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Effective pain relief in animals relies on the ability to discern pain and assess its severity. However, few objective measures exist to assess the presence and severity of pain in axolotls, and few resources are available regarding drugs and appropriate doses to provide pain relief in this species. This study evaluated behavioral tools for cageside pain assessment and validated a reproducible and reliable quantitative method to evaluate analgesic efficacy in axolotls. Animals were divided into control and treatment groups (n = 6 per group); treatment groups received buprenorphine through injection (50 mg/kg every 24 h for 48 h intracelomically) or butorphanol immersion (0.50 or 0.75 mg/L every 24 h for 48 h). Qualitative behavioral tests, adapted from other amphibian studies, included tapping on the home tank, directing water jets or physically touching specific anatomic points on the animal, and placing a novel object in the home tank. Quantitative methods used to produce noxious stimuli were the acetic acid test and von Frey aesthesiometers. Animals that were treated with analgesics did not demonstrate a significant difference compared with controls during behavioral assessment at 1, 6, 12, 25, 30, and 48 h after analgesia administration. The acetic acid test revealed a reproducible, concentration-dependent pain response. However, a significant difference in the AAT response was not observed between control and treated groups with the tested analgesics and doses.

Necropsy of a female adult pregnant harbor porpoise Phocoena phocoena revealed a multicentric plasmacytoma. The plasmacytoma infiltrated the cranial lung lobes, mediastinal lymph nodes and the spleen. Diagnosis was based on gross, histopathologic and immunohistochemical studies. Histopathology revealed a diffuse proliferation of atypical pleomorphic neoplastic round cells with plasmacytic features. Positive immunohistochemistry with anti-CD79a and anti-CD20 antibody markers and anti-multiple myeloma oncogene 1 (MUM-1) for plasmacytoma confirmed this neoplasm to be of B-cell origin. This is the first recorded case of a plasmacytoma in a harbor porpoise. Routine viral screening was negative via standard PCR for herpesvirus and reverse transcriptase PCR for morbillivirus. Retroviral screening was not performed.

Nanomedicine is an emerging field with great potential in disease theranostics. We generated sterically stabilized superparamagnetic iron oxide nanoparticles (s-SPIONs) with average core diameters of 10 and 25 nm and determined the in vivo biodistribution and clearance profiles. Healthy nude mice underwent an intraperitoneal injection of these s-SPIONs at a dose of 90 mg Fe/kg body weight. Tissue iron biodistribution was monitored by atomic absorption spectroscopy and Prussian blue staining. Histopathological examination was performed to assess tissue toxicity. The 10 nm s-SPIONs resulted in higher tissue-iron levels, whereas the 25 nm s-SPIONs peaked earlier and cleared faster. Increased iron levels were detected in all organs and body fluids tested except for the brain, with notable increases in the liver, spleen, and the omentum. The tissue-iron returned to control or near control levels within 7 days post-injection, except in the omentum, which had the largest and most variable accumulation of s-SPIONs. No obvious tissue changes were noted although an influx of macrophages was observed in several tissues suggesting their involvement in s-SPION sequestration and clearance. These results demonstrate that the s-SPIONs do not degrade or aggregate in vivo and intraperitoneal administration is well tolerated, with a broad and transient biodistribution. In an ovarian tumor model, s-SPIONs were shown to accumulate in the tumors, highlighting their potential use as a chemotherapy delivery agent.

B-cell lymphoma, a common morphologic variant of non-Hodgkin lymphoma, has been associated with persistent pollutants in humans, but this association is not well-characterized in top-level predators sharing marine resources with humans. We characterized and compared blubber contaminants and hormones of a pregnant harbor porpoise (Phocoena phocoena) with B-cell lymphoma, with those in two presumed healthy fishery by-caught porpoises with no lymphoma: a pregnant adult and female juvenile. Common historic use compounds, including polychlorinated biphenyls, polybrominated diphenyl ethers, and pesticides, were evaluated in blubber samples from three porpoises. In addition, blubber cortisol and progesterone levels (ng/g) were determined in all three animals. Total pollutant concentrations were highest in the juvenile porpoise, followed by the lymphoma porpoise and the nonlymphoma adult. Blubber cortisol concentrations were 191% greater in the pregnant with lymphoma porpoise compared with the pregnant no lymphoma porpoise, and 89% greater in the juvenile female compared with the pregnant no lymphoma porpoise. Although both adults were pregnant, progesterone levels were substantially greater (90%) in the healthy compared with the lymphoma adult. Health monitoring of top-level marine predators, such as porpoise, provides a sentinel measure of contaminants that serve as indicators of potential environmental exposure to humans.

