The Collaborative Cross as a Resource for Modeling Human Disease: CC011/Unc,a New Mouse Model for Spontaneous Colitis

Inflammatory bowel disease (IBD) is an immune-mediated condition driven by improper responses to intestinal microflora in the context of environmental and genetic background. GWAS in humans have identified many loci associated with IBD, but animal models are valuable for dissecting the underlying molecular mechanisms, characterizing environmental and genetic contributions and developing treatments. Mouse models rely on interventions such as chemical treatment or introduction of an infectious agent to induce disease. Here, we describe a new model for IBD in which the disease develops spontaneously in 20-week-old mice in the absence of known murine pathogens. The model is part of the Collaborative Cross and came to our attention due to a high incidence of rectal prolapse in an incompletely inbred line. Necropsies revealed a profound proliferative colitis with variable degrees of ulceration and vasculitis, splenomegaly and enlarged mesenteric lymph nodes with no discernible anomalies of other organ systems. Phenotypic characterization of the CC011/Unc mice with homozygosity ranging from 94.1 to 99.8 % suggested that the trait was fixed and acted recessively in crosses to the colitis-resistant C57BL/6J inbred strain. Using a QTL approach, we identified four loci, Ccc1, Ccc2, Ccc3 and Ccc4 on chromosomes 12, 14, 1 and 8 that collectively explain 27.7 % of the phenotypic variation. Surprisingly, we also found that minute levels of residual heterozygosity in CC011/Unc have significant impact on the phenotype. This work demonstrates the utility of the CC as a source of models of human disease that arises through new combinations of alleles at susceptibility loci.     

Papiine herpesvirus 2 as a Predictive Model for Drug Sensitivity of Macacine herpesvirus 1 (Monkey B Virus)

Monkey B virus (Macacine herpesvirus 1; BV) is endemic in macaques. BV (a BSL4 agent) is the primary zoonotic concern for persons working with macaques in research, and human BV infections frequently are fatal. We assessed the use of a BSL2 baboon herpesvirus (Papiine herpesvirus 1; HVP2) for predicting the drug sensitivity of BV by comparing the sensitivity of the 2 viruses to 12 antiherpetic drugs. Plaque reduction assays showed that 4 drugs (HBPG, BVdU, PFA, and BrdU) were ineffective against both viruses. Of the 8 effective drugs, both viruses were most sensitive to TFT, whereas sensitivity to the remaining 7 drugs varied between BV and HVP2 as well as between strains of HVP2. In addition, the efficacy of 5 drugs (ACV, PCV, GCV, CDV, and EDU) was tested by using a murine model. ACV and EDU were completely ineffective against both HVP2 and BV, and high doses of PCV only delayed death by a few days. GCV and CDV both protected mice against death, and CDV also prevented the development of neurologic symptoms. When the initiation of drug therapy was delayed until after virus gained access to the CNS, both GCV and CDV were ineffective. The similarity of the drug sensitivities of HVP2 and BV in both models validates the use of HVP2 as a BSL2 level model that can be used to predict drug sensitivity of BV. The greater efficacy of CDV relative to GCV suggests the potential for use of CDV in the treatment of zoonotic BV infections.Abbreviations: ACV, acyclovir; AraA, 9-β-D-arabinofuranosyl-adenine; BrdU, 5-bromo-2′-deoxyuridine; BV, monkey B virus; BVdU, (E)-5-(2-bromovinyl)-2′-deoxyuridine; CDV, cidofovir; EDU, 5-ethyl-2′-deoxyuridine; GCV, ganciclovir; HBPG, 9-(4-hydroxybutyl)-N²-phenylguanine; HSV, herpes simplex virus; HVP2, Herpesvirus papio 2; IUdR, 5-iodo-2’-deoxyuridine; PCV, penciclovir; PFA, phosphonoformic acid; TFT, trifluorothymidineMacacine herpesvirus 1 (monkey B virus; BV) is an alpha-herpesvirus of macaque monkeys and is closely related to human herpes simplex virus (HSV) types 1 and 2.^8,11,27 Although BV primarily causes asymptomatic or mild, self-limiting disease in healthy macaques, the virus is extremely neurovirulent when transmitted via bites or scratches to other nonmacaque primate species, including humans. Although human infections are not common, approximately 80% of untreated patients die of BV infection, and survivors frequently continue to suffer from neurologic sequelae. As a consequence of its lethality in humans, BV is classified as a BSL4 pathogen³ and is the single most serious zoonotic concern for veterinary and research personnel who work with macaques. The increasing popularity of ecotourism to monkey temples in Southeast Asia, where tourists and wild, BV-infected macaque populations come into direct contact, represents another potential concern for zoonotic BV infections.^9,12,13,21The antiviral drugs recommended for use in treating BV infections all were originally developed for treatment of HSV infections.^4,18 Because the genes encoding the enzymes targeted by these drugs are conserved between these viruses, BV is sensitive to many of these anti-HSV drugs. However, compared with HSV, BV is less sensitive to these drugs.^2,10,14 Although more effective drugs are needed for the treatment of BV infections, the biohazardous nature of and facility requirements associated with studying a BSL4 agent severely limit research on BV. A potential solution to this problem is using a closely related virus whose biologic and molecular properties are very similar to those of BV as a surrogate or model system in which preliminary research can be conducted safely, leaving only confirmative testing to be done with infectious BV.Baboons carry an alpha-herpesvirus (Papiine herpesvirus 2; HVP2) that is biologically and genetically very similar to BV and HSV.^7,8,15,26 In mice, most HVP2 isolates are extremely neurovirulent and closely reflect the pathogenesis of BV in mice^20,23 At the antigenic level, HVP2 and BV are so similar that HVP2 has found use as an alternative antigen for diagnostic BV serology.^17,25,28 Despite the virus''s similarity to BV, HVP2 infections have never been reported in humans. Consequently, HVP2 is rated as a BSL2 pathogen and, as such, HVP2 can be used under BSL2/ABSL2 containment. This study was conducted to assess the potential use of HVP2 as a surrogate model system for predicting the sensitivity of BV to antiviral drugs.     

