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Biomedical subjects

E M Mishkin

Publications and source records attributed to E M Mishkin.

At least 19 recordsLinked to original sources

Combined systemic and mucosal immunization with microsphere-encapsulated inactivated simian immunodeficiency virus elicits serum, vaginal, and tracheal antibody responses in female rhesus macaques.

We determined the efficacy of immunization with microsphere-encapsulated whole inactivated simian immunodeficiency virus (SIV) by combined systemic and mucosal administration to protect female rhesus macaques against vaginal challenge with homologous rhesus PBMC-grown SIVmac251. Animals in one group were primed and boosted intramuscularly. Two groups were primed intramuscularly and boosted either intratracheally or orally. A final group was primed by vaccinia/rgp140 scarification and subdivided for either intratracheal or oral boosting. Strong ELISA titers of circulating SIV-specific IgG and modest IgA responses were elicited in the animals primed intramuscularly. Intratracheal boosting in the intramuscularly primed macaques resulted in high bronchial alveolar wash (BAW) IgG and less pronounced IgA. SIV-specific vaginal wash (VW) IgG was also present in the intramuscular/intramuscular and intramuscular/intratracheal groups. Vaccinia/rgp140 priming gave low ELISA titers to whole SIV, and failed to elicit mucosal antibody regardless of the booster route. No animal in any group developed serum neutralizing antibody to homologous SIVmac251. On vaginal challenge none of the immunized groups was infected at a lesser frequency than the unimmunized controls. These data suggest that the use of microspheres in a combined parenteral and mucosal regimen is an effective method of eliciting IgG and IgA antibody at mucosal surfaces.

Animals↗

Induction of mucosal antibody responses by microsphere-encapsulated formalin-inactivated simian immunodeficiency virus in a male urethral challenge model.

Male rhesus macaques were immunized mucosally with microsphere-encapsulated formalin-inactivated simian immunodeficiency virus (SIV) particles in a test of immunogenicity and protection against mucosal SIV challenge. Tracheal boosting of animals that had been primed intramuscularly resulted in strong serum ELISA titers to SIV, and evidence of local IgA responses in broncho-alveolar washes. The bulk of the antibody response was against non-envelope epitopes. No neutralizing antibody was observed, and intraurethral challenge with cell-free rhesus-grown virus showed no evidence of protection against infection. Microsphere-based immunization efficiently raises local and system responses, but the resulting immunity to SIV is apparently not sufficient to protect against mucosal challenge.

Animals↗

Th1/Th2-like immunity and resistance to herpes simplex labialis.

To investigate potential immunologic mechanisms of resistance to recurrent herpes simplex labialis, we assayed serum antibody titers and cultured peripheral blood mononuclear cell (PBMC) cytokine production among patients with a history of frequent episodes (H+S+), herpes simplex virus (HSV)-seropositive individuals without a history of herpes labialis (H-S+) and HSV-seronegative persons (H-S-). In addition, H+S+ patients were exposed to experimental ultraviolet radiation (UVR) on the lips and the immunologic assay results compared among those who developed experimental lesions and those who did not. H+S+ patients were found to have higher median serum titers of HSV antibody and trends to lower levels of HSV-specific PBMC IFN-gamma and IL-2 than H-S+ control patients (123 vs 66, P = 0.04; 424 vs 548 pg/ml, P = 0.08; 14 vs 26 pg/ml, P = 0.14, respectively). Correlation of the results with the occurrence of experimental lesions showed the inverse: the subgroup of H+S+ patients with UVR-induced lesions had lower titers of antibody and trends to higher levels of IFN-gamma and IL-2 than H+S+ patients who could not be induced (93 vs 149, P = 0.02; 501 vs 347 pg/ml, P = NS; 26 vs 11 pg/ml, P = NS, respectively). The size and duration of UVR-induced lesions showed positive correlations with IFN-gamma and IL-2 levels (r = 0.60-0.67, P = 0.02-0.04). Although the small number of patients limited the power of this study, the overall pattern of the findings suggests that a Th1-like cytokine response (IFN-gamma and IL-2 production) may be associated with resistance to naturally occurring episodes of herpes labialis. The development and severity of experimental UVR-induced herpes labialis appears to be regulated differently and may involve an immunopathologic mechanism.

Antibodies, Viral↗

Immunomodulatory effects of HSV2 glycoprotein D in HSV1 infected mice: implications for immunotherapy of recurrent HSV infection.