Approximately 50% of the world's population is infected with Helicobacter pylori , leading to chronic inflammation, which increases the risk for gastric adenocarcinoma. MyD88 is a key adaptor molecule in inflammatory pathways involved in interleukin 1 (IL-1)/IL-18/Toll-like receptor signaling and has been shown to have divergent effects in carcinogenesis. The role of MyD88 in Helicobacter -induced gastric malignancy is unknown. Using a mouse model of Helicobacter -induced gastric cancer, we assessed the role of MyD88 in cancer development by evaluating gastric histopathology, apoptosis, proliferation, and cytokine expression. Infection of MyD88-deficient ( Myd88 −/− ) mice with Helicobacter resulted in early and rapid advancement to gastric dysplasia as early as 25 weeks postinfection. The progression of Helicobacter -induced disease to precancerous and cancerous lesions in the absence of MyD88 signaling was accompanied by increased gastric epithelial apoptosis and proliferation. In addition, inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), gamma interferon (IFN-γ), IL-6, and IL-1β were highly expressed in association with the development of gastric dysplasia. These data suggest that MyD88 signaling retards development and progression to cancer during Helicobacter infection. This is the first study to show evidence of MyD88 protection in an infection-driven inflammation-associated cancer model.

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-1 β, Il-10 , Cxcl1 , and iNos , in the clodronate-treated H. bilis -infected Rag2 −/− mice compared to the H. bilis -infected Rag 2 −/− 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.

Background: Helicobacter pylori is a spiral-shaped Gram-negative microaerophilic bacterium associated with a number of gastrointestinal disorders, including gastritis, peptic ulcers, and gastric cancer. Several studies have implicated a Th17 response as a key to protective immunity against Helicobacter. Materials and Methods: Wild type (WT) and MyD88-deficient (MyD88−/−) mice in the C57BL/6 background were infected with H. felis for 6 and 25 weeks and colonization density and host response evaluated. Real-time PCR was used to determine the expression of cytokines and antimicrobial peptides in the gastric tissue of mice. Results: mRNA expression levels of the Th17 cytokines interleukin-17A (IL-17A) and IL-22 were markedly up-regulated in WT compared with MyD88−/− mice both at 6 and at 25 weeks in response to infection with H. felis, indicating that induction of Th17 responses depends on MyD88 signaling. Furthermore, reduction in the expression of Th17-dependent intestinal antimicrobial peptide lipocalin-2 was linked with increased bacterial burden in the absence of MyD88 signaling. Conclusion: We provide evidence showing that MyD88-dependent signaling is required for the host to induce a Th17 response for the control of Helicobacter infection.

Author Manuscript: 2010 October 15, Helicobacter pylori infection causes severe dysplasia manifested as gastrointestinal intraepithelial neoplasia (GIN) after 28 weeks post–H. pylori infection (WPI) in cancer-prone, hypergastrinemic male INS-GAS mice. We examined the efficacy of the nonsteroidal anti-inflammatory drug sulindac (400 ppm in drinking water) alone, the CCK2/gastrin receptor antagonist YM022 (45 mg/kg/wk) alone, and sulindac or YM022 combined with H. pylori eradication therapy to prevent H. pylori–associated gastric cancer in male INS-GAS mice. Treatments started at 22 WPI, and mice were euthanized at 28 WPI. In uninfected mice, all treatments significantly delayed development of spontaneous GIN (P < 0.05). In H. pylori–infected mice, sulindac alone or YM022 alone had no protective effect on H. pylori–associated GIN. Importantly, sulindac exacerbated the severity of H. pylori–associated gastritis despite decreased gastric prostaglandin E2 levels. However, sulindac combined with H. pylori antimicrobial eradication reduced the incidence of GIN (P < 0.05), whereas YM022 combined with antimicrobial eradication did not reduce GIN. In infected mice, sulindac or YM022 treatment did not alter gastric expression of the proinflammatory cytokines Ifn-γ and Tnf-α and mucosal cell proliferation. Sulindac or YM022 combined with antimicrobial eradication down-regulated mRNA levels of Ifn-γ and Tnf-α and mucosal cell proliferation (P < 0.05). We conclude that sulindac enhances H. pylori gastritis and may promote inflammation-mediated gastric carcinogenesis. The combination of sulindac and antimicrobial H. pylori eradication was beneficial for reducing proinflammatory cytokine mRNA in the stomach and preventing progression from severe dysplasia to gastric cancer in H. pylori–infected INS-GAS mice. [Cancer Res 2009;69(20):8166–74], National Institutes of Health (U.S.) (Grant R01AI37750), National Institutes of Health (U.S.) (Grant P01CA26731), National Institutes of Health (U.S.) (Grant P30ES02109), National Institutes of Health (U.S.) (Grant R01CA093405-07A1)

