High vitamin D3 diet administered during active colitis negatively affects bone metabolism in an adoptive T cell transfer model.

Decreased bone mineral density (BMD) represents an extraintestinal complication of inflammatory bowel disease (IBD). Vitamin D3 has been considered a viable adjunctive therapy in IBD. However, vitamin D3 plays a pleiotropic role in bone modeling and regulates the bone formation-resorption balance, depending on the physiological environment, and supplementation during active IBD may have unintended consequences. We evaluated the effects of vitamin D3 supplementation during the active phase of disease on colonic inflammation, BMD, and bone metabolism in an adoptive IL-10-/- CD4⁺ T cell transfer model of chronic colitis. High-dose vitamin D3 supplementation for 12 days during established disease had negligible effects on mucosal inflammation. Plasma vitamin D3 metabolites correlated with diet, but not disease, status. Colitis significantly reduced BMD. High-dose vitamin D3 supplementation did not affect cortical bone but led to a further deterioration of trabecular bone morphology. In mice fed a high vitamin D3 diet, colitis more severely impacted bone formation markers (osteocalcin and bone alkaline phosphatase) and increased bone resorption markers, ratio of receptor activator of NF-κB ligand to osteoprotegrin transcript, plasma osteoprotegrin level, and the osteoclast activation marker tartrate-resistant acid phosphatase (ACp5). Bone vitamin D receptor expression was increased in mice with chronic colitis, especially in the high vitamin D3 group. Our data suggest that vitamin D3, at a dose that does not improve inflammation, has no beneficial effects on bone metabolism and density during active colitis or may adversely affect BMD and bone turnover. These observations should be taken into consideration in the planning of further clinical studies with high-dose vitamin D3 supplementation in patients with active IBD.

Assessment of acridine orange and SYTO 16 for in vivo imaging of the peritoneal tissues in mice.

The effect of peritoneal injection of acridine orange and SYTO 16 in mice was investigated. Images of peritoneal tissues stained with these dyes and obtained through a confocal micro-endoscope are presented. Seventy-five Balb/c mice were split into five groups and given peritoneal injections of dye or saline. The proportions of negative outcomes in each group were compared using confidence intervals and the Fisher's exact statistical test. A statistically significant increase in adverse events due to dye injection was not observed. These data provide an initial investigation into the safety of acridine orange and SYTO 16 for in vivo imaging.

Detection of Reovirus type 3 by use of fluorogenic nuclease reverse transcriptase polymerase chain reaction.

Reovirus type 3 (Reo-3) can infect numerous rodent species and induces the clinical syndrome 'oily skin disease' in neonatal mice, and is a common contaminant of biological materials. The reverse transcriptase polymerase chain reaction (RT-PCR) assay has proven useful for the detection of Reo-3 in rodents and contaminated biological materials. Fluorogenic nuclease reverse transcriptase polymerase chain reaction assays (fnRT-PCR) combine RT-PCR with an internal fluorogenic hybridization probe, thereby potentially enhancing specificity and eliminating post-PCR processing. Therefore, an fnRT-PCR assay specific for Reo-3 was developed by targeting primer and probe sequences to a unique region of the Reo-3 M3 gene. The fnRT-PCR detected both strains of Reo-3 (Dearing and Abney), but did not detect Reovirus types 1 or 2, other viruses in the family Reoviridae, or other RNA viruses that naturally infect rodents. The fnRT-PCR detected less than 1 fg of target template and detected viral RNA in tissues obtained from mice experimentally infected with Reo-3. The assay also displayed comparable sensitivity when compared to the mouse antibody production test commonly used to detect viral contamination of biological materials. In conclusion, this fnRT-PCR assay offers a potentially high-throughput diagnostic assay for detecting Reo-3 RNA in infected mice and contaminated biological materials.

Comparative analysis of IFN-gamma B7.1 and antisense TGF-beta gene transfer on the tumorigenicity of a poorly immunogenic metastatic mammary carcinoma.

