Factors Affecting Hematologic and Serum Biochemical Parameters in Healthy Common Marmosets (Callithrix jacchus)

Physiologic changes during development, aging, and pregnancy may affect clinical parameters. Previously available reference values have been based on samples that may include wild and captive marmosets, with little representation of geriatric or pregnant animals. Establishing reference values under various conditions would support better recognition of pathologic conditions in marmosets. One hundred and forty-seven (70 males and 77 females) healthy marmosets from a research colony were included in this study. Exclusion criteria were abnormal physical exam findings at the time of blood sampling, chronic medications, or clinical or pathologic evidence of disease. Reference intervals were calculated for serum chemistry and hematology. Using metadata, samples were classified based on age, sex, colony source and pregnancy status. Multiple tests indicated significant differences with varying effect sizes, indicating that developing reference intervals based on metadata can be useful. Across all the comparisons, medium or large effect sizes were observed most frequently in blood urea nitrogen (BUN), calcium, total protein, alkaline phosphatase (ALP), weight and serum albumin. We report normative clinical pathologic data for captive common marmosets through all life stages and reproductive status. Significant differences were observed in most parameters when stratifying data based on age, sex, colony source, or pregnancy, suggesting that developing reference intervals considering this information is important for clinicians.     

Alkyl hydroperoxide reductase is required for Helicobacter cinaedi intestinal colonization and survival under oxidative stress in BALB/c and BALB/c interleukin-10-/- mice

Helicobacter cinaedi, a common human intestinal bacterium, has been implicated in various enteric and systemic diseases in normal and immunocompromised patients. Protection against oxidative stress is a crucial component of bacterium-host interactions. Alkyl hydroperoxide reductase C (AhpC) is an enzyme responsible for detoxification of peroxides and is important in protection from peroxide-induced stress. H. cinaedi possesses a single ahpC, which was investigated with respect to its role in bacterial survival during oxidative stress. The H. cinaedi ahpC mutant had diminished resistance to organic hydroperoxide toxicity but increased hydrogen peroxide resistance compared with the wild-type (WT) strain. The mutant also exhibited an oxygen-sensitive phenotype and was more susceptible to killing by macrophages than the WT strain. In vivo experiments in BALB/c and BALB/c interleukin-10 (IL-10)(-/-) mice revealed that the cecal colonizing ability of the ahpC mutant was significantly reduced. The mutant also had diminished ability to induce bacterium-specific immune responses in vivo, as shown by immunoglobulin (IgG2a and IgG1) serum levels. Collectively, these data suggest that H. cinaedi ahpC not only contributes to protecting the organism against oxidative stress but also alters its pathogenic properties in vivo.     

Characterization and immunogenicity of pyrogenic mitogens SePE-H and SePE-I of Streptococcus equi.

Two pyrogenic mitogens, SePE-H and SePE-I, were characterized in Streptococcus equi, the cause of equine strangles. SePE-H and SePE-I have molecular masses of 27.5 and 29.5 kDa, respectively, and each is almost identical to its counterpart in Streptococcus pyogenes M1. Both genes are adjacent to a gene encoding a phage muramidase of 49.7 kDa and are located immediately downstream from a phage genomic sequence almost identical to a similar phage sequence in S. pyogenes M1. Strong mitogenic responses were elicited by both proteins from horse peripheral blood mononuclear cells. However, although both were pyrogenic for rabbits, only SePE-I was pyrogenic in ponies. Convalescent sera contained antibody to each mitogen and horses recovered from strangles or immunized with SePE-I were resistant to the pyrogenic effect of SePE-I. The immunogenicity of SePE-I suggests that it should be included in new generation strangles vaccines. In isolates of S. equi sepe-I and sepe-H were consistently present but they were absent from the closely related Streptococcus zooepidemicus, suggesting that phage mediated transfer was an important event in the formation of the clonal, more virulent, S. equi from its putative S. zooepidemicus ancestor.     

Vaccine potential of novel surface exposed and secreted proteins of Streptococcus equi

Streptococcus equi, a clonal descendent of an ancestral S. zooepidemicus, causes equine strangles, a highly contagious purulent lymphadenitis of the head and neck. The aim of this study was to evaluate as vaccine components novel surface exposed or secreted S. equi proteins identified in an expression gene library with sera from resistant horses. Six proteins expressed by S. equi CF32 but not by S. zooepidemicus 631 were used to vaccinate one group of eight ponies. A second pony group was immunized with five adhesin and other proteins encoded by genes of Linkage Gr 1. All ponies made strong serum antibody responses to each protein as measured by ELISA but none were resistant to subsequent comingling challenge with S. equi CF32. These results in combination with evidence that recovered horses rapidly clear intranasally inoculated S. equi and do not make detectable serum antibody responses to its surface proteins suggest that acquired immune-mediated tonsillar clearance and not serum antibody must be stimulated by an effective strangles vaccine.     

