Chronic Intestinal Infection Research Articles

Statin pleiotropy prevents rho kinase-mediated intestinal epithelial barrier compromise induced byBlastocystiscysteine proteases

Blastocystis is an enteric parasite that causes acute and chronic intestinal infections, often non-responsive to conventional antibiotics. The effects of Blastocystis infections on human epithelial permeability are not known, and molecular mechanisms of Blastocystis-induced intestinal pathology remain unclear. This study was conducted to determine whether Blastocystis species alters human intestinal epithelial permeability, to assess whether these abnormalities are rho kinase (ROCK)-dependent, and to investigate the therapeutic potential of the HMG-CoA reductase inhibitor Simvastatin in altered intestinal epithelial barrier function. The effect of metronidazole resistant (Mz(r)) Blastocystis isolated from a symptomatic patient on human colonic epithelial monolayers (Caco-2) was assessed. Modulation of enterocyte myosin light chain phosphorylation, transepithelial fluorescein isothiocyanate-dextran fluxes, transepithelial resistance, cytoskeletal F-actin and tight junctional zonula occludens-1 (ZO-1) by parasite cysteine proteases were measured in the presence or absence of HMG-CoA reductase and ROCK inhibition. Blastocystis significantly decreased transepithelial resistance, increased epithelial permeability, phosphorylated myosin light chain and reorganized epithelial actin cytoskeleton and ZO-1. These alterations were abolished by inhibition of enterocyte ROCK, HMG-CoA reductase and parasite cysteine protease. Our findings suggest that cysteine proteases of Mz(r) Blastocystis induce ROCK-dependent disruption of intestinal epithelial barrier function and correlates with reorganization of cytoskeletal F-actin and tight junctional ZO-1. Simvastatin prevented parasite-induced barrier-compromise, suggesting a therapeutic potential of statins in intestinal infections.

ZYM-201 Sodium Succinate Ameliorates Streptozotocin-Induced Hyperlipidemic Conditions

ZYM-201 is a methyl ester of a novel triterpenoid glycoside. It is isolated from SANGUISORBA OFFICINALIS, a widely used medicinal plant in Korea, China, and Japan, that is prescribed for various diseases such as diarrhea, chronic intestinal infections, duodenal ulcers, and bleeding. In this study, the antihyperlipidemic effect of the salt form (sodium succinate) of ZYM-201 was examined using streptozotocin (STZ)-treated hyperglycemic rats. Oral administration of ZYM-201 sodium succinate (3 to 10 mg/kg) resulted in recovery of the increased serum levels of triglyceride (TG) and total cholesterol (TC) back to normal levels. Elevated levels of serum lipoproteins, such as high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL), were also significantly restored by this compound without altering 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity. Finally, ZYM-201 sodium succinate displayed antioxidative properties, including suppression of lipid peroxide and hydroxyl radical generation and upregulation of superoxide dismutase (SOD) activity. Therefore, our data strongly suggest that ZYM-201 sodium succinate can be used as a remedy for the treatment of diabetes-derived hyperlipidemic disorders such as atherosclerosis and vascular diseases.

Cellular immune responses in Echinostoma caproni experimentally infected mice

The Echinostoma caproni-mice system is extensively used as an experimental model for the study of the factors involved in the establishment of chronic intestinal helminth infections. Although several parameters of the immunobiology of the host-parasite system have been studied in detail, the current knowledge of the cellular responses in these infections is still scarce. In the present paper, we analyze the kinetics of the circulating CD3(+) and CD19(+) cell populations and the different T-cell phenotype profiles in mice experimentally infected with E. caproni. Whereas the CD3(+) populations remained stable during the complete experiment, a marked increase in CD19(+) cells was observed from 4 weeks post-infection and beyond. Similarly, a marked increase in CD8(+) cell populations was observed in the 2 week post-infection. Our results show that E. caproni infection in mice alters the peripheral lymphoid cell populations, which may be important to determine the course of the infection. In this sense, CD8(+) cells can be essential in relation to their role as a source of IFN-γ.