Cancer progression is attributed in part to immune evasion strategies that include lack of co-stimulation, down-regulation of cell surface MHC molecules, and secretion of immunosuppressive factors, such as transforming growth factor-beta (TGF-beta). Gene therapy has been employed to counter these mechanisms of immune evasion by transference of B7.1, IFN-gamma or antisense TGF-beta genes into tumor cells, resulting in cell surface expression of B7.1, upregulation of MHC class I and class II molecules, or elimination of tumor-derived TGF-beta, respectively. Although each of these transgenes has been shown to alter tumorigenicity in murine models, a direct comparison of their efficacy has not been performed. In this study, we have employed a very aggressive, poorly immunogenic and highly metastatic mammary model, 4T1, to compare the efficacy of B7.1, IFN-gamma and antisense TGF-beta gene transfer in stimulating an anti-tumor response. We demonstrate that both IFN-gamma and antisense TGF-beta gene expression significantly reduced the tumorigenicity of these cells compared to mock transduced cells, with IFN-gamma having a greater effect. In contrast, B7.1 gene transfer did not affect the tumorigenicity of 4T1 cells. The anti-tumor response directed against antisense TGF-beta-expressing 4T1 tumors was mediated by CD4+ and CD8+ T cells. However, CD8+ T cells, and not CD4+ T cells, appeared to mediate the anti-tumor response against IFN-gamma-expressing tumors. Treatment of tumor-bearing animals with IFN-gamma or antisense TGF-beta gene-modified tumor cell vaccines reduced the number of clonogenic metastases to the lungs and liver compared to treatment with mock-transduced cells. Finally, in a residual disease model in which the primary tumor was excised and mice were vaccinated with irradiated tumor cells, treatment of mice with vaccinations consisting of 4T1 cells expressing both antisense TGF-beta and IFN-gamma genes was the most effective in prolonging survival.

Invasion and metastasis of a mammary tumor involves TGF-beta signaling.

Several studies have correlated escape from TGF-beta-mediated cell cycle arrest with the tumorigenic phenotype. Most often, this escape from growth control has been linked to dysfunctional TGF-beta receptors or defects in the TGF-beta-mediated SMAD signaling pathway. In this report, we found that highly metastatic 4T1 mammary carcinoma cells express functional TGF-beta receptors capable of initiating SMAD-mediated transcription, yet are not growth inhibited by TGF-beta1. We further observed that TGF-beta directly contributes to the metastatic behavior of this cell line. Exposure to TGF-beta caused 4T1 cells to undergo morphological changes associated with the metastatic phenotype and invade more readily through collagen coated matrices. Furthermore, expression of a dominant negative truncated type II receptor diminished TGF-beta signaling and significantly restricted the ability of 4T1 cells to establish distant metastases. Our results suggest that regardless of 4T1 resistance to TGF-beta-mediated growth inhibition, TGF-beta signaling is required for tumor invasion and metastases formation.

Detection of rodent parvoviruses by use of fluorogenic nuclease polymerase chain reaction assays.

Polymerase chain reaction (PCR) assays have proven useful for detection of rodent parvoviruses in animals and contaminated biological materials. Fluorogenic nuclease PCR assays combine PCR with an internal fluorogenic hybridization probe, eliminating post-PCR processing and potentially enhancing specificity. Consequently, three fluorogenic nuclease PCR assays were developed, one that detects all rodent parvoviruses, one that specifically detects minute virus of mice (MVM), and one that specifically detects mouse parvovirus 1 (MPV) and hamster parvovirus (HaPV). When rodent parvoviruses and other rodent DNA viruses were evaluated, the rodent parvovirus assay detected only rodent parvovirus isolates, whereas the MVM and MPV/HaPV assays detected only the MVM or MPV/ HaPV isolates, respectively. Each assay detected the equivalent of 10 or fewer copies of target template, and all fluorogenic nuclease PCR assays exceeded the sensitivities associated with previously reported PCR assays and mouse antibody production testing. In addition, each fluorogenic nuclease PCR assay detected the targeted parvovirus DNA in tissues obtained from mice experimentally infected with MVM or MPV. Results of these studies indicate that fluorogenic nuclease PCR assays provide a potentially high-throughput, PCR-based method to detect rodent parvoviruses in infected mice and contaminated biological materials.

