegleria fowleri amoebae, and herpes simplex virus-infected cells was examined. Delta-9-THC inhibited tumoricidal and amoebicidal activity in a dose-related manner. Antiviral activity was decreased when mice received 25 and 50 mg/kg delta-9-THC. The cannabinoid did not directly suppress the activation of macrophages as determined by levels of 5'-nucleotidase activity and did not inhibit splenic T-lymphocytes of BCG-recipient mice from producing interferon gamma. Nomarski optics microscopy, scanning electron microscopy, and radiolabeling binding studies demonstrated that macrophages from delta-9-THC-treated mice retained their capacity to attach to their targets. These results suggest that delta-9-THC suppresses cell contact-dependent amoebicidal, tumoricidal, and antiviral activities of activated macrophages at a stage following effector cell-target cell conjugation.

5'-Nucleotidase↗
PubMed1993

Delta 9-tetrahydrocannabinol inhibition of tumor necrosis factor-alpha: suppression of post-translational events.

Delta 9-tetrahydrocannabinol (delta 9-THC), the major psychoactive component of marijuana, has been shown to suppress macrophage soluble cytolytic activity. The purpose of this study was to determine whether delta 9-THC inhibited this function by affecting tumor necrosis factor-alpha (TNF-alpha). The RAW264.7 macrophage cell line was used as an in vitro bacterial lipopolysaccharide-inducible system for production of TNF-alpha. Macrophage-conditioned medium of RAW264.7 macrophages treated with delta 9-THC was shown to be deficient in tumoricidal activity. Immunoprecipitation experiments demonstrated that the macrophage-conditioned medium of cultures treated with drug contained lower levels of TNF-alpha. Northern analysis indicated that delta 9-THC had no effect on the levels of TNF-alpha messenger RNA. However, radiolabel pulsing and pulse-chase experiments revealed that the intracellular conversion of the 26-kD presecreted form of TNF-alpha to the 17-kD secreted form was inhibited by the drug. These results indicate that delta 9-THC suppresses soluble macrophage tumoricidal activity, at least in part, by decreasing the intracellular conversion of presecretory TNF-alpha to its 17-kD secretory form.

Animals↗
PubMed1992

delta 9-Tetrahydrocannabinol suppresses macrophage extrinsic antiherpesvirus activity.

The effect of delta 9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, on macrophage intrinsic and extrinsic antiherpesvirus activities was examined. THC had no effect on the capacity of the macrophage-like cells RAW264.7, J774A.1, and P388D1 to take up virus. In addition, replication of virus within macrophages did not occur regardless of drug treatment, indicating that THC had no effect on macrophage intrinsic antiviral activity. In contrast, the cannabinoid exerted a dose-dependent inhibition of macrophage extrinsic antiviral activity. This activity describes that macrophage function by which these cells suppress virus replication within xenogeneic cells in an interferon-independent manner. The inhibitory effect of THC on extrinsic antiviral activity was greatest on RAW264.7 and J774A.1 cells, followed by P388D1 cells. These macrophage-like cells regained their extrinsic antiviral activity in a time-related fashion following removal of the drug. These results indicate that THC inhibits macrophage extrinsic antiherpesvirus activity, but has no effect on intrinsic antiviral activity. However, the suppressive effect of THC on extrinsic antiviral activity is reversible upon removal of the drug.

Animals↗
PubMed1992

Delta 9-tetrahydrocannabinol decreases cytotoxic T lymphocyte activity to herpes simplex virus type 1-infected cells.

The purpose of this study was to examine the effect of delta 9-tetrahydrocannabinol (delta 9-THC), the major psychoactive component of marijuana, on T lymphocyte functional competence against herpes simplex virus Type 1 (HSV1) infection. Spleen cells from C3H/HeJ (H-2k) mice primed with HSV1 and exposed to delta 9-THC were examined for anti-HSV1 cytolytic T lymphocyte (CTL) activity. Flow cytometry was used to determine whether delta 9-THC altered T cytotoxic (Lyt-2+) and T helper (L3T4+) lymphocyte numbers or cell ratios. Nomarski optics microscopy was used to determine whether effector lymphocytes from drug-treated mice were able to bind to virally infected L929 (H-2k) target cells. Cytotoxicity assays demonstrated that CTL from mice exposed to delta 9-THC were deficient in anti-HSV1 cytolytic activity. delta 9-THC in vivo treatment had little effect on the number of T lymphocytes expressing the Lyt-2 or L3T4 antigens. Nomarski optics microscopy revealed that the CTL from the drug-treated mice were able to bind specifically to the HSV1-infected targets. However, delta 9-THC in vivo exposure affected CTL cytoplasmic polarization toward the virus-infected target cell. CTL granule reorientation toward the effector cell-target cell interface following cell conjugation occurred at a lower frequency in co-cultures containing CTL from drug-treated mice. These results suggest that delta 9-THC elicits dysfunction in CTL by altering effector cell-target cell postconjugation events.