Chronic Helminth Infection Promotes Immune Regulation In Vivo through Dominance of CD11cloCD103− Dendritic Cells

Gastrointestinal helminth infections are extremely prevalent in many human populations and are associated with downmodulated immune responsiveness. In the experimental model system of Heligmosomoides polygyrus, a chronic infection establishes in mice, accompanied by a modulated Th2 response and increased regulatory T cell (Treg) activity. To determine if dendritic cell (DC) populations in the lymph nodes draining the intestine are responsible for the regulatory effects of chronic infection, we first identified a population of CD11c(lo) nonplasmacytoid DCs that expand after chronic H. polygyrus infection. The CD11c(lo) DCs are underrepresented in magnetic bead-sorted preparations and spared from deletion in CD11c-diptheria toxin receptor mice. After infection, CD11c(lo) DCs did not express CD8, CD103, PDCA, or Siglec-H and were poorly responsive to TLR stimuli. In DC/T cell cocultures, CD11c(lo) DCs from naive and H. polygyrus-infected mice could process and present protein Ag, but induced lower levels of Ag-specific CD4(+) T cell proliferation and effector cytokine production, and generated higher percentages of Foxp3(+) T cells in the presence of TGF-β. Treg generation was also dependent on retinoic acid receptor signaling. In vivo, depletion of CD11c(hi) DCs further favored the dominance of the CD11c(lo) DC phenotype. After CD11c(hi) DC depletion, effector responses were inhibited dramatically, but the expansion in Treg numbers after H. polygyrus infection was barely compromised, showing a significantly higher regulatory/effector CD4(+) T cell ratio compared with that of CD11c(hi) DC-intact animals. Thus, the proregulatory environment of chronic intestinal helminth infection is associated with the in vivo predominance of a newly defined phenotype of CD11c(lo) tolerogenic DCs.

Chronic intestinal Mycobacteria infection: discrimination via VOC analysis in exhaled breath and headspace of feces using differential ion mobility spectrometry**Data were partly presented at the German–Austrian Workshop on Breath Gas Analysis, Greifswald (Germany), June 07–09, 2010 and at the 20th Annual Congress of the European Respiratory Society, Barcelona (Spain), September 18–22, 2010.

Differential ion mobility spectrometry (DMS) is a method to detect volatile organic compounds (VOC) in the ppt range. This study assessed whether VOC analysis using DMS could discriminate subjects with an experimentally induced chronic intestinal infection caused by Mycobacteria from non-infected controls. The animal model consisted of two groups of goats orally infected with two different doses of Mycobacterium avium subspecies paratuberculosis (MAP) and one group of non-infected healthy controls (each group: n = 6). Using DMS, exhaled breath and headspace of feces were analyzed on-line on an individual basis 9 months after inoculation of MAP. Data analysis included peak detection, cluster analysis, selection of discriminating VOC features (Mann–Whitney U test), and classification using a support-vector-machine. Taking the background of ambient air conditions into account, VOC analysis of exhaled breath as well as of feces revealed significant differences between chronically infected animals and non-infected controls. In both specimens, increasing as well as decreasing VOC features could be attributed to infection. Discrimination between infected and non-infected animals was sharper analyzing exhaled breath compared to headspace of feces. In exhaled breath, at least two VOC features were found to increase in a dose-dependent manner with increasing doses of MAP inoculated. Results of this study provide strong evidence that DMS analysis of exhaled breath has the potential to become a valuable tool for non-invasive assessment of VOC specifically related to certain diseases or infections.

Hsp70 vaccination-induced antibodies recognize B cell epitopes in the cell wall of Mycobacterium avium subspecies paratuberculosis

Mycobacterium avium subspecies paratuberculosis (MAP) causes a chronic intestinal infection of ruminants and has been associated with the etiology of human Crohn's disease. A MAP Hsp70/DDA subunit vaccine previously showed a significant reduction in fecal shedding of MAP in cattle, concomitant with pronounced antibody production against MAP Hsp70, rather than T cell reactivity. Our hypothesis is that if Hsp70-specific antibodies are able to confer protection, the first requisite would be that the Hsp70 molecule is accessible for antibodies in intact MAP bacteria. In the current study monoclonal antibodies identified MAP Hsp70 B cell epitopes. Two linear epitopes were also recognized by antibodies of vaccinated calves and goats. These epitopes showed to be accessible by antibodies in the bacterial cell wall and in intestinal lesional tissue during natural infection. These results indicate that vaccination-induced antibodies can bind intact bacteria and have the potential to contribute to the protective effect of Hsp70/DDA subunit vaccination against bovine paratuberculosis.