Effect of mouse strain and age on detection of mouse parvovirus 1 by use of serologic testing and polymerase chain reaction analysis.

BACKGROUND AND PURPOSE: Detection of mouse parvovirus 1 (MPV) depends on use of serologic and polymerase chain reaction (PCR) assays. These assays were evaluated for their ability to detect virus-specific antibodies or viral DNA in multiple strains and ages of mice inoculated with MPV. METHODS: Twelve-week-old ICR, BALB/c, C3H, C57BL/6, and DBA/2 mice and four- and eight-week-old ICR mice were inoculated with MPV. Serum was harvested four weeks after inoculation and analyzed by use of recombinant non structural protein 1 (rNS1) enzyme-linked immunosorbent assay (ELISA), minute virus of mice (MVM) ELISA, and MPV indirect fluorescent antibody (IFA), MVM IFA, and MPV hemagglutination inhibition (HAI) assays. Select tissues were harvested and analyzed by use of an MPV-specific PCR assay. RESULTS: The number of mice in each group with detectable MPV-specific antibodies or MPV DNA varied with mouse strain, mouse age when inoculated, and viral dose. Seroconversion in mice inoculated at 12 weeks of age was detected almost exclusively by use of the MPV IFA and MPV HAI assays, whereas seroconversion in almost all mice inoculated at 4 and 8 weeks of age was detected by use of all immunoassays except the MVM ELISA. Viral DNA was detected by use of PCR analysis in all strains and ages of mice except DBA/2 mice. CONCLUSIONS: Mouse strain and age have important roles in seroconversion to nonstructural and structural MPV antigens and persistence of viral DNA in mouse tissues. Therefore, diagnostic serologic testing and PCR analysis should be considered within the context of mouse strain and age at the time of MPV exposure, especially when sentinel mice are used for surveillance.

Transmission of vesicular stomatitis virus from infected to noninfected black flies co-feeding on nonviremic deer mice.

Vesicular stomatitis is an economically important arboviral disease of livestock. Viremia is absent in infected mammalian hosts, and the mechanism by which insects become infected with the causative agents, vesicular stomatitis viruses, remains unknown. Because infected and noninfected insects potentially feed on the same host in nature, infected and noninfected black flies were allowed to feed on the same host. Viremia was not detected in the host after infection by a black fly bite, but because noninfected black flies acquired the virus while co-feeding on the same host with infected black flies, it is concluded that a viremic host is not necessary for an insect to be infected with the virus. Thus co-feeding is a mechanism of infection for an insect-transmitted virus.

Interferon gamma and antisense transforming growth factor beta transgenes synergize to enhance the immunogenicity of a murine mammary carcinoma.

Transforming growth factor beta (TGFbeta) is an immunosuppressive cytokine that contributes to the immunological escape of tumor cells. In a previous study we demonstrated that inhibition of TGFbeta production by EMT6 murine mammary tumor cells expressing an antisense TGF-beta transgene reduces their tumorigenicity. On the basis of this observation we hypothesized that down-regulation of TGFbeta production coupled with interferon gamma (IFNgamma) stimulation would induce an immune response superior to that generated by either strategy alone. In this study, EMT6 tumor cells expressing antisense TGFbeta were transduced with the murine IFNgamma gene. Tumor cells expressing either or both transgenes grew more slowly than mock-transduced tumors. Dual-transgene-expressing tumor cells were more immunogenic than tumor cells expressing either transgene alone. Studies in mice depleted of T cell subsets indicated that CD8+ T cells are the primary effectors of the antitumor activity observed. These results suggest that down-regulation of immunosuppression combined with cytokine-mediated immune augmentation is a useful strategy to improve antitumor immunity.

Rodent parvovirus infections.

Parvoviruses are among the most common infectious agents of laboratory rodents and major impediments to rodent-based research. The original prototypic rodent parvoviruses-minute virus of mice, rat virus, and H-1 virus-have recently been joined by biologically and antigenically distinct parvoviruses in mice, rats, and hamsters. Recognition of the increased diversity of rodent parvoviruses presents new challenges for determining the impact of parvovirus infection on research and for detecting, preventing, and eliminating infection. This review summarizes current knowledge about rodent parvoviruses and parvovirus infections, highlighting recent research on newly isolated virus strains.