Animals↗
PubMed1991

Chronic marijuana smoke alters alveolar macrophage morphology and protein expression.

Male rhesus monkeys were subjected to chronic exposure to marijuana smoke. High dose animals (HI) were exposed 7 days/week to 1 MJ cigarette/day; low dose animals (LO) were exposed on 2 consecutive weekend days to 1 MJ cigarette/day; placebo animals (EM) were exposed to 1 ethanol-extracted MJ cigarette/day for 7 days/week; sham animals (SH) were exposed to sham smoking conditions 7 days/week. This regimen was maintained for 1 year and was followed by a 7 month rest period. Alveolar macrophages of animals exposed to the LO and HI dose smoking regimens exhibited irregular cell surface morphology, increased vacuolization, and a spherical conformation upon adherence to plastic. Gel protein profiles of purified macrophages from HI and LO animals showed marked differences in both constitutive and bacterial lipopolysaccharide-elicited protein expression when compared with those of macrophages from the EM or SH animals. These results indicate that chronic THC exposure alters macrophage morphology and protein expression to external stimuli even after a 7 month rest period.

Animals↗
PubMed1989

Delta-9-tetrahydrocannabinol inhibits macrophage protein expression in response to bacterial immunomodulators.

The objective of this study was to determine the effect of delta-9-tetrahydrocannabinol (delta-9-THC), the major psychoactive component of marihuana, on macrophage protein expression in response to bacterial immunomodulators. Peritoneal macrophages of (B6C3)F1 mice receiving Propionibacterium acnes exhibited a novel protein profile when compared to resident or vehicle-treated macrophages. In contrast, macrophages from mice treated with P. acnes in concert with delta-9-THC exhibited profiles for which the majority of protein species reverted to patterns seen in profiles of resident or vehicle-treated macrophages. Treatment of the murine macrophage line P388D1 (DBA/2) in vitro with bacterial lipopolysaccharide (LPS) resulted in the hyperproduction of a subset of proteins ranging from 73 to 18 kD relative molecular weight. Coexposure of the P388D1 cells to LPS and 10(-7) M to 10(-5) M delta-9-THC resulted in a dose-related depletion of these proteins. These results suggest that delta-9-THC suppresses the expression of proteins elicited by macrophage bacterial immunomodulators.

Adjuvants, Immunologic↗
PubMed1987

Interaction of delta-9-tetrahydrocannabinol with rat B103 neuroblastoma cells.

The effect of delta-9-tetrahydrocannabinol (delta-9-THC) on the growth kinetics and morphology of rat B103 neuroblastoma cells was assessed. Delta-9-THC in doses ranging from 10(-4) to 10(-7) M inhibited cellular growth in a dose-dependent fashion as evidenced by delay in doubling time, decrease in saturation density, and decrease in efficiency of plating. The inhibition in cellular growth was paralleled by dose-related alterations in cell morphology. Modifications included rounding of cells, retraction of neurites, blebbing of the cell surface, and exfoliation of the plasma membrane. Cytoplasmic alterations included distension of the endoplasmic reticulum, Golgi apparatus, and perinuclear space, and macrovacuolization. Intracytoplasmic laminated inclusions and vesicular clusters were suggestive of membrane repair in drug-treated cells. These morphological changes were accompanied by cytoskeletal rearrangement in the absence of significant alteration in the concentration of total cytoskeletal protein. Autoradiographic examination of the intracellular fate of 3H-delta-9-THC demonstrated that the drug was confined to the cytoplasmic compartment and often associated with macrovacuoles. These results suggest that delta-9-THC interacts with cellular membranes, thereby altering neuroblastoma cell growth and behavior.

Animals↗
PubMed1987

Effect of micromolar concentrations of delta-9-tetrahydrocannabinol on herpes simplex virus type 2 replication in vitro.

The effect of micromolar concentrations of delta-9-tetrahydrocannabinol (delta-9-THC) on the in vitro replication and biosynthesis of herpes simplex virus type 2 (HSV2) was determined. A 100-fold increase in extracellular virus was recorded for infected Vero cells pretreated with 10(-6) M or 10(-5) M drug when compared to infected vehicle-treated controls. However, no significant differences were observed in the production of total infectious virus for any of the vehicle or drug-treated cultures. Immunofluorescence of virus-infected cells revealed that delta-9-THC did not alter the intracellular compartmentalization of virus-specified proteins. Analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of isotopically labeled, cell-associated virus-specified proteins revealed that delta-9-THC had no major effect on the production of early nonstructural proteins but decreased the synthesis of late structural proteins. Scanning electron microscopy and light microscopy revealed blebs on the surface and macrovacuoles in the cytoplasm of both infected and uninfected cells treated with drug. These results suggest that delta-9-THC at micromolar concentrations selectively targets the host cell with the consequence of perturbation of cellular membranes. The alteration of cellular membranes may account for the enhanced virus release and for the decreased expression of virus-specified, cell-associated late structural proteins.