Aag-initiated base excision repair promotes ischemia reperfusion injury in liver,brain, and kidney

Inflammation is accompanied by the release of highly reactive oxygen and nitrogen species (RONS) that damage DNA, among other cellular molecules. Base excision repair (BER) is initiated by DNA glycosylases and is crucial in repairing RONS-induced DNA damage; the alkyladenine DNA glycosylase (Aag/Mpg) excises several DNA base lesions induced by the inflammation-associated RONS release that accompanies ischemia reperfusion (I/R). Using mouse I/R models we demonstrate that Aag^−/− mice are significantly protected against, rather than sensitized to, I/R injury, and that such protection is observed across three different organs. Following I/R in liver, kidney, and brain, Aag^−/− mice display decreased hepatocyte death, cerebral infarction, and renal injury relative to wild-type. We infer that in wild-type mice, Aag excises damaged DNA bases to generate potentially toxic abasic sites that in turn generate highly toxic DNA strand breaks that trigger poly(ADP-ribose) polymerase (Parp) hyperactivation, cellular bioenergetics failure, and necrosis; indeed, steady-state levels of abasic sites and nuclear PAR polymers were significantly more elevated in wild-type vs. Aag^−/− liver after I/R. This increase in PAR polymers was accompanied by depletion of intracellular NAD and ATP levels plus the translocation and extracellular release of the high-mobility group box 1 (Hmgb1) nuclear protein, activating the sterile inflammatory response. We thus demonstrate the detrimental effects of Aag-initiated BER during I/R and sterile inflammation, and present a novel target for controlling I/R-induced injury.Ischemia reperfusion (I/R)-induced tissue injury is one of the most common examples of acute, sterile inflammation-induced tissue damage. Health events that incur I/R include ischemic stroke, acute liver or kidney failure, myocardial infarction, various forms of circulatory shock, sickle cell disease, and organ transplantations. These conditions are frequently accompanied by profound morbidity and mortality worldwide (1). The need for effective approaches to manage patients with I/R-induced organ damage is highlighted by the fact that current treatment is primarily supportive care (1).During I/R, a burst of reactive oxygen and nitrogen species (RONS) occurs within the first moments of reperfusion, and this burst is thought to be primarily responsible for collateral tissue damage (2). Furthermore, RONS are also generated during the preceding ischemia, despite the low oxygen tension (3). I/R alters cellular metabolism and redox state, and induces so-called “sterile inflammation” by activating the innate immune system. The activation of macrophages and recruitment of neutrophils to sites of I/R by intravascular danger signals results in the release of an arsenal of RONS capable of inducing DNA damage and lipid peroxidation, thus causing major collateral tissue damage. Base excision repair (BER), initiated by various DNA glycosylases, is critical for the repair of RONS-associated DNA damage, including oxidized, deaminated, and etheno (ε)-adducted DNA bases (4, 5). BER involves the orchestration of the following enzymatic steps: damaged bases are excised by DNA glycosylase, followed by cleavage of the DNA backbone at the resulting abasic site; DNA ends are trimmed to generate a 3′OH and 5′P; the gap is then filled by DNA polymerase and the remaining nick sealed by DNA ligase to complete BER (6, 7) (Fig. 1A). The murine alkyladenine DNA glycosylase (Aag; also known as Mpg) acts efficiently on 1,N⁶-ethenodeoxyadenosine (ε-A) lesions and deaminated adenosine (hypoxanthine, Hx) (8, 9), and with lower efficiency on 8-oxoguanine (8-oxoG) (10), all of which are induced directly or indirectly by RONS (11). Aag therefore seemed likely to provide resistance to I/R-induced toxicity. In support of this hypothesis, we previously found that Aag^−/− mice suffer more inflammation-associated intestinal tissue damage and colon carcinogenesis than WT mice (5, 12); with this in mind, we set out to examine whether the Aag DNA glycosylase modulates I/R-induced tissue injury in three distinct tissue environments—namely liver, brain, and kidney. To our surprise, we find that Aag-initiated BER actually exacerbates, rather than attenuates, I/R tissue injury in all three organs.Open in a separate windowFig. 1.Aag deficiency protects liver against I/R-induced tissue damage. (A) The BER pathway initiated by various DNA glycosylases consists of four key enzymatic steps: (i) glycosylase-mediated excision of a damaged base generating an abasic site; (ii) incision of the sugar-phosphate backbone at the abasic site by APE, yielding a 3′-OH adjacent to a 5′-dRP moiety; (iii) 5′-dRP removal and DNA synthesis by DNA polymerase β (Pol β); and (iv) sealing of the DNA nick by DNA ligase (LIG). (B) Serum ALT concentrations after 90 min of ischemia and 24 h of reperfusion (n = 3 in sham groups and n = 7–8 in I/R groups). (C) Representative images of necrotic areas 24 h after I/R in Aag^−/− (n = 7) or WT livers (n = 8); liver sections (4 μm) were stained with H&E. (D) Mean necrotic area in ischemic liver samples and (E) serum LDH concentrations 24 h after reperfusion (n = 3 in sham groups and n = 7–8 in I/R groups); necrosis analyzed by ImageJ (necrotic areas indicated by green color). (F) Serum ALT in control mice (no surgery) or after 60 min of ischemia plus either 6- or 24-h reperfusion in WT and Aag^−/− mice. Error bars represent the mean ± SEM of at least three independent experiments.     