We have previously described a synergistic interaction between hypergastrinemia and Helicobacter felis infection on gastric corpus carcinogenesis in FVB/N mice housed under specific-pathogen-free conditions. However, gastrin-deficient (GAS-KO) mice on a mixed C57BL/6/129Sv genetic background maintained in conventional housing were reported to develop spontaneous gastric antral tumors. Therefore, we investigated the role of gastrin in Helicobacter-associated gastric carcinogenesis in H. felis-infected mice on a uniform C57BL/6 background housed in specific-pathogen-free conditions. Hypergastrinemic transgenic (INS-GAS) mice, GAS-KO mice, and C57BL/6 wild-type mice were infected with H. felis for either 12 or 18 months. At 12 months postinfection, INS-GAS mice had mild corpus dysplasia, while B6 wild-type mice had either severe gastritis or metaplasia, and GAS-KO mice had only mild to moderate gastritis. At 18 months postinfection, both INS-GAS and B6 wild-type mice had both severe atrophic gastritis and corpus dysplasia, while GAS-KO mice had severe gastritis with mild gastric atrophy, but no corpus dysplasia. In contrast, both GAS-KO and B6 wild-type mice had mild to moderate antral dysplasia, while INS-GAS mice did not. H. felis antral colonization remained stable over time among the three groups of mice. These results point to a distinct effect of gastrin on carcinogenesis of both the gastric corpus and antrum, suggesting that gastrin is an essential cofactor for gastric corpus carcinogenesis in C57BL/6 mice.

Inflammatory bowel disease is a chronic inflammatory response of the gastrointestinal tract mediated in part by an aberrant response to intestinal microflora. Expression of IL-23 subunits p40 and p19 within cells of the innate immune system plays a central role in the development of lower bowel inflammation in response inflammatory challenge. The NF-κB subunit c-Rel can regulate expression of IL-12/23 subunits suggesting that it could have a critical role in mediating the development of chronic inflammation within the lower bowel. In this study, we have analyzed the role of c-Rel within the innate immune system in the development of lower bowel inflammation, in two well-studied models of murine colitis. We have found that the absence of c-Rel significantly impaired the ability of Helicobacter hepaticus to induce colitis upon infection of RAG-2-deficient mice, and ameliorated the ability of CD4+CD45RBhigh T cells to induce disease upon adoptive transfer into RAG-deficient mice. The absence of c-Rel interfered with the expression of IL-12/23 subunits both in cultured primary macrophages and within the colon. Thus, c-Rel plays a critical role in regulating the innate inflammatory response to microflora within the lower bowel, likely through its ability to modulate expression of IL-12/23 family members.

Helicobacter hepaticus strain 3B1 (H. hepaticus) contains a genomic island of approximately 71 kb, HHGI1, with some of the common features shared among known bacterial pathogenicity islands. In this study, we characterized the pathogenic potential of HHGI1 by infecting B6.129-IL10 tm1Cgn (IL10-/-) mice with an isogenic mutant (namely HhPAId1) lacking 19 predicted genes within HHGI1. In contrast to H. hepaticus (P

Adenomatous polyposis coli ( APC ) mutations are linked to human and mouse colorectal cancers. The Apc multiple intestinal neoplasia (Min) mouse mutation causes adenomas to develop throughout the small and large intestines. The BALB-Min (C.B6- Apc Min /+ ) congenic strain was generated by backcrossing into BALB/c the Apc Min allele from C57BL/6J- Apc Min /+ mice. BALB-Min mice have a low tumor multiplicity (27.4 small intestine tumors/mouse) and a relatively long life span (>1 year) that makes them amenable to long-term studies. To investigate the interplay of the adaptive immune system and intestinal tumorigenesis, the immunodeficient compound mutant strain BALB-RagMin (C.Cg- Rag2 −/− Apc Min /+ ) was generated. BALB-RagMin mice had a significant increase in tumors in the small, but not large, intestine relative to their BALB-Min counterparts (43.0 versus 24.0 tumors/mouse, respectively). The results suggest that the adaptive immune system plays a role in either the elimination or the equilibrium phase of cancer immunoediting in the small intestine in this model. We investigated the effect of the enterohepatic bacterial pathogen Helicobacter hepaticus on liver and intestine tumorigenesis in BALB-RagMin mice. H. hepaticus -infected BALB-RagMin mice developed moderate hepatitis, moderate typhlitis, and mild colitis. There were no differences in small intestine and cecal tumor multiplicity, regionality, or size relative to that in uninfected mice. However, H. hepaticus -infected BALB-RagMin mice had a significant increase in colon tumor incidence relative to uninfected BALB-RagMin mice (23.5% versus 1.7%, respectively). The data suggest that H. hepaticus , which is present in many research colonies, promotes colon tumorigenesis in the BALB-RagMin mouse and that it has the potential to confound colon tumorigenesis studies.