Inhibitory effect of Sanguisorba officinalis ethanol extract on NO and PGE2 production is mediated by suppression of NF-κB and AP-1 activation signaling cascade

Sanguisorba officinalis, a well known valuable medicinal plant in Korea, China and Japan used traditionally for the treatment of inflammatory and metabolic diseases such as diarrhea, chronic intestinal infections, duodenal ulcers, and bleeding. Recent studies have revealed that its aqueous or ethanolic extracts exhibit a variety of pharmacological activities such as anti-oxidative, anti-cancer, anti-lipid peroxidation, anti-atherogenic, and vasorelaxant effects. Systematic studies on the anti-inflammatory effect of this plant and its molecular mechanisms have not yet been fully investigated. Ethanol extract of Sanguisorba officinalis (So-EE) the lipopolysaccharide (LPS)-stimulated macrophages and production of inflammatory mediators were employed to assess these properties. So-EE significantly suppressed the production of nitric oxide (NO) and prostaglandin (PG) E(2) from LPS-activated RAW264.7 cells and peritoneal macrophages in a dose-dependent manner. This extract effectively diminished the mRNA levels of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2, implying that the blockade is generated at the transcriptional level. So-EE strongly blocked the activation and translocation of NF-κB and AP-1 by suppressing the upstream kinases including inhibitor of κBα (IκBα), IκBα kinase (IKK), Akt (protein kinase B), phosphoinositide-dependent kinase 1 (PDK1), p85/phosphoinositide-3-kinase (PI3K), and mitogen activated protein kinase (MAPK) such as extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). Moreover, So-EE suppressed the phosphorylation of Src, its kinase activity, and complex formation between Src and p85. This study suggests that So-EE has a potent anti-inflammatory activity mediated by NF-κB, and AP-1 inhibitory properties linked to the suppression of Src and MAPK activation.

A matter of timing: Early, not chronic phase intestinal nematode infection restrains control of a concurrent enteric protozoan infection

Infections with parasitic worms are often long lasting and associated with modulated immune responses. We analyzed the influence of the nematode Heligmosomoides polygyrus bakeri dwelling in the small intestine on concurrent protozoan infection with Eimeria falciformis residing in the cecum. To dissect the effects of a nematode infection in the early versus chronic phase, we infected animals with E. falciformis 6 or 28 days post H. p. bakeri infection. Only a concurrent early nematode infection led to an increased replication of the protozoan parasite, whereas a chronic worm infection had no influence on the control of E. falciformis. Increased protozoan replication correlated with the reduced production of IFN-γ, IL-12/23, CCL4, CXCL9 and CXCL10, reduced migration of T cells and increased expression of Foxp3 at the site of protozoan infection. This was accompanied by a stronger nematode-specific Th2 response in gut-draining LN. Protection of mice against challenge infections with the protozoan parasite was not altered. Hence, the detrimental effect of a nematode infection on the control of a concurrent protozoan infection is transient and occurs only in the narrow time window of the early phase of infection.

Chronic Intestinal Helminth Infections Are Associated with Immune Hyporesponsiveness and Induction of a Regulatory Network

Helminth infections have been associated with protection against allergy and autoimmune diseases. We investigated the effects of chronic infections with Ascaris lumbricoides and Trichuris trichiura (measured twice over a 5-year period) on cytokine and antibody responses. We collected blood from 1,060 children aged 4 to 11 years living in a poor urban area of Brazil and measured Th1 (gamma interferon [IFN-gamma]) and Th2 (interleukin-5 [IL-5] and IL-13) cytokines and the regulatory cytokine IL-10 in unstimulated and stimulated (with mitogen or A. lumbricoides antigens) cultures of peripheral blood leukocytes and levels of total IgE and anti-A. lumbricoides IgG4 and IgE in serum. Intestinal helminth infections were associated with an increased proportion of children producing IL-5 in response to A. lumbricoides and producing IL-10 spontaneously, especially among coinfected and chronically infected children. Helminth infections were associated with a generalized suppression of cytokine responses to mitogen. Levels of total IgE and anti-A. lumbricoides IgG4 and IgE were especially elevated in chronically infected children. In conclusion, intestinal helminth infections were associated with a typical Th2 immune response profile and with the induction of immune hyporesponsiveness that was associated with greater frequencies of the production of spontaneous IL-10.