Systemic Macrophage Depletion Inhibits Helicobacter bilis-Induced Proinflammatory Cytokine-Mediated Typhlocolitis and Impairs Bacterial Colonization Dynamics in a BALB/c Rag2?/? Mouse Model of Inflammatory Bowel Disease

Helicobacter bilis, an enterohepatic helicobacter, is associated with chronic hepatitis in aged immunocompetent inbred mice and inflammatory bowel disease (IBD) in immunodeficient mice. To evaluate the role of macrophages in H. bilis-induced IBD, Rag2^−/− BALB/c or wild-type (WT) BALB/c mice were either sham dosed or infected with H. bilis Missouri strain under specific-pathogen-free conditions, followed by an intravenous injection of a 0.2-ml suspension of liposomes coated with either phosphate-buffered saline (control) or clodronate (a macrophage depleting drug) at 15 weeks postinfection (wpi). At 16 wpi, the ceca of H. bilis-infected Rag2^−/− mice treated with control liposomes had significantly higher histopathological lesional scores (for cumulative typhlitis index, inflammation, edema, epithelial defects, and hyperplasia) and higher counts of F4/80⁺ macrophages and MPO⁺ neutrophils compared to H. bilis-infected Rag2^−/− mice treated with clodronate liposomes. In addition, cecal quantitative PCR analyses revealed a significant suppression in the expression of macrophage-related cytokine genes, namely, Tnfa, Il-1β, Il-10, Cxcl1, and iNos, in the clodronate-treated H. bilis-infected Rag2^−/− mice compared to the H. bilis-infected Rag2^−/− control mice. Finally, cecal quantitative PCR analyses also revealed a significant reduction in bacterial colonization in the clodronate-treated Rag2^−/− mice. Taken together, our results suggest that macrophages are critical inflammatory cellular mediators for promoting H. bilis-induced typhlocolitis in mice.     

17 β-estradiol suppresses Helicobacter pylori-induced gastric pathology in male hypergastrinemic INS-GAS mice

Helicobacter pylori-associated gastric cancer is male predominant and animal studies suggest that sex hormones influence gastric carcinogenesis. We investigated the effects of 17β-estradiol (E2) or castration on H.pylori-induced gastritis in male INS-GAS/FVB/N (Tg(Ins1-GAS)1Sbr) mice. Comparisons were made to previously evaluated sham (n = 8) and H.pylori-infected (n = 8), intact male INS-GAS mice which had developed severe corpus gastritis accompanied by atrophy, hyperplasia, intestinal metaplasia and dysplasia of the epithelium within 16 weeks postinfection (all P < 0.01). Castration at 8 weeks of age had no sparing effect on lesions in uninfected (n = 5) or H.pylori-infected mice (n = 7) but all lesion subfeatures were attenuated by E2 in H.pylori-infected mice (n = 7) (P < 0.001). Notably, inflammation was not reduced but glandular atrophy, hyperplasia, intestinal metaplasia and dysplasia were also less severe in uninfected, E2-treated mice (n = 7) (P < 0.01). Attenuation of gastric lesions by E2 was associated with lower messenger RNA (mRNA) expression of interferon (IFN)-γ (P < 0.05) and interleukin (IL)-1β (P < 0.004), and higher IL-10 (P < 0.02) as well as decreased numbers of Foxp3(+) regulatory T cells when compared with infected intact males. Infected E2-treated mice also developed higher Th2-associated anti-H.pylori IgG1 responses (P < 0.05) and significantly lower Ki-67 indices of epithelial proliferation (P < 0.05). E2 elevated expression of mRNA for Foxp3 (P < 0.0001) and IL-10 (P < 0.01), and decreased IL-1β (P < 0.01) in uninfected, intact male mice compared with controls. Therefore, estrogen supplementation, but not castration, attenuated gastric lesions in H.pylori-infected male INS-GAS mice and to a lesser extent in uninfected mice, potentially by enhancing IL-10 function, which in turn decreased IFN-γ and IL-1β responses induced by H.pylori.