Mary K. Reinhard

Comparative murine norovirus studies reveal a lack of correlation between intestinal virus titers and enteric pathology

Human noroviruses are significant emerging pathogens, causing the majority of non-bacterial gastroenteritis outbreaks worldwide. The recent discovery of 30 murine norovirus strains is beginning to facilitate a detailed investigation of norovirus pathogenesis. Here, we have performed an in vivo comparative analysis of two murine norovirus strains, MNV-1 and MNV-3. In immunocompetent mice, MNV-1 caused modest intestinal pathology whereas MNV-3 was attenuated compared to MNV-1. Surprisingly though, MNV-3 reached higher titers in intestinal tissue than MNV-1. MNV-3 also displayed attenuation in mice deficient in the critical interferon signaling molecule STAT-1, demonstrating that MNV-3 attenuation is not a result of increased interferon sensitivity. Importantly, MNV-3-infected mice lost weight and developed gastric bloating and diarrhea in STAT1(-/-) mice, from which all animals recovered. This disease profile recapitulates several key features of acute gastroenteritis experienced by people infected with a human norovirus.

Centrally administered angiotensin-(1–7) increases the survival of stroke-prone spontaneously hypertensive rats

What is the central question of this study? Activation of angiotensin‐converting enzyme 2, resulting in production of angiotensin‐(1–7) and stimulation of its receptor, Mas, exerts beneficial actions in a number cardiovascular diseases, including ischaemic stroke. A potential beneficial role for angiotensin‐(1–7) in haemorrhagic stroke has not previously been reported. What is the main finding and its importance? Central administration of angiotensin‐(1–7) into stroke‐prone spontaneously hypertensive rats, a model of haemorrhagic stroke, increases lifespan and improves the neurological status of these rats, as well as decreasing microglial numbers in the striatum (implying attenuation of cerebral inflammation). These actions of angiotensin‐(1–7) have not previously been reported and identify this peptide as a potential new therapeutic target in haemorrhagic stroke.

Different inflammatory responses are associated with Ureaplasma parvum-induced UTI and urolith formation

BackgroundEpidemiologic studies show a strong association between Ureaplasmas and urogenital tract disease in humans. Since healthy humans can be colonized with Ureaplasmas, its role as a pathogen remains controversial. In order to begin to define the role of the host in disease, we developed a rodent model of urinary tract infection (UTI) using Fischer 344 (F344) rats. Animals were inoculated with sterile broth, 101, 103, 105, 107, or 109 log CFU of a rat-adapted strain of Ureaplasma parvum.ResultsInfected animals exhibited two distinct profiles, asymptomatic UTI and UTI complicated with struvite urolithiasis. Inoculum dose of U. parvum affected the incidence of UTI, and 50% to 57% of animals inoculated with ≥ 107 CFU of U. parvum remained infected (p < 0.04). However, inoculum dose did not influence immune response to U. parvum. Asymptomatic UTI was characterized by a minimal immune response that was predominantly monocytic and lymphocytic, with limited lesions, and elevated urinary levels of IFN-γ, IL-18 and MCP-1 (P ≤ 0.02). UTI complicated with struvite formation was characterized by an exaggerated immune response that was mostly neutrophilic (P ≤ 0.0001), with lesions that showed extensive uroepithelial hyperplasia (P ≤ 0.0001), and a predominance of IL-1α, IL-1β, and GRO/KC in the urine (P ≤ 0.02). Animals with asymptomatic UTI also had a significantly high rate of kidney infection (P ≤ 0.0005).ConclusionComplications associated with U. parvum infection are primarily dependent upon host-specific factors rather than Ureaplasma microbial load. The immune response in F344 rats is similar to that which occurs in humans with ureaplasmal associated disease. Therefore, this model of infection is a useful tool for elucidating U. parvum-host interactions that confer UTI and disease.

Myxoma Virus Expressing Interleukin-15 Fails To Cause Lethal Myxomatosis in European Rabbits

ABSTRACT Myxoma virus (MYXV) is a poxvirus pathogenic only for European rabbits, but its permissiveness in human cancer cells gives it potential as an oncolytic virus. A recombinant MYXV expressing both the tdTomato red fluorescent protein and interleukin-15 (IL-15) (vMyx-IL-15-tdTr) was constructed. Cells infected with vMyx-IL-15-tdTr secreted bioactive IL-15 and had in vitro replication kinetics similar to that of wild-type MYXV. To determine the safety of this virus for future oncolytic studies, we tested its pathogenesis in European rabbits. In vivo, vMyx-IL-15-tdTr no longer causes lethal myxomatosis. Thus, ectopic IL-15 functions as an antiviral cytokine in vivo, and vMyx-IL-15-tdTr is a safe candidate for animal studies of oncolytic virotherapy.