CMV-Enterokolitis bei einer erwachsenen lebertransplantierten Patientin als Ursache rezidivierender Invaginationen?

Intestinal intussusception in the adult is often idiopathic but also known to be associated with chronic inflammatory bowel disease, coeliac disease, tumours or previous abdominal operations. A 22-year-old women after liver transplantation due to Crigler Najar Syndrome suffered from repeated episodes of abdominal pain. The diagnosis of repeated self-limited intestinal intussusceptions was made by computed tomography and ultrasonography. A laparoscopy revealed no cause for the intussusceptions. During a new episode of abdominal pain caused again by an intussusception a colonoscopy was performed that showed aspects of a discreet colitis. In the biopsies CMV was detected by qualitative PCR, while blood tests for CMV pp65 antigen were negative. A therapy with gancyclovir was initiated which lead to remission of the patient's symptoms. A colonoscopy six weeks later showed a completely normal colon, while in the biopsies CMV was not detectable. After a follow-up of one year the patient has not suffered from any further episodes. This case demonstrates the role of chronic intestinal CMV infection as a possible causative factor for repeated intussusceptions in immunosuppressed patients. Whenever possible a PCR for CMV in colon biopsies should be carried out to detect an intestinal CMV infection because as shown in our case results for immunohistopathology and CMV pp65 can be negative despite a chronic infection.

Susceptibility to paratuberculosis infection in cattle is associated with single nucleotide polymorphisms in Toll-like receptor 2 which modulate immune responses against Mycobacterium avium subspecies paratuberculosis

Paratuberculosis is a chronic intestinal infection in ruminants, caused by Mycobacterium avium subspecies paratuberculosis ( Map). To study the role of host genetics in disease susceptibility, the Toll-like receptor 2 (TLR2) gene, selected based on its potential role in immunity to mycobacterial infections, was analyzed for single nucleotide polymorphisms (SNP) and their potential association with disease. For SNP discovery and to study SNP association with disease, a case–control study including 24 cows from farms with paratuberculosis was conducted. Sequence analysis of the TLR2 genes from 12 paratuberculosis-infected animals and 12 age-matched healthy herd mates revealed 21 different SNP. The TLR2-1903 T/C SNP was significantly associated with resistance to Map. This and four additional TLR2 SNP were studied in a subsequent observational field study with 553 cows from farms with paratuberculosis. The allelic distribution of the TLR2-1903 T/C SNP was confirmed to be significantly different between the infected and non-infected animals. For the TLR2-1903 T/C SNP the odds ratio was calculated, and similar to the dominance model in the association study, the CT and CC genotypes were compared to the TT genotype. Cows with the TLR2-1903 T/C mutation (i.e., the CT and CC genotypes) were at 1.7 (95% CI: 1.2, 2.8) times the odds of being Map-infected compared to cows with the TT genotype. In in vitro functional assays, monocyte-derived macrophages from animals with a TLR2-1903 TT genotype produced more IL12p40 and IL1β when stimulated with Map compared to cells derived from TLR2-1903 CT and CC genotypes. Also, T cell proliferative responses to mycobacterial antigens were higher in animals with a TLR2-1903 TT genotype. In conclusion, we have found a significant association between SNP TLR2-1903 T/C in the bovine TLR2 gene and bovine paratuberculosis infection. This SNP and other genetic markers could be useful in marker-assisted breeding strategies as an additional tool in paratuberculosis control strategies. In addition, the functional studies suggest that genetic polymorphisms in bovine TLR2 which result in higher macrophage activity may contribute to enhanced T cell activation and a lower susceptibility to paratuberculosis in cattle.

Regulatory T Cells in children with intestinal parasite infection

Chronic intestinal parasite infection can induce both persistent immune activation and defective responsiveness of T cells. This study aimed to assess the number and function of T regulatory (Treg) cells in children with intestinal parasite infection. We have studied the peripheral blood from 93 children, 53 of them parasitized with protozoa, helminths, or both; the remainder were non parasitized, healthy controls. The number and function of CD4(+) CD25(high) and CD4(+) Foxp3(+) cells were similar in parasitized and control children. In contrast, there was a significant increase in the levels of CD3(+) CD69(+), CD4(+) CTLA-4(+), and CD8(+) CD28(-) T cells in helminth infected children. Moreover, some of these patients showed a diminished response to CD3/CD28 stimulation in comparison with the control children. Our data strongly suggest that whilst Treg cells are not affected by intestinal parasite infection, CD3(+) CD69(+), CD4(+) CTLA-4(+) and CD8(+) CD28(-) lymphocytes may play an important, but as yet undetermined role in the diminished immune competence observed in parasitized children.