Immunological analyses in this laboratory and others have suggested that a nonrecurrent HSV seropositive immune status is more closely correlated with a type 1 T helper cell (Th1) response characterized by elevated levels of interferon-gamma and IL2 rather than high titers of virus-specific antibodies. Effective intervention with an immunotherapeutic vaccine may require modulation of the regulatory network of T helper cells such that there is selective restimulation and expansion of the Th1 response. We have established a murine model for assessing the immunomodulatory capacity of an HSV glycoprotein subunit vaccine in animals with pre-existing herpes immunity. Animals were infected with varying doses of HSV1 and then administered glycoprotein D (gD) vaccine adjuvanted with aluminum phosphate at 3-week intervals. Observed changes in serological and cellular responses indicated that administration of subunit vaccine adjuvanted with aluminum phosphate could shift a dominant Th1 response, induced by sensitization with live HSV, towards a Th2 profile of activity. These data suggest that use of aluminum based adjuvants will not selectively stimulate Th1-associated responses and alternative adjuvants may be required for effective use of subunit vaccine in an immunotherapeutic indication in humans.

Adjuvants, Immunologic↗

IgG subtype is correlated with efficiency of passive protection and effector function of anti-herpes simplex virus glycoprotein D monoclonal antibodies.

IgG subclasses differ in their effector functions in a variety of in vitro assays. To assess the effect of antibody subclass differences on in vivo protective efficacy against herpes simplex virus (HSV), a series of subclass switch mutants was made from an anti-HSV glycoprotein D monoclonal antibody. Purified antibody was examined for the ability to protect against HSV-2 challenge in mice. IgG2a was found to be more effective than IgG1. This correlated both with activity in antibody-dependent cell-mediated cytotoxicity and with efficiency of complement-mediated neutralization. These data suggest that optimization of passive immunization against HSV requires consideration of antibody subclass.

Animals↗

Baculovirus-expressed herpes simplex virus type 2 glycoprotein D is immunogenic and protective against lethal HSV challenge.

Herpes simplex virus type 2 glycoprotein D (gD2) was cloned and expressed in the baculovirus-Spodoptera frugiperda system. Milligram quantities of glycoprotein were recovered from suspension culture and subjected to purification by ion-exchange and immunoaffinity chromatography. The resultant purified gD existed as a homogeneous 57,500 MW monomeric species demonstrating reactivity with anti-gD monoclonal antibodies including those directed at a non-sequential neutralizing epitope of gD. Immunization of Balb/c mice with doses of 0.1-10.0 micrograms of AlPO4-absorbed gD resulted in elicitation of humoral and cellular responses to both HSV1 and HSV2 as well as to purified gD1 and gD2. Immunized mice receiving an infectious dose of 1 x 10(6) p.f.u. of HSV2 via the footpad route were significantly protected against infection at all doses tested when compared with unimmunized AlPO4 and uninoculated control animals.

Animals↗

Effects of in vivo depletion of immunocyte populations on herpes simplex virus glycoprotein D vaccine-induced resistance to HSV2 challenge.

BALB/c mice, preimmunized with a protective dose of native herpes simplex virus type 1 glycoprotein D (ngD1) vaccine, were depleted of selected immunocyte populations in vivo using monoclonal antibodies directed at Thy1+, L3T4+, or Lyt2+ cells. Following immunization and depletion, animals were inoculated with varied challenge levels of herpes simplex virus type 2 (HSV2) in the footpad and were monitored for disease. Both depleted undepleted gD-immunized mice were significantly protected when compared with placebo controls. T-cell-independent protection in Thy1 and L3T4-depleted ngD1-immunized animals was effective at low and moderate levels of HSV2 challenge levels, high levels of HSV2 giving high symptom scores in immunized and depleted mice. Depletion of Lyt2+ cells had no significant effect on the outcome of HSV2 infection. Depleted and nondepleted animals also were assessed in parallel for cellular and humoral responsiveness to ngD1 and to HSV antigens in vitro. Lymphoproliferative responses were abrogated in gD-immunized mice treated with anti-Thy1 or anti-L3T4, anti-Lyt2 treatment having little effect. Postimmunization T-cell depletion did not undermine ELISA or neutralizing antibody responses. These findings suggest that at low to moderate levels of virus challenge vaccine-elicited antibody plays a primary role in limiting the severity of infection, T-cell-mediated protective responses being of enhanced significance only at high levels of virus challenge.