Rat Strains Differ in Susceptibility to Ureaplasma parvum-Induced Urinary Tract Infection and Struvite Stone Formation

ABSTRACT Individuals with struvite uroliths are susceptible to recurrent urinary tract infections (UTI), sepsis, and renal disease. Unfortunately, little is known about the host-specific factors that predispose to this disease. In order to develop a rodent model that can address this problem, we inoculated female Fischer 344 (F344), Lewis (LEW), Sprague-Dawley (SD), and Wistar (WIS) rats with a host-adapted strain of Ureaplasma parvum. Animals were necropsied at 2 weeks postinoculation; 100% of F344, 42% of SD, 10% of LEW, and 10% of WIS rats remained infected. Severe bladder lesions and struvite calculi were seen in 64% of F344 rats; in other rat strains, bladder lesions were mild or absent. F344 rats with struvite uroliths had the highest urinary levels of proinflammatory cytokines, such as GRO/KC, interleukin-1α (IL-1α), and IL-1β. F344 rats without struvite stones at necropsy had milder bladder lesions and significantly lower urinary levels of proinflammatory cytokines but a more prominent inflammatory response than did other rat strains. Based on our results, struvite stone formation is linked to a robust inflammatory response that does not resolve UTI but instead promotes damage to surrounding tissues.

PKR regulates proliferation, differentiation and survival of murine hematopoietic stem/progenitor cells

Protein kinase R (PKR) is an interferon (IFN)-inducible, double-stranded RNA-activated kinase that initiates apoptosis in response to cellular stress. To determine the role of PKR in hematopoiesis, we developed transgenic mouse models that express either human PKR (TgPKR) or a dominant-negative PKR (TgDNPKR) mutant specifically in hematopoietic tissues. Significantly, peripheral blood counts from TgPKR mice decrease with age in association with dysplastic marrow changes. TgPKR mice have reduced colony-forming capacity and the colonies also are more sensitive to hematopoietic stresses. Furthermore, TgPKR mice have fewer hematopoietic stem/progenitor cells (HSPCs), and the percentage of quiescent (G0) HSPCs is increased. Importantly, treatment of TgPKR bone marrow (BM) with a PKR inhibitor specifically rescues sensitivity to growth factor deprivation. In contrast, marrow from PKR knockout (PKRKO) mice has increased potential for colony formation and HSPCs are more actively proliferating and resistant to stress. Significantly, TgPKR HSPCs have increased expression of p21 and IFN regulatory factor, whereas cells from PKRKO mice display mechanisms indicative of proliferation such as reduced eukaryotic initiation factor 2α phosphorylation, increased extracellular signal-regulated protein kinases 1 and 2 phosphorylation, and increased CDK2 expression. Collectively, data reveal that PKR is an unrecognized but important regulator of HSPC cell fate and may play a role in the pathogenesis of BM failure.

Host Genetic Background Impacts Disease Outcome During Intrauterine Infection with Ureaplasma parvum

Ureaplasma parvum, an opportunistic pathogen of the human urogenital tract, has been implicated in contributing to chorioamnionitis, fetal morbidity, and fetal mortality. It has been proposed that the host genetic background is a critical factor in adverse pregnancy outcome as sequela to U. parvum intra-amniotic infection. To test this hypothesis we assessed the impact of intrauterine U. parvum infection in the prototypical TH1/M1 C57BL/6 and TH2/M2 BALB/c mouse strain. Sterile medium or U. parvum was inoculated into each uterine horn and animals were evaluated for intra-amniotic infection, fetal infection, chorioamnionitis and fetal pathology at 72 hours post-inoculation. Disease outcome was assessed by microbial culture, in situ detection of U. parvum in fetal and utero-placental tissues, grading of chorioamnionitis, and placental gene expression of IL-1α, IL-1β, IL-6, TNF-α, S100A8, and S100A9. Placental infection and colonization rates were equivalent in both strains. The in situ distribution of U. parvum in placental tissues was also similar. However, a significantly greater proportion of BALB/c fetuses were infected (P<0.02). C57BL/6 infected animals predominantly exhibited mild to moderate chorioamnionitis (P<0.0001), and a significant reduction in placental expression of IL-1α, IL-1β, IL-6, TNF-α, S100A8, and S100A9 compared to sham controls (P<0.02). Conversely, severe protracted chorioamnionitis with cellular necrosis was the predominant lesion phenotype in BALB/c mice, which also exhibited a significant increase in placental expression of IL-1α, IL-1β, IL-6, TNF-α, S100A8, and S100A9 (P<0.01). Fetal pathology in BALB/c was multi-organ and included brain, lung, heart, liver, and intestine, whereas fetal pathology in C57BL/6 was only detected in the liver and intestines. These results confirm that the host genetic background is a major determinant in ureaplasmal induced chorioamnionitis with fetal infection and fetal inflammatory response.