Internalization-dependent recognition ofMycobacterium aviumssp.paratuberculosisby intestinal epithelial cells

Mycobacterium avium ssp. paratuberculosis (MAP) is the causative agent of Johne's disease, a highly prevalent chronic intestinal infection in domestic and wildlife ruminants. The microbial pathogenesis of MAP infection has attracted additional attention due to an association with the human enteric inflammatory Crohn's disease. MAP is acquired by the faecal-oral route prompting us to study the interaction with differentiated intestinal epithelial cells. MAP was rapidly internalized and accumulated in a late endosomal compartment. In contrast to other opportunistic mycobacteria or M. bovis, MAP induced significant epithelial activation as indicated by a NF-kappaB-independent but Erk-dependent chemokine secretion. Surprisingly, MAP-induced chemokine production was completely internalization-dependent as inhibition of Rac-dependent bacterial uptake abolished epithelial activation. In accordance, innate immune recognition of MAP by differentiated intestinal epithelial cells occurred through the intracellularly localized pattern recognition receptors toll-like receptor 9 and NOD1 with signal transduction via the adaptor molecules MyD88 and RIP2. The internalization-dependent innate immune activation of intestinal epithelial cells is in contrast to the stimulation of professional phagocytes by extracellular bacterial constituents and might significantly contribute to the histopathological changes observed during enteric MAP infection.

Heligmosomoides bakeri: a model for exploring the biology and genetics of resistance to chronic gastrointestinal nematode infections

The intestinal nematode Heligmosomoides bakeri has undergone 2 name changes during the last 4 decades. Originally, the name conferred on the organism in the early 20th century was Nematospiroides dubius, but this was dropped in favour of Heligmosomoides polygyrus, and then more recently H. bakeri, to distinguish it from a closely related parasite commonly found in wood mice in Europe. H. bakeri typically causes long-lasting infections in mice and in this respect it has been an invaluable laboratory model of chronic intestinal nematode infections. Resistance to H. bakeri is a dominant trait and is controlled by genes both within and outside the MHC. More recently, a significant QTL has been identified on chromosome 1, although the identity of the underlying genes is not yet known. Other QTL for resistance traits and for the accompanying immune responses were also defined, indicating that resistance to H. bakeri is a highly polygenic phenomenon. Hence marker-assisted breeding programmes aiming to improve resistance to GI nematodes in breeds of domestic livestock will need to be highly selective, focussing on genes that confer the greatest proportion of overall genetic resistance, whilst leaving livestock well-equipped genetically to cope with other types of pathogens and preserving important production traits.

Suppression of airway inflammation by a natural acute infection of the intestinal epithelium

Although chronic intestinal helminth infections may suppress allergen-induced airway pathology by inducing a combination of modified T-helper (Th) 2 and immunosuppressive cytokines, a similar capacity of natural acute intestinal infections has remained untested, despite their global prevalence. Here, we show that allergic airway phenotypes including eosinophilia, eotaxin mRNA, and Th2 cytokines are significantly suppressed in animals that were infected by and that have cleared the intestinal parasite Eimeria vermiformis. Unlike in helminth-infected animals, regulation requires temporal coincidence of infection with sensitization; depends on interferon-γ; and is not associated with an enhanced antigen-specific immunoglobulin G1 response. Moreover, regulation was effective following allergen sensitization in different anatomical sites, and in young and adult mice. These data highlight a transient anatomical dissemination of “functional immunologic dominance” following infection of the gut mucosa. They strongly support the hypothesis that airway allergies are naturally suppressed by both acute and chronic mucosal pathogens, but by different mechanisms.