Animals↗
PubMed1987

Delta-9-tetrahydrocannabinol inhibits the splenocyte proliferative response to herpes simplex virus type 2.

The present investigation was undertaken to determine the effect of in vivo Delta-9-tetrahydrocannabinol (Delta-9-THC) treatment on immune responsiveness to secondary exposure to herpes simplex virus type 2 (HSV2) antigens in vitro. A splenocyte proliferative assay, employing HSV2-infected mouse embryo fibroblasts as target cells, was used to measure immune responsiveness. Administration of 50 mg/kg or 100 mg/kg Delta-9-THC to B6C3F1 mice in concert with HSV2 infection resulted in suppression of the proliferative response to HSV2 cell-surface antigens expressed on virus-infected mouse embryo fibroblasts. Similarly, in vitro treatment of HSV2-infected cells with Delta-9-THC (10(-7) M to 10(-5) M) resulted in a dose-dependent suppression of proliferative responsiveness of splenocytes of non-drug-treated HSV2-sensitized mice. These results suggest that Delta-9-THC inhibits immune responsiveness of B6C3F1 mice to homotypic challenge with HSV2. This inhibition may be resultant of drug action on both effector immunocytes and target HSV2 antigen-bearing cells.

Animals↗
PubMed1987

Inhibition of cell-associated herpes simplex virus type 2 glycoproteins by delta 9-tetrahydrocannabinol.

This study was conducted to define the effect of micromolar concentrations of delta 9-tetrahydrocannabinol (delta 9-THC) on the biosynthesis and expression of herpes simplex virus type 2 (HSV2)-specified glycoproteins. Dose-related reductions in all species of virus glycoproteins were recorded by one-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography of [14C]glucosamine-labeled infected Vero cells treated with 10(-7) to 10(-5) M delta 9-THC. A drug dose-related depletion of the mature HSV2 major envelope glycoprotein complex (119-kDa average molecular weight), accompanied by accumulation of immature unglycosylated species, was demonstrated by two-dimensional SDS-PAGE in concert with Western immunoblotting or autoradiography. Light and electron microscopy immunoperoxidase staining revealed that delta 9-THC effected depletion of 119-kDa determinants from the infected cell surface. This depletion occurred concomitantly with accumulation of 119-kDa components at the perinucleus. However, the expression of 119-kDa glycoproteins on the virion envelope was not affected. These results indicate that delta 9-THC inhibits the synthesis, maturation, and cellular transport of HSV2-specified glycoproteins. Decreased expression of virus glycoproteins on the infected cell surface may affect host immune responsiveness to HSV2.

Animals↗
PubMed1986

Association of human hepatocellular membrane fusions with non-A, non-B hepatitis.

Liver biopsies from patients with alcoholic hepatitis, chemical hepatitis, or viral hepatitis types A, B, or non-A, non-B were examined by electron microscopy. Circular, fused, cytoplasmic membranes were observed in hepatocytes of 17% of patients with hepatitis type B and 92% of patients with hepatitis type non-A, non-B. The membrane alterations were not observed in hepatocytes of patients with the other types of hepatitis. The greater frequency of altered cytoplasmic membranes in hepatocytes of patients with non-A, non-B hepatitis was shown to be statistically significant (p less than 0.05) when compared to that in patients with viral hepatitis type B.

Animals↗
PubMed1986

Delta-9-tetrahydrocannabinol enhances release of herpes simplex virus type 2.

This study was undertaken to determine the effect of micromolar concentrations of delta-9-tetrahydrocannabinol (Delta-9-THC) on herpes simplex virus type 2 (HSV-2) replication in vitro. Virus-infected Vero cells pretreated for 24 h with 10(-5) M- or 10(-6) M-Delta-9-THC yielded 100-fold increases in infectious extracellular virus. Transmission electron microscopy of drug-treated cells revealed plasma membrane dissolution, distension of the smooth and rough endoplasmic reticulum, and the appearance of macrovacuoles in the cytoplasm containing aggregates of virus. These results suggest that Delta-9-THC enhances the release of HSV-2 by perturbing cellular membranes in virus-infected cells.

Animals↗