Molecular characterization of newly recognized rodent parvoviruses.

Several autonomous rodent parvoviruses distinct from the prototypic rodent parvoviruses have been isolated. These include variants of a mouse parvovirus (MPV), a hamster isolate designated hamster parvovirus (HaPV), and a variant strain of minute virus of mice (MVM) designated MVM-Cutter or MVM(c). In this study, the DNA sequence of the coding regions of the viral genome and the predicted protein sequences for each of these new isolates were determined and compared to the immunosuppressive and prototypic strains of MVM [MVM(i) and MVM(p)], the rodent parvovirus H-1, and LuIII, an autonomous parvovirus of uncertain host origin. Sequence comparisons showed that the MPV isolates were almost identical, HaPV was very similar to MPV, and MVM(c) was most similar to MVM(i) and MVM(p). Haemagglutination inhibition assays revealed that MPV and HaPV represent two serotypes distinct from previously characterized rodent parvovirus serotypes while MVM(c) belongs to the MVM serotype. Each of the newly isolated rodent parvoviruses was shown to encapsidate a predominantly negative-sense 5 kb DNA genome and to encode two nonstructural proteins (NS1 and NS2) and two structural viral proteins (VP1 and VP2). These studies indicate that MPV and HaPV are autonomous parvoviruses distinct from previously characterized parvoviruses and MVM(c) is a variant strain of MVM distinct from MVM(i) and MVM(p).

Detection of newly recognized rodent parvoviruses by PCR.

Several autonomous parvovirus isolates distinct from the prototypic rodent parvoviruses have recently been identified. These include variants of a mouse orphan parvovirus (MOPV) and a hamster isolate designated hamster orphan parvovirus (HOPV). In this study, a PCR primer set specific for these newly identified rodent parvoviruses was designed on the basis of DNA sequence comparisons of these isolates with other autonomous parvoviruses. The specificity of the primer set was determined by testing viral preparations of seven different parvoviruses and eight other viruses known to infect rodents. The PCR assay amplified the expected 260-bp product only in the presence of DNA from MOPV, HOPV, or LuIII a parvovirus of unknown species origin. The assay was able to detect as little as 10 pg of MOPV viral DNA or 1 pg of HOPV viral DNA, and it was able to detect MOPV in tissues from naturally infected mice and HOPV in tissues from experimentally infected hamsters. In contrast, the 260-bp product was not amplified from tissues of MOPV-negative mice or mock-infected hamsters. Our findings indicate that this PCR assay provides a rapid, specific, and sensitive method for the detection of MOPV in mice, HOPV in hamsters, and MOPV and HOPV in cell culture systems and that it may also be useful for the detection of LuIII contamination of cell culture systems.

Detection of H-1 parvovirus and Kilham rat virus by PCR.

H-1 virus and Kilham rat virus (KRV) are autonomous parvoviruses which generally cause subclinical infections in rats and can cause persistent infections in cell cultures. In this study, primer sets specific for either H-1 or KRV were designed on the basis of DNA sequence comparisons of the rodent parvoviruses. The specificities of the H-1 and KRV-specific primer sets were determined by testing viral preparations of seven different parvoviruses and nine other viruses known to infect rodents. The H-1-specific PCR assay amplified the expected 254-bp product only in the presence of H-1 viral DNA and was able to detect as little as 100 fg of H-1 viral DNA. The KRV-specific PCR assay generated the expected 281-bp product only when KRV viral DNA was used as the template and was able to detect as little as 10 pg of KRV viral DNA. Each assay was able to detect its respective virus in tissues from rats experimentally infected with H-1 or KRV. In contrast, no product was amplified by either assay with tissues from mock-infected rats. Our findings indicate that these PCR assays provide rapid, specific, and sensitive methods for the detection of H-1 or KRV infection in rats and cell culture systems.