Common Variable Immunodeficiency Disorders in Children: Delayed Diagnosis Despite Typical Clinical Presentation

To characterize common variable immunodeficiency disorder (CVID) in childhood. We retrospectively investigated clinical findings in 32 children with primary CVID by questionnaire and file review. Clinical presentation included recurrent or chronic respiratory tract infections (88%), sinusitis (78%), otitis media (78%), and intestinal tract infections (34%), mainly with encapsulated bacteria. Meningitis was found in 25%, sepsis in 16%, and pyelonephritis in 16% of patients. Poliomyelitis after vaccination occurred in 2 patients and opportunistic infections occasionally. Allergic disorders were present in 38%, and autoimmune disease in 31% of patients. Eighty percent of the patients underwent surgical procedures because of recurrent infections. Growth retardation was seen in 28% of patients, and 16% showed retarded mental development. Bronchiectasis developed in 34%, and lymphoid proliferative disease in 13%. Incidence of allergic and autoimmune diseases was increased in first-degree relatives with normal immunologic findings. Mean time between symptoms and induction of immunoglobulin substitution therapy was 5.8 years (0.2-14.3). CVID in children presents with comparable symptoms and disorders as in adults. We found a significant influence on growth and development. The marked delay of diagnosis may be due to overlap with common pediatric disorders, while also reflecting insufficient awareness of these disorders.

Nosocomial Gastroenteritis in Children With and Without Rotavirus Infection

To the Editors: Rotavirus (RV) is the most common cause of severe gastroenteritis in children and the proportion of childhood gastroenteritis hospitalizations attributed to RV has recently increased from 22% to 39%.1 RV gastroenteritis is also one of the leading nosocomial infections in the pediatric age group.2 Although the typical symptoms in nosocomial RV gastroenteritis are well recognized,2 information regarding the differences of clinical presentation and evolution of nosocomial gastroenteritis with and without RV infection is scarce. We performed a retrospective cohort study on nosocomial infection in a pediatric university hospital in Salvador, Brazil, between October 2002 and September 2005. Nosocomial gastroenteritis was defined as >3 watery or looser-than-normal stools and/or a single episode of forceful vomiting within a 24-hour period after the first 72 hours of hospitalization. RV antigen was searched for in stool specimens by a rapid latex assay (ROTA Rich, Richmond Diagnostics, Spain). Data were collected from an epidemiologic surveillance form of the Nosocomial Infection Control Service and laboratory database; details regarding the child's illness were collected from the patient charts. All hospitalized patients were actively surveyed to detect occurrence of nosocomial infection. Fever was defined as axillary temperature >37.5°C. The study was approved by the Ethics Committee of the Federal University of Bahia. Overall, 125 cases of nosocomial gastroenteritis were identified, of which 66 had the rotavirus assay performed and the patient chart accessible for review. Therefore, the study group comprised 66 cases, of which 49 (74%) were RV positive. None of the patients had chronic gastrointestinal tract disease, immunodeficiency, or bacterial intestinal infection. The median age was 7 (mean, 11 ± 13; range, 0.3–105) months and there were 39 (59%) males. There was no difference in age (month) (10 ± 8 vs. 16 ± 27) or male gender (57% vs. 65%) distribution when patients with and without RV infection were compared, as well as in the length of signs and symptoms or duration of hospitalization (data not shown). Vomiting (69% vs. 41%, P = 0.04, OR 3.2, 95% CI 1.04–10.1), dehydration (37% vs. 6%, P = 0.02, OR 9.3, 95% CI 1.1–76), the combination of symptoms fever, vomiting, and diarrhea (49% vs. 18%, P = 0.02, OR 4.5, 95% CI 1.1–18) and fever, diarrhea, and dehydration (29% vs. 0%, P = 0.01, OR 1.5, 95% CI 1.2–1.8) were associated with RV infection. All patients were discharged from hospital after improvement. Vomiting and fever have been recognized as prominent symptoms in RV-associated hospitalized cases.3 Among community-acquired RV infected hospitalized children, the combination of fever, vomiting, and diarrhea was the most frequent clinical presentation (63%).4 We found similar features among our patients with nosocomial RV gastroenteritis. This finding may be explained by the nosocomial RV acquisition because of circulation of community-acquired RV in the hospital.3 Despite the association with dehydration, the length of hospitalization was not different between patients with and without RV because of early diagnosis and rapid rehydration. Licia L. Moreira, RN Infection Control Service of the Professor Hosannah de Oliveira Pediatric Center Federal University of Bahia Salvador, Brazil Eduardo M. Netto, MD, PhD Infectious Diseases Unit University Hospital Federal University of Bahia Salvador, Brazil Cristiana M. Nascimento-Carvalho, MD, PhD Department of Pediatrics School of Medicine Federal University of Bahia Salvador, Brazil