Helicobacter pylori infection results in chronic gastritis, which may progress to gastric cancer. In this study, we investigated the efficacy of H. pylori eradication in preventing the progression of gastritis to gastric cancer in H. pylori–infected transgenic INS-GAS mice. H. pylori infection induced severe dysplasia and gastric cancer classified as high-grade and low-grade gastrointestinal intraepithelial neoplasia (GIN) in INS-GAS mice at 28 weeks postinfection (WPI). H. pylori eradication therapy using omeprazole, metronidazole, and clarithromycin was administered p.o. at 8, 12, or 22 WPI. Compared with untreated infected mice, H. pylori eradication at 8, 12, and 22 WPI significantly reduced the severity of dysplasia (P < 0.01). Moreover, H. pylori eradication at 8 WPI completely prevented the development of GIN (P < 0.001). Although not as effective as early antimicrobial treatment, prevention of progression to high-grade GIN was achieved by H. pylori eradication at 12 and 22 WPI (P < 0.05). Consistent with reduced gastric pathology, H. pylori eradication at all time points significantly down-regulated gastric Interferon-γ, tumor necrosis factor-α, inducible nitric oxide synthase, and Reg 1 mRNA levels (P < 0.05) and reduced epithelial proliferation in the corpus (P < 0.01) compared with untreated infected mice. We concluded that H. pylori eradication prevented gastric cancer to the greatest extent when antibiotics are given at an early point of infection, but that eradication therapy given at a later time point delayed the development of severe dysplastic lesions. [Cancer Res 2008;68(9):3540–8]

In human studies, low vitamin C intake has been associated with more severe Helicobacter pylori gastritis and a higher incidence of gastric cancer. However, vitamin C supplementation has not been definitively shown to protect against gastric cancer. Using vitamin C-deficient B6.129P2-Gulo(tm1Umc/mmcd) (gulo(-/-)) mice lacking L-gulono-gamma-lactone oxidase, we compared gastric lesions and Th1 immune responses in H. pylori-infected gulo(-/-) mice supplemented with low (33 mg/L) or high (3,300 mg/L) vitamin C in drinking water for 16 or 32 weeks. Vitamin C levels in plasma and gastric tissue correlated with the vitamin C supplementation levels in gulo(-/-) mice. H. pylori infection resulted in comparable gastritis and premalignant lesions in wildtype C57BL/6 and gulo(-/-) mice supplemented with high vitamin C, but lesions were less severe in gulo(-/-) mice supplemented with low vitamin C at 32 weeks post infection. The reduced gastric lesions in infected gulo(-/-) mice supplemented with low vitamin C correlated with reduced Th1-associated IgG2c, gastric IFN-gamma and TNF-alpha mRNA and higher H. pylori colonization levels. These results in the H. pylori-infected gulo(-/-) mouse model suggest that although supplementation with a high level of vitamin C achieved physiologically normal vitamin C levels in plasma and gastric tissue, this dose of vitamin C did not protect gulo(-/-) mice from H. pylori-induced premalignant gastric lesions. In addition, less severe gastric lesions in H.pylori infected gulo(-/-) mice supplemented with low vitamin C correlated with an attenuated Th1 inflammatory response.

