Mucosal and Parenteral Vaccination against Acute and Latent Murine Cytomegalovirus (MCMV) Infection by Using an Attenuated MCMV Mutant

We used a live attenuated murine cytomegalovirus (MCMV) mutant to analyze mechanisms of vaccination against acute and latent CMV infection. We selected MCMV mutant RV7 as a vaccine candidate since this virus grows well in tissue culture but is profoundly attenuated for growth in normal and severe combined immunodeficient (SCID) mice (V. J. Cavanaugh et al., J. Virol. 70:1365–1374, 1996). BALB/c mice were immunized twice (0 and 14 days) subcutaneously (s.c.) with tissue culture-passaged RV7 and then challenged with salivary gland-passaged wild-type MCMV (sgMCMV) intraperitoneally (i.p.) on day 28. RV7 vaccination protected mice against challenge with 10⁵ PFU of sgMCMV, a dose that killed 100% of mock-vaccinated mice. RV7 vaccination reduced MCMV replication 100- to 500-fold in the spleen between 1 and 8 days after challenge. We used the capacity to control replication of MCMV in the spleen 4 days after challenge as a surrogate for protection. Protection was antigen specific and required both live RV7 and antigen-specific lymphocytes. Interestingly, RV7 was effective when administered s.c., i.p., perorally, intranasally, and intragastrically, demonstrating that attenuated CMV applied to mucosal surfaces can elicit protection against parenteral virus challenge. B cells and immunoglobulin G were not essential for RV7-induced immunity since B-cell-deficient mice were effectively vaccinated by RV7. CD8 T cells, but not CD4 T cells, were critical for RV7-induced protection. Depletion of CD8 T cells by passive transfer of monoclonal anti-CD8 (but not anti-CD4) antibody abrogated RV7-mediated protection, and RV7 vaccination was less efficient in CD8 T-cell-deficient mice with a targeted mutation in the β2-microglobulin gene. Although gamma interferon is important for innate resistance to MCMV, it was not essential for RV7 vaccination since gamma interferon receptor-deficient mice were protected by RV7 vaccination. Establishment of and/or reactivation from latency by sgMCMV was decreased by RV7 vaccination, as measured by diminished reactivation of MCMV from splenic explants. We found no evidence for establishment of splenic latency by RV7 after s.c. vaccination. We conclude that RV7 administered through both systemic and mucosal routes is an effective vaccine against MCMV infection. It may be possible to design human CMV vaccines with similar properties.     

Replication of Murine Cytomegalovirus in Differentiated Macrophages as a Determinant of Viral Pathogenesis

Blood monocytes or tissue macrophages play a pivotal role in the pathogenesis of murine cytomegalovirus (MCMV) infection, providing functions beneficial to both the virus and the host. In vitro and in vivo studies have indicated that differentiated macrophages support MCMV replication, are target cells for MCMV infection within tissues, and harbor latent MCMV DNA. However, this cell type presumably initiates early, antiviral immune responses as well. In addressing this paradoxical role of macrophages, we provide evidence that the proficiency of MCMV replication in macrophages positively correlates with virulence in vivo. An MCMV mutant from which the open reading frames M139, M140, and M141 had been deleted (RV10) was defective in its ability to replicate in macrophages in vitro and was highly attenuated for growth in vivo. However, depletion of splenic macrophages significantly enhanced, rather than deterred, replication of both wild-type (WT) virus and RV10 in the spleen. The ability of RV10 to replicate in intact or macrophage-depleted spleens was independent of cytokine production, as this mutant virus was a poor inducer of cytokines compared to WT virus in both intact organs and macrophage-depleted organs. Macrophages were, however, a major contributor to the production of tumor necrosis factor alpha and gamma interferon in response to WT virus infection. Thus, the data indicate that tissue macrophages serve a net protective role and may function as "filters" in protecting other highly permissive cell types from MCMV infection. The magnitude of virus replication in tissue macrophages may dictate the amount of virus accessible to the other cells. Concomitantly, infection of this cell type initiates the production of antiviral immune responses to guarantee efficient clearance of acute MCMV infection.     

Atomic force microscopy of mammalian sperm chromatin

We have used the atomic force microscope (AFM) to image the surfaces of intact bull, mouse and rat sperm chromatin and partially decondensed mouse sperm chromatin attached to coverglass. High resolution AFM imaging was performed in air and saline using uncoated, unfixed and unstained chromatin. Images of the surfaces of intact chromatin from all three species and of an AFM-dissected bull sperm nucleus have revealed that the DNA is organized into large nodular subunits, which vary in diameter between 50 and 100 nm. Other images of partially decondensed mouse sperm chromatin show that the nodules are arranged along thick fibers that loop out away from the nucleus upon decondensation. These fibers appear to stretch or unravel, generating narrow smooth fibers with thicknesses equivalent to a single DNA-protamine complex. High resolution AFM images of the nodular subunits suggest that they are discrete, clipsoid-shaped DNA packaging units possibly only one level of packaging above the protamine-DNA complex.     