Characterization of a Helicobacter hepaticus putA Mutant Strain in Host Colonization and Oxidative Stress

Helicobacter hepaticus is a gram-negative, spiral-shaped microaerophilic bacterium associated with chronic intestinal infection leading to hepatitis and colonic and hepatic carcinomas in susceptible strains of mice. In the closely related human pathogen Helicobacter pylori, L-proline is a preferred respiratory substrate and is found at significantly high levels in the gastric juice of infected patients. A previous study of the proline catabolic PutA flavoenzymes from H. pylori and H. hepaticus revealed that Helicobacter PutA generates reactive oxygen species during proline oxidation by transferring electrons from reduced flavin to molecular oxygen. We further explored the preference for proline as a respiratory substrate and the potential impact of proline metabolism on the redox environment in Helicobacter species during host infection by disrupting the putA gene in H. hepaticus. The resulting putA knockout mutant strain was characterized by oxidative stress analysis and mouse infection studies. The putA mutant strain of H. hepaticus exhibited increased proline levels and resistance to oxidative stress relative to that of the wild-type strain, consistent with proline's role as an antioxidant. The significant increase in stress resistance was attributed to higher proline content, as no upregulation of antioxidant genes was observed for the putA mutant strain. The wild-type and putA mutant H. hepaticus strains displayed similar levels of infection in mice, but in mice challenged with the putA mutant strain, significantly reduced inflammation was observed, suggesting a role for proline metabolism in H. hepaticus pathogenicity in vivo.

ATP release by infected bovine monocytes increases the intracellular survival of Mycobacterium avium subsp. paratuberculosis

Mycobacterium avium subsp. paratuberculosis is the etiologic agent of Johne's disease, a chronic intestinal infection in ruminants. Adenosine 5'-Triphosphate (ATP) has been reported to induce killing of several Mycobacterium species in human and murine macrophages. We investigated whether ATP secreted from M. avium subsp. paratuberculosis-infected bovine monocytes affects intracellular survival of the bacilli. Bovine monocytes constitutively secreted ATP during an 8-day incubation period in vitro; however, M. avium subsp. paratuberculosis infection did not enhance ATP release. Removal of extracellular ATP by the addition of apyrase increased the viability of infected monocytes, but surprisingly decreased the number of viable intracellular bacilli. In contrast to previous reports, addition of extracellular ATP (1mM) increased intracellular survival of M. avium subsp. paratuberculosis in bovine monocytes. Neither apyrase nor ATP altered production of reactive oxygen intermediates (ROI) or reactive nitrogen intermediates (RNI) by bovine monocytes. These results suggest that ATP release from infected bovine monocytes improves, rather than decreases, the intracellular survival of M. avium subsp. paratuberculosis.

An Increase in Epithelial Cell Apoptosis Is Associated with Chronic Intestinal Nematode Infection

It is well established that homeostasis of the intestinal epithelium becomes dysregulated during gastrointestinal helminth infection and is under immune control. An increase in both enterocyte proliferation and the subsequent generation of crypt hyperplasia are hallmarks of chronic infection with Trichuris muris, a large intestinal dwelling nematode. The effect of this parasitic infection on apoptosis induction in the large intestine and its regulation has been neglected. To address this, mice of resistant and susceptible phenotypes were infected with different doses of T. muris, and the levels of epithelial cell apoptosis were determined. It is clear that apoptosis is induced during chronic T. muris infection. This occurs mainly at the base of the cecal crypt, within the stem cell region. The level of apoptosis induced is independent of worm number, suggesting that it is not a consequence of worm-induced damage but rather a mechanism for controlling cell number within the crypt. Neutralization of both gamma interferon and tumor necrosis factor alpha caused a significant reduction in the levels of apoptosis, showing that proinflammatory cytokines generated in response to chronic infection play an important role in apoptosis induction in this system. It is proposed that the generation of proinflammatory cytokines during chronic T. muris infection may play a positive role, by promoting intestinal epithelial cell apoptosis, to counter infection-induced epithelial hyperplasia.