Defects within the innate immune system sensitize NF-κB-deficient (p50−/−; p65+/−) mice to Helicobacter hepaticus (Hh)-induced colitis. Because IL-10 plays a central role in the inhibition of Hh-induced colitis, we hypothesized that the ability of IL-10 to inhibit the innate inflammatory response to Hh may be compromised in NF-κB-deficient mice. To test this hypothesis, we evaluated the ability of an IL-10-Ig fusion protein with IL-10-like properties to inhibit Hh-induced colitis in RAG-2−/− (RAG) and p50−/−; p65+/−; RAG-2−/− (3X/RAG) mice. As expected, IL-10-Ig efficiently inhibited the development of colitis in RAG mice. In contrast, the ability of IL-10-Ig to inhibit colitis was compromised in 3X/RAG mice. The defect in response to IL-10-Ig appeared to be primarily the result of the absence of the p50/p105 subunit, because the ability of IL-10-Ig to inhibit colitis was also compromised in p50−/−; RAG-2−/− (p50/RAG) mice. Radiation chimeras demonstrated that the presence of p50/p105 within hemopoietic cells of the innate immune system was necessary for efficient inhibition of colitis by IL-10-Ig. Consistent with a defect in the suppressive effects of IL-10 in the absence of p50/p105, we found that the ability of IL-10 to control LPS-induced expression of IL-12 p40 was significantly compromised in macrophages lacking p50/p105. These results suggest that the absence of the p50/p105 subunit of NF-κB within hemopoietic cells of the innate immune system interferes with the ability of IL-10 to suppress inflammatory gene expression and Hh-induced colitis.

A general, chemical/chiroptical approach based on the CD exciton chirality method has been developed to determine the absolute configuration of α-hydroxy acids. This approach consists of amidation of the carboxyl group with ethanolamine followed by derivatization with the hydrophobic 10,15,20-triphenylporphyrinyl-5-benzoyl chromophore to form π,π-bisporphyrin derivatives which undergo intramolecular stacking. The sign of the observed bisignate couplet resulting from this stacking (in methylcyclohexane) is dictated by the preferred lower energy conformer and reflects the absolute configuration of the stereogenic center.

Complexation of 1,2-disubstituted dioxydiacetamides with Group II cations in MeOH-d4 results in downfield 1H NMR shifts. These cation induced shifts (CATIS) can be used to estimate the stoichiometry of cation/ligand binding. The shifts decrease in the order: Ca2+>Mg2+>Sr2+>Ba2+ while the order of binding is Ca2+>Sr2+>Ba2+⪢Mg2+. The shifts are independent of the nature of the anion except for Mg2+ where the CATIS decreases in the order: I−>Br−>Cl−>ClO4−.

[5]- and [6]helicenebisquinones can be prepared easily and in quantity by combining enol ethers of 1,4-diacetylbenzene or 2,7-diacetylnaphthalene with p-benzoquinone. Similar diethenyl aromatics that either have no ether functions or have them attached not to the double bonds, but to the aromatic rings, give the corresponding helicenes in only low yields and low purities. [6]Helicenebisquinone 11c is resolved into its enantiomers. An X-ray diffraction analysis of the adduct of one of these enantiomers and l-prolinol shows the absolute stereochemistry of 11c and the regiochemistry with which the amine adds to the quinone.

Polymorphisms of interleukin-1beta (IL-1beta) are associated with an increased risk of solid malignancies. Here, we show that stomach-specific expression of human IL-1beta in transgenic mice leads to spontaneous gastric inflammation and cancer that correlate with early recruitment of myeloid-derived suppressor cells (MDSCs) to the stomach. IL-1beta activates MDSCs in vitro and in vivo through an IL-1RI/NF-kappaB pathway. IL-1beta transgenic mice deficient in T and B lymphocytes develop gastric dysplasia accompanied by a marked increase in MDSCs in the stomach. Antagonism of IL-1 receptor signaling inhibits the development of gastric preneoplasia and suppresses MDSC mobilization. These results demonstrate that pathologic elevation of a single proinflammatory cytokine may be sufficient to induce neoplasia and provide a direct link between IL-1beta, MDSCs, and carcinogenesis.

A general, chemical/chiroptical approach based on the CD exciton chirality method has been developed to determine the absolute configuration of α-hydroxy acids. This approach consists of amidation of the carboxyl group with ethanolamine followed by derivatization with the hydrophobic 10,15,20-triphenylporphyrinyl-5-benzoyl chromophore to form π,π-bisporphyrin derivatives which undergo intramolecular stacking. The sign of the observed bisignate couplet resulting from this stacking (in methylcyclohexane) is dictated by the preferred lower energy conformer and reflects the absolute configuration of the stereogenic center.