Ribavirin: Biotechnological Synthesis and Effect on the Reproduction of Vaccinia Virus

The biotechnological method of synthesis of ribavirin, vidarabin, and 6-azauridine by the use of immobilized recombinant enzymatic preparations of nucleoside phosphorylase was improved. The effect of ribavirin and its combinations with the other synthesized nucleosides on the reproduction of Vaccinia virus was studied on the culture of Vero cells. The combination of ribavirin and vidarabin was shown to provide the antiviral effect at lesser concentrations than with these compounds taken separately.     

Nanoscale Investigation of Pathogenic Microbial Adhesion to a Biomaterial

Microbial infections of medical implants occur in more than 2 million surgical cases each year in the United States alone. These increase patient morbidity and mortality, as well as patient cost and recovery time. Many treatments are available, but none are guaranteed to remove the infection. In many cases, the device infections are caused by the adhesion of microbes to the implant, ensuing growth, pathogenesis, and dissemination. The purpose of this work is to examine the initial events in microbial adhesion by simulating the approach and contact between a planktonic cell, immobilized on an atomic force microscope (AFM) cantilever, and a biomaterial or biofilm substrate. The two model microbes used in this study, Candida parapsilosis (ATCC 90018) and Pseudomonas aeruginosa (ATCC 10145), were chosen for both their clinical relevance and their ease of acquisition and handling in the laboratory setting. Attractive interactions exist between C. parapsilosis and both unmodified silicone rubber and P. aeruginosa biofilms. Using C. parapsilosis cells immobilized on AFM cantilevers with a silicone substrate, we have measured attractive forces of 4.3 ± 0.25 nN in the approach portion of the force cycle. On P. aeruginosa biofilms, the magnitude of the attractive force decreases to 2.0 ± 0.40 nN and is preceded by a 2.0-nN repulsion at approximately 75 nm from the cell surface. These data suggest that C. parapsilosis may adhere to both silicone rubber and P. aeruginosa biofilms, possibly contributing to patient morbidity and mortality. Characterization of cell-biomaterial and cell-cell interactions allows for a quantitative link between the physicomechanical and physicochemical properties of implant materials and the nanoscale interactions leading to microbial colonization and infection.     

Radiation and nonradiation factors in thyroid pathology development for Chernobyl cleanup workers--residents of Mayak PA affected zone

Thyroid state study was been carried out for Chernobyl NPP accident liquidators living in Ozyorsk, the city, located in the nuclear facility affected zone (Mayak PA). In total 531 individuals were examined. Thyroid assessment was made basing on physical examination, on ultrasound study and on hormones measurement of thyroid stimulating hormone, free thyroxine and thyroid peroxidase antibody. Screening revealed thyroid pathology for 27% of clean-up workers. As a result of the study carried the following conclusions were made: thyroid disease prevalence among the Ozyorsk residents participated in the breakdown elimination after Chernobyl accident was not differ from the individuals who didn't take part in this work; the prevalence of thyroid disease in liquidators was significantly connected with non radiation risk factors: with gender (for women compared to men), RR = 4.0, 95% CI 2.9-5.5; with age (over 60 years old vs under 60 years old), RR for women was 1.7; 95% CI 1.0-3.0 and for men -1.9; 95% CI 1.1-3.2; with hereditary predisposition, RR for autoimmune thyroiditis was 3.3, 95% CI 1.4-7.9.     

Ecological and physiological effects of PaV1 infection on the Caribbean spiny lobster (Panulirus argus Latreille)

Pathogens can profoundly impact the ecology of the organisms they infect through changes in host behavior that influence demographic processes. For example, juvenile Caribbean spiny lobsters (Panulirus argus Latreille) infected with the PaV1 virus (Panulirus argus Virus 1) are avoided by their normally social conspecifics, which alters local spatial distributions and presumably rates of disease transmission. PaV1 infections are nearly always lethal, but prior to succumbing to the disease, infection may impact other host dynamics (e.g., movement, growth, or survival) that effect transmission of the virus. We used mark-recapture surveys and laboratory studies to determine the impact of PaV1 infection on lobster movement and physiological condition, and in turn, the effect of condition on susceptibility to infection. Significantly more healthy lobsters were recaptured than heavily diseased lobsters in 5-d mark-recapture surveys, indicating either greater emigration or greater mortality of infected lobsters. Results of a laboratory bioassay suggest that lobsters with early-stage infections moved at an equivalent rate to healthy lobsters, however, as infection progressed, lobsters inoculated with PaV1 moved less than healthy lobsters ultimately remaining sedentary. Infected lobsters captured in nature also had significantly lower hemolymph (blood) serum protein values, indicating poor physiological condition. However, poor condition did not predispose lobsters to greater risk of infection because prevalence was not different between starved lobsters and healthy lobsters challenged with PaV1. Although lobsters infected with PaV1 eventually become sedentary and of poor nutritional condition, during the early stages of infection they remain active and are thus capable of dispersing the virus throughout the population.