Heterogeneous interleukin-15 inducibilities in murine B16 melanoma and RM-1 prostate carcinoma by interferon-alpha treatment.

Long-term treatment of mouse cancer cells with interferon-alpha (IFN-alpha) converts parental B16 melanoma cells to B16alpha vaccine cells. Inoculation of syngeneic mice with UV-irradiated B16alpha vaccine cells triggers immunity to the parental B16 tumor that is mediated by host macrophages, T cells, and NK cells. Lymph node cells from mice inoculated with irradiated B16alpha vaccine cells, but not with irradiated parental cells, proliferate when cultured in vitro, suggesting long-term in vivo activation of lymphoid cells. Both IL-15 mRNA and IL-15 protein are highly induced in B16alpha vaccine cells. The bulk of the induced IL-15 is shown to be cell-associated, either cytoplasmic or membranous. The current study investigated the feasibility of applying the B16alpha vaccination protocol to generate a cancer vaccine against murine RM-1 prostate carcinoma. In comparison to B16alpha vaccine cells, long-term IFN-alpha-treated RM-1 cells (RM-1alpha vaccine cells) showed significant IL-15 mRNA induction but relatively low IL-15 protein up-regulation. When UV-irradiated, a 3-fold increase in intracellular IL-15 was observed in RM-1alpha vaccine cells, suggesting UV damage may have negated a possible control mechanism for IL-15 synthesis. Efficacy of in vivo vaccination of syngeneic mice with UV-irradiated RM-1alpha and B16alpha vaccine cells showed correlation between high IL-15 level and high vaccine efficacy in B16alpha cells compared to low IL-15 level and low vaccine efficacy in RM-1alpha cells. This supports the concept that the induction of IL-15 in tumor cells can be useful for creating whole-cell cancer vaccines.

Expression of matrix metalloproteinases 2 and 9 in experimental corneal injury and fungal keratitis.

PURPOSE: Levels of matrix metalloproteinases (MMPs) can be modulated during corneal infection, but little is known about MMP profiles during fungal keratitis. The purpose of this study was to determine the effect of corneal trauma and immunosuppressive treatment on the expression kinetics of MMP-2 and MMP-9 during experimental keratomycosis. METHODS: Corneas of immunocompetent and cyclophosphamide-treated adult BALB/c mice were topically inoculated with 1 x 10 culturable units of Fusarium solani or mock-inoculated with or without superficial corneal scarification. Eyes were scored daily for disease severity and processed for zymography after 1.5 hours, 6 hours, 1 day, 4 days, or 8 days. Gelatinase activity was densitometrically quantitated and normalized to MMP-2 and MMP-9 controls. RESULTS: MMP-9 levels in nontraumatized eyes transiently increased at 6 hours after fungal exposure, but this increase was inhibited by cyclophosphamide treatment. Corneal injury significantly induced early MMP-9 expression that returned to baseline levels within 4 days. Cyclophosphamide pretreatment reduced and delayed MMP-9 after scarification. Fusarium exposure dampened the MMP-9 response to corneal trauma in immunocompetent and cyclophosphamide-treated animals. Ocular levels of MMP-2 were not affected by scarification, fungal exposure, or immunosuppressive treatment. CONCLUSIONS: Ocular MMP-9 levels, but not MMP-2 levels, increased soon after corneal injury. A similar, although muted, MMP-9 response occurs during early filamentous fungal keratitis, with a kinetic profile similar to corneal disease progression. The early stage of ulcerative keratitis may involve selective regulation of corneal matrix metalloproteinases, suggesting an initial opportunity for therapeutic intervention.

IFN-alpha-induced murine B16 melanoma cancer vaccine cells: induction and accumulation of cell-associated IL-15.