Citrobacter rodentiumcauses epithelial hyperplasia and colitis and is used as a model for enteropathogenic and enterohemorrhagicEscherichia coliinfections. Little or no mortality develops in most inbred strains of mice, but C3H and FVB/N mice exhibit fatal outcomes of infection. Here we test the hypothesis that decreased intestinal transport activity duringC. rodentiuminfection results in fatality in C3H/HeOu and FVB/N mice. Susceptible strains were compared to resistant C57BL/6 mice and to inbred strains SWR and SJL of Swiss origin, which have not been previously characterized for outcomes ofC. rodentiuminfection. Mortality in susceptible strains C3H/HeOu and FVB/N was associated with significant fluid loss in feces, a remarkable downregulation of Slc26a3 and carbonic anhydrase IV (CAIV) message and protein expression, retention of chloride in stool, and hypochloremia, suggesting defects in intestinal chloride absorption. SWR, SJL, and C57BL/6 mice were resistant and survived the infection. Fluid therapy fully prevented mortality in C3H/HeOu and FVB/N mice without affecting clinical disease. Common pathogenic mechanisms, such as decreased levels of expression of Slc26a3 and CAIV, affect intestinal ion transport inC. rodentium-infected FVB and C3H mice, resulting in profound electrolyte loss, dehydration, and mortality. Intestinal chloride absorption pathways are likely a potential target for the treatment of infectious diarrhea.

Helicobacter cinaedi colonizes a wide host range, including rodents, and may be an emerging zoonotic agent. Colonization parameters, pathology, serology, and inflammatory responses to wild-type H. cinaedi (WT Hc ) were evaluated in B6.129P2- IL-10 tm1Cgn (IL-10 −/− ) mice for 36 weeks postinfection (WPI) and in C57BL/6 (B6) mice for 12 WPI. Because cytolethal distending toxin (CDT) may be a virulence factor, IL-10 −/− mice were also infected with the cdtB Hc and cdtB -N Hc isogenic mutants and evaluated for 12 WPI. Consistent with other murine enterohepatic helicobacters, WT Hc did not cause typhlocolitis in B6 mice, but mild to severe lesions developed at the cecocolic junction in IL-10 −/− mice, despite similar colonization levels of WT Hc in the cecum and colon of both B6 and IL-10 −/− mice. WT Hc and cdtB mutants also colonized IL-10 −/− mice to a similar extent, but infection with either cdtB mutant resulted in attenuated typhlocolitis and hyperplasia compared to infection with WT Hc ( P < 0.03), and only WT Hc infection caused dysplasia and intramucosal carcinoma. WT Hc and cdtB Hc mutant infection of IL-10 −/− mice elevated mRNA expression of tumor necrosis factor alpha, inducible nitric oxide synthase, and gamma interferon in the cecum, as well as elevated Th1-associated serum immunoglobulin G2a b compared to infection of B6 mice ( P < 0.05). Although no hepatitis was noted, liver samples were PCR positive at various time points for WT Hc or the cdtB Hc mutant in approximately 33% of IL-10 −/− mice and in 10 to 20% of WT Hc -infected B6 mice. These results indicate that WT Hc can be used to model inflammatory bowel disease in IL-10 −/− mice and that CDT contributes to the virulence of H. cinaedi .

We recently showed that L-Gln protects cultured gastric cells from ammonia-induced cell death and predicted that Gln may also protect during Helicobacter pylori infection in vivo. Thus, the aim of this study was to test whether supplemental dietary Gln protects against H. pylori-associated pathology. For this, C57BL/6 mice were fed a purified diet consisting of 20.3% protein (1.9% Gln), 66% carbohydrate, and 5% fat or 25.3% protein (5% supplemental L-Gln; 6.9% total Gln), 61% carbohydrate, and 5% fat. After a 2-wk prefeeding period, mice were divided into sham-(uninfected) or H. pylori-infected groups. Body weight and food consumption were recorded weekly. Tissue histopathology, H. pylori colonization, serum IgG, and pro- and antiinflammatory cytokine mRNA expression were determined at 6, 12, and 20 wk postinfection (wkPI). Inflammation, antiinflammatory cytokine, and interleukin-1beta mRNA expression were significantly greater at 6 wkPI in H. pylori-infected mice fed supplemental Gln compared with those fed the control diet. At 20 wkPI, however, inflammation and foveolar hyperplasia were significantly lower in H. pylori-infected mice fed supplemental Gln compared with those fed the control diet. Body weight gain, food consumption, H. pylori colonization, and serum IgG did not differ in H. pylori-infected mice fed supplemental Gln compared with the control diet. Our data demonstrate that H. pylori-infected mice fed supplemental dietary Gln have reduced H. pylori-associated pathology in vivo that is accompanied by beneficial changes in the immune response to H. pylori early in infection. Thus, Gln supplementation may be an alternative therapy for reducing H. pylori-associated pathology.