Field calibration of soil-core microcosms: Fate of a genetically altered rhizobacterium

Microcosms containing intact soil-cores are a potential tool for assessing the risks of the release of genetically engineered microorganisms (GEMs) to the environment. Before microcosms become a standard assessment tool, however, they must first be calibrated to ensure that they adequately simulate key parameters in the field. Four systems were compared: intact soil-core microcosms located in the laboratory at ambient temperature and in a growth chamber with temperature fluctuations that simulated average conditions in the field, field lysimeters, and field plots. These four systems were inoculated with rifampicin-resistantPseudomonas sp. and planted to winter wheat. Populations of thePseudomonas sp. in soil decreased more rapidly at ambient temperature, but population size at the three-leaf stage of wheat growth was the same in all four systems. Populations of thePseudomonas sp. on the rhizoplane of wheat were the same at the three-leaf stage in all four systems, and colonization with depth at the final boot stage-sampling was also similar. In general, microcosms incubated at ambient temperature in the laboratory or in the growth chamber were similar to those in the field with respect to survival of and colonization of the rhizoplane by the introducedPseudomonas sp.     

SENP1 and SENP2 affect spatial and temporal control of sumoylation in mitosis

Sumoylation of centromere, kinetochore, and other mitotic chromosome-associated proteins is essential for chromosome segregation. The mechanisms regulating spatial and temporal sumoylation of proteins in mitosis, however, are not well understood. Here we show that the small ubiquitin-related modifier (SUMO)–specific isopeptidases SENP1 and SENP2 are targeted to kinetochores in mitosis. SENP2 targeting occurs through a mechanism dependent on the Nup107-160 subcomplex of the nuclear pore complex and is modulated through interactions with karyopherin α. Overexpression of SENP2, but not other SUMO-specific isopeptidases, causes a defect in chromosome congression that depends on its precise kinetochore targeting. By altering SENP1 kinetochore associations, however, this effect on chromosome congression could be phenocopied. In contrast, RNA interference–mediated knockdown of SENP1 delays sister chromatid separation at metaphase, whereas SENP2 knockdown produces no detectable phenotypes. Our findings indicate that chromosome segregation depends on precise spatial and temporal control of sumoylation in mitosis and that SENP1 and SENP2 are important mediators of this control.