Eric Mishkin

Delta 9-tetrahydrocannabinol decreases alpha/beta interferon response to herpes simplex virus type 2 in the B6C3F1 mouse.

Abstract This study was undertaken to determine the effect of Δ9-tetrahydrocannabinol (Δ9-THC) on polyinosinic:polycytidylic acid [poly(I):poly(C)]-induced, and on herpes simplex virus type 2 (HSV-2)-induced, alpha/beta interferon in the B6C3F1 mouse. Animals were administered Δ9-THC, or the diluent, intraperitoneally for 4 consecutive days or at various time intervals prior to administration of the interferon inducer. Poly(I):poly(C) or HSV-2 was injected intravenously on Day 4. Animals receiving poly(I):poly(C) and treated with Δ9-THC at doses ranging from 5 to 100 mg/kg exhibited significantly lower titers of interferon than mice given poly(I):poly(C) and the diluent. Diminished interferon titers occurred in HSV-2-infected animals treated with Δ9-THC in doses exceeding 15 mg/kg when compared to virus-infected animals given the diluent. This suppression of early interferon persisted through 24 hr.

Assessing the immunopotency of Toll‐like receptor agonists in an in vitro tissue‐engineered immunological model

The in vitro Peripheral Tissue Equivalent (PTE) module is a three‐dimensional tissue‐engineered endothelial cell/collagen matrix culture system, which has been reported to reproduce in vivo physiological conditions and which generates dendritic cells (DC) autonomously. In the present study, we used the PTE module to investigate the immunopotency of Toll‐like receptor (TLR) agonists, including polyinosine‐polycytidylic acid, Gardiquimod, CpG 2006 and lipopolysaccharide. Application of TLR agonists in the PTE module induced a wide range of cytokines, including interleukins 1α/β, 6, 8 and 10 and tumour necrosis factor‐α. Compared with traditional peripheral blood mononuclear cell (PBMC) cultures, the PTE module produced twofold to 100‐fold higher levels of cytokine secretion, indicating that it can be a highly sensitive assay system. This increased sensitivity is the result of the natural synergy between the leucocytes and the endothelium. Furthermore, the application of TLR agonists, such as lipopolysaccharide and Gardiquimod, to the PTE module enhanced DC differentiation and promoted DC maturation, as indicated by up‐regulated expression of CD83, CD86 and CCR7(CD197). In addition, functional assays indicated PTE‐derived DC treated with Gardiquimod, a TLR‐7 agonist, significantly augmented anti‐tetanus toxoid antibody production. Interestingly, replacing PBMC with purified myeloid cells (CD33+) significantly reduced the responsiveness of the PTE module to TLR stimulation. The reduced sensitivity was partly the result of the removal of plasmacytoid DC that participated in the response to TLR stimulation and sensitization of the PTE module. Overall, the in vitro PTE module clearly demonstrated the effects of TLR agonists on DC generation, maturation and antigen‐presenting capacity, and may serve as a sensitive and predictive test bed for the evaluation of adjuvant candidates.

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.

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.

Inhibition of Cell-Associated Herpes Simplex Virus Type 2 Glycoproteins by Δ9-Tetrahydrocannabinol

Abstract This study was conducted to define the effect of micromolar concentrations of Δ9-tetrahydrocannabinol (Δ9-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 Δ9-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 immuno-blotting or autoradiography. Light and electron microscopy immunoperoxidase staining revealed that Δ9-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 Δ9-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.