Cytotoxic-T-lymphocyte-associated antigen 4 (CTLA-4) expressed at high levels by CD4+CD25+CD45RBlowregulatory T cells (Treg) is essential to their homeostatic and immunoregulatory functions. However, its relevance to anti-inflammatory roles of Treg in the context of colitogenic innate immune response during pathogenic bacterial infections has not been examined. We showed earlier in Rag2-deficient 129/SvEv mice that Treg cells are capable of suppressing colitis and colon cancer triggered byHelicobacter hepaticus, a widespread murine enterohepatic pathogen. Using this model, we now examined the effects of antibody blockade of CTLA-4 on Treg function during innate immune inflammatory response. Consistent with our previous findings, we found that a single adoptive transfer of Treg cells prior to infection prevented colitis development despite persistentH. hepaticusinfection in recipient mice. However, when infected mice were injected with anti-CTLA-4 antibody along with Treg cell transfer, they developed a severe acute colitis with poor body condition that was not observed in Rag2−/−mice without Treg cell transfer. Despite high numbers of Foxp3+Treg cells, evident by immunohistochemical analyses in situ, the CTLA-4 antibody-treated mice had severely inflamed colonic mucosa and increased rather than decreased expression levels of cytokines gamma interferon and interleukin-2. These findings indicate that antibody blockade of CTLA-4 clearly abrogates Treg cell ability to suppress innate immune-driven colitis and suggest that Treg cell CTLA-4 cognate interactions may be necessary to maintain homeostasis among cells of innate immunity.

Chronic inflammation increases cancer risk. While it is clear that cell signaling elicited by inflammatory cytokines promotes tumor development, the impact of DNA damage production resulting from inflammation-associated reactive oxygen and nitrogen species (RONS) on tumor development has not been directly tested. RONS induce DNA damage that can be recognized by alkyladenine DNA glycosylase (Aag) to initiate base excision repair. Using a mouse model of episodic inflammatory bowel disease by repeated administration of dextran sulfate sodium in the drinking water, we show that Aag-mediated DNA repair prevents colonic epithelial damage and reduces the severity of dextran sulfate sodium-induced colon tumorigenesis. Importantly, DNA base lesions expected to be induced by RONS and recognized by Aag accumulated to higher levels in Aag-deficient animals following stimulation of colonic inflammation. Finally, as a test of the generality of this effect we show that Aag-deficient animals display more severe gastric lesions that are precursors of gastric cancer after chronic infection with Helicobacter pylori. These data demonstrate that the repair of DNA lesions formed by RONS during chronic inflammation is important for protection against colon carcinogenesis.

Gastric Helicobacter spp. are associated with chronic inflammation and neoplastic transformation in humans as well as domestic and laboratory species. The present study examined the association of Helicobacter heilmannii (Hhe) infection in pet cats with feline gastric mucosa associated lymphoid tissue (MALT) lymphoma. Tissues were collected via gastric biopsy or at necropsy from 47 pet cats with clinical signs of gastrointestinal disease, including vomiting and inappetance, and classified as gastritis (14/47), lymphoma (31/37), or normal (2/47). Tissues positive for argyrophilic organisms with Warthin–Starry stain (29/47) were assessed by fluorescent in situ hybridization (FISH) for the presence of Hhe strains 1–4 as well as with a fifth probe that detected Helicobacter salomonis, Helicobacter bizzozeronii, or Helicobacter felis. A significant association of positive Warthin–Starry status with Hhe infection was found in cases of sick cats (22/29; p < 0.05 by Chi-square; χ2 = 7.034). Interestingly, a significant association between Hhe status and a diagnosis of lymphoblastic or lymphocytic lymphoma was observed as well in a subset of 24 Warthin–Starry positive lymphoma cases: of lymphoblastic lymphoma cases, 13/17 were positive for Hhe ( p < 0.05; χ2 = 4.854). Hhe strains 2 and 4 were most commonly found (18/29 and 17/29, respectively) among sick cats, although a higher than expected number of cats was also positive for Hhe1, which initial reports have described as rare in cats and common in humans. The association found between a positive Hhe status with the presence of feline gastric lymphoma, especially lymphoblastic lymphoma, argues for the need to conduct prospective studies to better identify the frequency and strain distribution of Hhe infection in both healthy and clinically ill cats, particularly those cats with gastric lymphoma.

