Role of discoidin domain receptor 1 in dysregulation of collagen remodeling by cyclosporin A.

The anti-transplant rejection drug cyclosporin A (CsA) causes loss of collagen homeostasis in rapidly remodeling connective tissues, such as human gingiva. As a result of CsA treatment, collagen degradation by fibroblasts is inhibited, which leads to a net increase of tissue collagen and gingival overgrowth. Since fibrillar collagen is the primary ligand for the discoidin domain receptor 1 (DDR1), we hypothesized that CsA perturbs DDR1-associated functions that affect collagen homeostasis. For these experiments, human fibroblasts obtained from gingival explants or mouse 3T3 fibroblasts (wild type, over-expressing DDR1 or DDR1 knockdown) or mouse GD25 cells (expressing DDR1 but null for ß1 integrin), were treated with vehicle (dimethyl sulfoxide) or with CsA. The effect of CsA on cell binding to collagen was examined by flow cytometry; cell-mediated collagen remodeling was analyzed with contraction, compaction and migration assays. We found that CsA inhibited cell binding to collagen, internalization of collagen, contraction of collagen gels and cell migration over collagen in a DDR1-dependent manner. CsA also enhanced collagen compaction around cell extensions. Treatment with CsA strongly reduced surface levels of ß1 integrins in wild type and DDR1 over-expressing 3T3 cells but did not affect ß1 integrin activation or focal adhesion formation. We conclude that CsA inhibition of collagen remodeling is mediated through its effects on both DDR1 and cell surface levels of the ß1 integrin.

Antibody prophylaxis and therapy for flavivirus encephalitis infections.

The outbreak of West Nile (WN) encephalitis in the United States has rekindled interest in developing direct methods for prevention and control of human flaviviral infections. Although equine WN vaccines are currently being developed, a WN vaccine for humans is years away. There is also no specific therapeutic agent for flaviviral infections. The incidence of human WN virus infection is very low, which makes it difficult to target the human populations in need of vaccination and to assess the vaccine's economic feasibility. It has been shown, however, that prophylactic application of antiflaviviral antibody can protect mice from subsequent virus challenge. This model of antibody prophylaxis using murine monoclonal antibodies (MAbs) has been used to determine the timing of antibody application and specificity of applied antibody necessary for successful prophylaxis. The major flaviviral antigen is the envelope (E) glycoprotein that binds cellular receptors, mediates cell membrane fusion, and contains an array of epitopes that elicit virus-neutralizing and nonneutralizing antibodies. The protective efficacy of an E-glycoprotein-specific MAb is directly related to its ability to neutralize virus infectivity. The window for successful application of prophylactic antibody to prevent flaviviral encephalitis closes at about 4 to 6 days postinfection concomitant with viral invasion of the brain. Using murine MAbs to modify human disease results in a human antimouse antibody (HAMA) response that eventually limits the effectiveness of subsequent murine antibody applications. To reduce the HAMA response and make these MAbs more generally useful for humans, murine MAbs can be "humanized" or human MAbs with analogous reactivities can be developed. Antiflaviviral human or humanized MAbs might be practical and cost-effective reagents for preventing or modifying flaviviral diseases.

Immunity of swine to Ascaris suum.

Swine were hyperimmunized to Ascaris suum by giving multiple oral inoculations of embryonated eggs. Sera and lymphocyte lysate from these pigs were administered parenterally to 4-week-old pigs. The latter animals were no more resistant to larval migration than control pigs receiving sera or lymphocyte lysate from non-immunized pigs. Other pigs were infected with transmissible gastroenteritis (TGE) virus, allowed to recover and challenged with embryonated ascarid eggs. They likewise were no more resistant to ascarid larval migration than control pigs.

Antigens in perienteric fluid of Ascaris suum as detected through antibodies in pigs orally inoculated with fully embryonated eggs.

Perienteric fluid (Pf) of adult Ascaris suum was fractionated by ammonium sulfate precipitation, gel filtration or DEAE--cellulose chromatography, and sucrose density gradient centrifugation. Anti-sera (anti-EE) from pigs which were inoculated orally with fully embryonated eggs (EE) of A. suum were used in an indirect radioimmunoassay (IRIA) to determine which fractions of Pf reacted positively in the analyses at the lowest protein concentration. These fractions were considered to contain more potent antigens. Comparative IRIA were performed employing antisera (anti-Pf) produced by injecting Pf into pigs. Six out of 35 fractions reacted positively at less than or equal to 0.2 micrograms protein when anti-EE was used in the IRIA. Twenty-two out of 35 fractions reacted positively at less than or equal to 0.2 micrograms protein when anti-Pf was used. Five of the 6 fractions reacting positively with anti-EE also reacted with anti-Pf. A 76000 dalton component appears to be one of the major proteins in the fractions which react positively with anti-EE while components from 10000-138000 daltons were present in the various fractions reacting positively with anti-Pf at less than or equal to 0.2 micrograms protein.

Immune responses of swine to oral inoculation with embryonated eggs of Ascaris suum.

Responses of swine to oral inoculation with embryonated eggs of Ascaris suum were monitored, using lymphocyte blastogenesis assays, indirect radioimmunoassays, and peripheral eosinophil counts (EC). Transient cell-mediated immune responses of peripheral lymphocytes were detected by lymphocyte blastogenesis assay as early as postinoculation day (PID) 2, but were rarely positive for consecutive samples taken at 2-day intervals. Humoral antibodies were first detected at PID 6 to 17 by indirect radioimmunoassays in the various experiments. Positive cutaneous delayed hypersensitivity reactions were observed when pigs were tested at 6 to 7 weeks after inoculation. Histopathologic examination verified infiltration of lymphocytes into the lesions. The EC increased as early as PID 4 to 7 and showed a secondary increase after the 2nd oral inoculation of eggs to as high as 11,400/mm3 (44% of the total WBC). Subsequently, EC decreased rapidly 14 days after the last inoculation of eggs.

Indirect solid-phase microradioimmunoassay for detection of Ascaris suum antibodies in swine sera.

Antibodies to Ascaris suum have been detected and quantitated in sera of swine orally inoculated with eggs of A suum, using an indirect solid-phase microradioimmunoassay. Antibody titer of 1:640 to 1:10,240 were quantitated, using purified and crude ascarid antigens in the assays. Swine sera from 71 of 72 farm herds located in 37 counties in Nebraska had at least 1 serum of the 7 tested with a titer equal to or greater than 1:20. A total of 437 sera were positive and 67 negative (ie, less than 1:20 titer). Six positive sera selected at random had titers of 1:640 to 1:5,120.