Bacteriophage administration significantly reduces Shigella colonization and shedding by Shigella-challenged mice without deleterious side effects and distortions in the gut microbiota

We used a mouse model to establish safety and efficacy of a bacteriophage cocktail, ShigActive™, in reducing fecal Shigella counts after oral challenge with a susceptible strain. Groups of inbred C57BL/6J mice challenged with Shigella sonnei strain S43-NalAcR were treated with a phage cocktail (ShigActive™) composed of 5 lytic Shigella bacteriophages and ampicillin. The treatments were administered (i) 1 h after, (ii) 3 h after, (iii) 1 h before and after, and (iv) 1 h before bacterial challenge. The treatment regimens elicited a 10- to 100-fold reduction in the CFUs of the challenge strain in fecal and cecum specimens compared to untreated control mice, (P < 0.05). ShigActiveTM treatment was at least as effective as treatment with ampicillin but had a significantly less impact on the gut microbiota. Long-term safety studies did not identify any side effects or distortions in overall gut microbiota associated with bacteriophage administration. Shigella phages may be therapeutically effective in a “classical phage therapy” approach, at least during the early stages after Shigella ingestion. Oral prophylactic “phagebiotic” administration of lytic bacteriophages may help to maintain a healthy gut microbiota by killing specifically targeted bacterial pathogens in the GI tract, without deleterious side effects and without altering the normal gut microbiota.

Tnk1/Kos1 Knockout Mice Develop Spontaneous Tumors

Tnk1/Kos1 is a non-receptor protein tyrosine kinase implicated in negatively regulating cell growth in a mechanism requiring its intrinsic catalytic activity. Tnk1/Kos1 null mice were created by homologous recombination by deleting the catalytic domain. Both Tnk1(+/-) and Tnk1(-/-) mice develop spontaneous tumors, including lymphomas and carcinomas, at high rates [27% (14 of 52) and 43% (12 of 28), respectively]. Tnk1/Kos1 expression is silenced in tumors that develop in Tnk1(+/-) mice but not in adjacent uninvolved tissue, and silencing occurs in association with Tnk1 promoter hypermethylation. Tissues and murine embryonic fibroblasts derived from Tnk1/Kos1-null mice exhibit proportionally higher levels of basal and epidermal growth factor-stimulated Ras activation that results from increased Ras-guanine exchange factor (GEF) activity. Mechanistically, Tnk1/Kos1 can directly tyrosine phosphorylate growth factor receptor binding protein 2 (Grb2), which promotes disruption of the Grb2-Sos1 complex that mediates growth factor-induced Ras activation, providing dynamic regulation of Ras GEF activity with suppression of Ras. Thus, Tnk1/Kos1 is a tumor suppressor that functions to down-regulate Ras activity.

Enhanced alveolar bone loss in a model of non-invasive periodontitis in rice rats.

OBJECTIVE The rice rat (Oryzomys palustris) develops periodontitis-like lesions when fed a diet rich in sucrose and casein (H-SC). We aimed to establish whether this model can accurately mimic the development of human periodontitis. MATERIALS AND METHODS For this purpose, 28-day-old rice rats (15/group) were assigned to standard (STD) or H-SC diets and sacrificed after 6, 12, and 18 weeks. Jaws were processed for morphometric, histometric, histologic, histomorphometric, and micro-CT analyses. RESULTS We found a progressive increase in horizontal alveolar bone loss (ABL) with age in maxillae of rats fed the STD diet as determined by morphometry. The H-SC diet exacerbated horizontal ABL at the palatal surface at 12 and 18 weeks. Furthermore, increased vertical ABL was detected in mandibles and maxillae of rats fed the H-SC diet for 12 and/or 18 weeks by histometry and micro-CT. Remarkably, the H-SC diet significantly increased bone remodeling at the interproximal alveolar bone of mandibles from rats fed for 6 weeks, but not in those fed for longer periods. CONCLUSIONS These findings indicate that the H-SC diet induced a transient increase in alveolar bone remodeling, which is followed by ABL characteristic of moderate periodontitis.