Helicobacter hepaticus strain 3B1 (H. hepaticus) contains a genomic island of approximately 71 kb, HHGI1, with some of the common features shared among known bacterial pathogenicity islands. In this study, we characterized the pathogenic potential of HHGI1 by infecting B6.129-IL10 tm1Cgn (IL10-/-) mice with an isogenic mutant (namely HhPAId1) lacking 19 predicted genes within HHGI1. In contrast to H. hepaticus (P0.001), HhPAId1 did not cause typhlocolitis and hyperplasia in IL10-/- mice. Colonization levels of HhPAId1 were significantly higher in the cecum (P0.007) and similar in the colon (P=0.27) when compared to H. hepaticus by 13 or 16 weeks post inoculation (WPI). The magnitude of the Th1-associated IgG2c response against HhPAId1 was less than that against H. hepaticus (P0.004). There was no significant difference in Th2-associated IgG1 responses against these two strains. Cecal and colonic mRNA levels of proinflammatory cytokines IFN-gamma, TNF-alpha and IL-17a in the HhPAId1-infected mice were significantly lower than those in the H. hepaticus-infected mice (P0.05) at 13 WPI. These results demonstrate that genes in the HHGI1 contribute to the pathogenicity of H. hepaticus, at least in part via up-regulation of proinflammatory mediators IFN-gamma, TNF-alpha and IL-17a.

Chronic idiopathic colitis is a common clinical entity in young captive rhesus monkeys. Eight isolates, cultured from five monkeys in colony 1 with endemic diarrhea and three from colony 2 without diarrhea, were grown under microaerobic conditions on selective agar and were classified by full 16S rRNA sequence, biochemical, and phenotypic analysis as a novel helicobacter, “ Helicobacter macacae ” (proposed name). All eight strains of H. macacae had 99.5% identical 16S rRNA sequences.

A nonempirical exciton chirality circular dichroic (CD) method for determining the absolute configurations of primary monoamines with amino group directly linked to the stereogenic center is described. Conventional exciton chirality CD method cannot be applied to these compounds since they lack the two sites for attaching the interacting chromophores. This was solved by covalently linking the monoamine to a trifunctional bidentate carrier moiety 1. Treatment of the carrier/monoamine conjugate with the porphyrin tweezer 4 consisting of two pentanediol-linked zinc porphyrins gives rise to 1:1 host-guest macrocyclic complexes that exhibit exciton-coupled CD spectra. The sign of the CD couplet can then be correlated with the absolute configuration of the monoamine as follows: a clockwise arrangement of the L, M, and S (large, medium, small) groups in the Newman projection of the monoamine with the amino group in the rear gives rise to a positive CD couplet, and vice versa; the assignments of L, M, S groups are based on conformational energies (A values). This method is applicable to cyclic and acyclic aliphatic amines, aromatic amines, amino esters, amides, and cyclic amino alcohols, and can be performed at the several microgram level.

Determination of molecular chirality is becoming increasingly important due to the sophistication in mode of action studies of biological reactions and the importance of chiral drugs. The exciton coupled CD, based on through space coupling, is a versatile microscale method with superb sensitivity that can be applied to a variety of problems including biopolymers and clusters in lipids. Applications of the exciton coupled CD method to the following problems exemplify its versatility. 1. Quaternary ammonium salts as chromophores for exciton-coupled CD.1' Exciton coupled circular dichroism is a nonempirical submicrogram scale method for determining absolute configurations and conformations of organic molecules in solution based on the interactions of chromophores which are preexisting in the substrate and/or introduced through derivatizations such as 0- or N-acylation.213 However, the method could not be applied to one major group, i.e., tertiary amines which comprise numerous natural products, particularly alkaloids, and synthetic drugs that exhibit conspicuous biological activities. We have succeeded in extending the CD method to quinuclidines (tertiary amines) through quaternary ammonium salt formation, and have applied this new procedure to the important class of hypocholesterolemic quinuclidines. Of the three conformers conceivable for the salt (l), the conformer shown determines the sign of the split Cotton effect (CE). When the phenylbenzyl group is in the north east sector the transition moments ,of the two chromophores are close to being parallel, hence the chirality is negligible; when it is in the north west sector, the distance between the two chromophores is more remote than that depicted, hence the resulting positive bisignate CD will be of smaller amplitude. OMe