Long-term treatment of mouse cancer cells with interferon-alpha (IFN-alpha) converts parental B16 melanoma cells to B16alpha vaccine cells. Inoculation of syngeneic mice with B16alpha vaccine cells triggers immunity to the parental B16 tumor that is mediated by host macrophages, T cells, and natural killer (NK) cells. Lymph node cells from mice inoculated with irradiated B16alpha vaccine cells, but not with irradiated parental cells, proliferate when cultured in vitro, suggesting long-term in vivo activation of lymphoid cells. Long-term IFN-alpha treatment of B16alpha vaccine cells induced both interleukin-15 (IL-15) mRNA and IL-15 protein. The bulk of the induced IL-15 remained cell associated, either cytoplasmic or associated with the cell membrane. Immunofluorescence microscopy studies showed that the cell-associated IL-15 was broadly distributed throughout the cytoplasm. These observations suggest that long-term IFN-alpha treatment may induce primarily the truncated isoform of IL-15. Vaccination with irradiated B16alpha vaccine cells may promote tumor immunity by releasing high levels of cell-associated IL-15 when spontaneously lysed or directly killed by innate immune cells. The release of accumulated cell-associated IL-15 may then trigger a host T cell response to tumor antigens and cause host development of immunity to the B16 tumor cells.

Candida albicans strain-dependent virulence and Rim13p-mediated filamentation in experimental keratomycosis.

PURPOSE: To compare the virulence of wild-type Candida albicans strains in a murine model of corneal candidiasis and to investigate the role of fungal filamentation in disease progression. METHODS: Scarified corneas of immunocompetent or cyclophosphamide-treated BALB/c mice were topically inoculated with one of three human isolates of C. albicans, a homozygous mutant of the pH-dependent filamentation gene rim13 or a mutant reference strain control. Mock-inoculated eyes served as negative controls. Corneal disease was categorized daily for 8 days with quantitative fungal culturing of eyes at 6 hours, 1 day, 4 days, and 8 days after infection and histopathologic examination at 1 day and 4 days after infection. RESULTS: Corneal disease severity differed significantly among wild-type strains (P < or = 0.02). The rim13(-/-) mutant Tn7-rim13 was fully attenuated, whereas the mutant control DAY286 was fully virulent. Pretreatment of mice with cyclophosphamide increased susceptibility to wild-type C. albicans and partially rescued the attenuated phenotype of the genetically deficient rim13(-/-) fungal mutant. All strains replicated with similar kinetics in vitro, and wild-type strains had similar clearance from infected eyes. Histopathologic findings correlated with disease severity. CONCLUSIONS: Wild-type strains of C. albicans that differ significantly in ocular pathogenicity correlate with the ability of yeast to produce pseudohyphae and hyphae and to invade corneal tissue. Full attenuation of the fungal rim13(-/-) mutant is the first direct demonstration of a hyphal morphogenesis-related gene as a specific virulence factor for C. albicans during corneal infection.

Development of an interferon-based cancer vaccine protocol: application to several types of murine cancers.

A protocol for the development of cancer vaccines is presented. The protocol is based upon the long-term in vitro treatment of cancer cells with interferon (IFN)-alpha to create cancer vaccine cells. This protocol has been used to develop cancer vaccines in mice against B16 melanoma, RM-1 prostate cancer, and P388 lymphocytic leukemia. A detailed description of the protocol is presented. Important considerations that are discussed include the method of selection of potential cancer vaccine cells that would make good models for cancer vaccines for human cancers, the effects of in vitro IFN-alpha treatment concentration on the efficacy of generated cancer vaccine cells, the differential ability of cancer cells to become efficacious cancer vaccine cells in response to IFN-alpha treatment, the determination of the effectiveness of ultraviolet-light killing of various cancer cell types for generating cancer vaccine cells, and the methods of evaluation of statistical significance of the data obtained. Potential problems also are addressed.

Immunosuppression affects the severity of experimental Fusarium solani keratitis.

We have established a mouse model of corneal fusariosis that permits the evaluation of fungal infection and pathogenesis. Corneas of immunocompetent and cyclophosphamide-treated adult BALB/c mice were topically inoculated with Fusarium solani after corneal scarification. Eyes were scored for corneal involvement daily for 8 days and at 2 weeks after infection. Eyes were enucleated at various time points for quantitative fungal recovery and histopathological examination. An inoculum-dose response was observed in cyclophosphamide-treated mice, and fungi were recovered from the infected eyes by quantitative microbial culturing. Treatment with cyclophosphamide increased disease severity and delayed fungal clearance. Fungal hyphae, inflammatory cells, and stromal edema were histologically evident within corneal tissue and correlated with disease severity. Although the mouse cornea resists fungal infections, F. solani keratitis could be induced in immunosuppressed mice after surface scarification, which resulted in infection and clinical disease that could be evaluated both in vivo and in vitro.