Animals↗

Native herpes simplex virus glycoprotein D vaccine: immunogenicity and protection in animal models.

Mice, guinea pigs, and rhesus monkeys were immunized with immunoaffinity-purified native glycoprotein D (gD) derived from herpes simplex virus type 1 (HSV1). The native glycoprotein has evoked significant in vivo responses even at low doses. Thus, mice immunized with doses as low as 1 microgram were significantly protected from the morbidity and mortality of lethal HSV2 challenge and from establishment of latent HSV2 infection. Protection was dose-related and correlated with prechallenge serum neutralizing antibody titres to HSV. Similarly, immunized guinea-pigs demonstrated significant reductions in the frequency, severity and duration of genital lesions induced by HSV2 vaginal challenge. In long term immunogenicity studies, immunized rhesus monkeys exhibited significant serum neutralizing antibody responses to both HSV1 and HSV2. In vitro stimulation of monkey peripheral blood leucocytes with purified gD resulted in a significant cellular proliferative response. The results obtained in these animal models with a gD subunit vaccine provide an appropriate foundation for the initiation of human studies.

Animals↗

Native HSV glycoprotein D subunit vaccine: analysis of in vitro T-cell activation and antigen presentation.

Native herpes simplex virus (HSV) glycoprotein D (ngD1) subunit vaccine, a potential human vaccine candidate, was examined to determine responsive murine lymphocyte populations in vitro. This vaccine preparation has been shown to protect against HSV challenge in mice and guinea pigs and to elicit humoral and cellular responses in rodents and primates. Immunized BALB/c mice were used in splenocyte lymphoproliferative studies to analyze the cellular response. After in vivo sensitization, the in vitro proliferative response observed appears to be resultant of Class II-restricted T-cell division in response to gD presented in the context of macrophage-expressed Ia.

Animals↗

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↗

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↗

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↗

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↗

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↗

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↗

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

This study was undertaken to determine the effect of delta 9-tetrahydrocannabinol (delta 9-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 delta 9-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 delta 9-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 delta 9-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.

Animals↗

Effect of delta 9-tetrahydrocannabinol on herpes simplex virus type 2 vaginal infection in the guinea pig.

The present investigation was undertaken to determine whether delta 9-tetrahydrocannabinol (delta 9-THC) decreases host resistance to herpes simplex virus type 2 vaginal infection in the guinea pig. The guinea pig was selected as the host since it has been shown to express a spectrum of primary herpes genitalis which is similar to that in humans. Animals were administered delta 9-THC or vehicle intraperitoneally on Days 1-4, 8-11, and 15-18. Herpes simplex virus was introduced intravaginally on Day 2. Host resistance to virus infection was assessed by comparing frequency and severity of lesions, virus shedding, and animal mortalities. Virus-infected animals treated with drug at doses of 4 and 10 mg/kg exhibited significantly greater severity of genital disease during the 30-day period of study when compared to virus-inoculated vehicle controls. A direct relationship was noted between dose of delta 9-THC and cumulative mortalities on Day 14 following primary infection. These results indicate that delta 9-THC decreases host resistance to herpes simplex virus type 2 vaginal infection in the guinea pig.

Animals↗

Effects of environmental calcium on fecundity and cercarial production of Biomphalaria glabrata (Say) infected with Schistosoma mansoni Sambon.

Biomphalaria glabrata were reared in stock culture and subjected to either 7-day or 60-day acclimation periods in complex CaCO3 media with calcium values ranging from 1.5 mg/L to 75 mg/L. Following 60-day acclimation, snails from series I were each exposed to 8 miracidia of Schistosoma mansoni. Snails of series II were each exposed to a single miracidium. Snails of both experimental regimens were observed for mortality, growth, rate of infection, and number of cercariae shed. Series I snails were also monitored for fecundity during acclimation and following miracidial exposure. Calcium levels of 1.5 and 75 mg/L resulted in significant snail mortality. Shell growth and rates of infection were positively correlated with calcium maintenance level. Snails with high fecundity prior to miracidial exposure subsequently shed more cercariae. In contrast, post-exposure (PE) fecundity of snails reared in media with up to 30 mg/L Ca++ were negatively correlated with calcium level, infection rate, and number of cercariae shed. Maximal cercarial emergence occurred at 30 mg/L Ca++. These results suggest that environmental calcium affects both the distribution patterns of snail hosts of human schistosomes and the productivity of intramolluscan schistosome infection.

Animals↗