Quantification of incidental needle and suture contamination during strabismus surgery.

PURPOSE: To quantify the level of bacterial contamination of needles and sutures immediately after use in strabismus surgery. METHODS: Strabismus surgery was performed on 56 eyes from 31 pediatric strabismus surgical cases. Preoperative site preparation included instillation of 5% povidone-iodine in the conjunctival fornices in all cases. A total of 124 needles and 127 sutures were cultured immediately after final scleral passage. RESULTS: Seventeen of the 31 cases (54.8%) produced at least one positive specimen. Nineteen per cent of the needles and 25.2% of the sutures were culture positive. The bulk of positive specimens (96.7% of needles, 91.3% of sutures) produced 3 or less colony forming units, corresponding to 7 or less total viable organisms per needle or sutures in accordance with the dilution scheme. Coagulase-negative staphylococci overwhelmingly predominated. CONCLUSIONS: Needles and sutures used in strabismus surgery can become contaminated during surgery despite preoperative povidone-iodine preparation. The number of viable bacterial contaminants is usually below the level known to consistently produce experimental endophthalmitis. However, a few needles and sutures carried a high contaminant load, suggesting the possibility that contaminated needles and sutures could potentially cause postoperative intraocular infection.

Activity of voriconazole against corneal isolates of Scedosporium apiospermum.

PURPOSE: To determine the in vitro antifungal activity of voriconazole, a new triazole, compared with other polyene and imidazole antifungal agents against corneal isolates of Scedosporium apiospermum. METHODS: Macro-broth dilution susceptibility testing was performed on five isolates of S. apiospermum obtained from patients with keratomycosis to determine the minimal inhibitory concentrations (MICs) for amphotericin B, natamycin, ketoconazole, itraconazole, and voriconazole. The use of oral voriconazole in the management of a patient with posttraumatic S. apiospermum keratitis is described. RESULTS: S. apiospermum is generally resistant to commonly used topical ophthalmic antifungal agents. The MIC of voriconazole was 0.5 microg/mL, a concentration lower than that of other imidazoles. CONCLUSION: Voriconazole has promising activity against and may prove useful in the management of fungal keratitis.

Experimental keratomycosis in a mouse model.

PURPOSE: To establish a murine model of corneal candidiasis that permits molecular evaluation of fungal adherence and invasion. METHODS: Corneas of immunocompetent, methylprednisolone-treated, and cyclophosphamide-treated adult NIH Swiss and BALB/c mice were topically mock inoculated or inoculated with 10-fold increasing amounts between 100 and 100 million colony-forming units (CFU) of Candida albicans after unilateral corneal scarification. Mock-inoculated eyes served as the control. Eyes were scored daily on a 12-point scale to categorize corneal inflammation and were enucleated for quantitative fungal cultures, analysis by polymerase chain reaction (PCR), and histopathologic examination. RESULTS: At least 100 CFU of C. albicans initiated measurable corneal infection, but 1 million or more colony-forming units were needed to induce consistent keratitis. Treatment with methylprednisolone increased disease severity in infected BALB/c mice and fungal persistence in both BALB/c and NIH Swiss mice. Treatment with cyclophosphamide increased disease severity and fungal persistence in both strains of mice. Infectious organisms were recovered by quantitative culture, and candidal DNA was detectable by PCR. C. albicans, inflammatory cells, and stromal necrosis were histologically evident within ocular tissue. CONCLUSIONS: Although mice are innately resistant to Candida infection after corneal inoculation, moderate to severe keratomycosis can be established in immunocompromised mice by the route of corneal scarification. Although differences between mouse strains and among immunosuppressive regimens remain to be explored, this murine model provides the basis for understanding the pathogenesis of fungal infections of the cornea.