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Background & Aims: Intestinal epithelial cells aid in mucosal defense by providing a physical barrier against entry of pathogenic bacteria and secreting antimicrobial peptides (AMPs). Autophagy is an important component of immune homeostasis. However, little is known about its role in specific cell types during bacterial infection in vivo. We investigated the role of autophagy in the response of intestinal epithelial and antigen-presenting cells to Salmonella infection in mice., Methods: We generated mice deficient in Atg16l1 in epithelial cells (Atg16l1(f/f) × Villin-cre) or CD11c(+) cells (Atg16l1(f/f) × CD11c-cre); these mice were used to assess cell type-specific antibacterial autophagy. All responses were compared with Atg16l1(f/f) mice (controls). Mice were infected with Salmonella enterica serovar typhimurium; cecum and small-intestine tissues were collected for immunofluorescence, histology, and quantitative reverse-transcription polymerase chain reaction analyses of cytokines and AMPs. Modulators of autophagy were screened to evaluate their effects on antibacterial responses in human epithelial cells., Results: Autophagy was induced in small intestine and cecum after infection with S typhimurium, and required Atg16l1. S typhimurium colocalized with microtubule-associated protein 1 light chain 3β (Map1lc3b or LC3) in the intestinal epithelium of control mice but not in Atg16l1(f/f) × Villin-cre mice. Atg16l1(f/f) × Villin-cre mice also had fewer Paneth cells and abnormal granule morphology, leading to reduced expression of AMPs. Consistent with these defective immune responses, Atg16l1(f/f) × Villin-cre mice had increased inflammation and systemic translocation of bacteria compared with control mice. In contrast, we observed few differences between Atg16l1(f/f) × CD11c-cre and control mice. Trifluoperazine promoted autophagy and bacterial clearance in HeLa cells; these effects were reduced upon knockdown of ATG16L1., Conclusions: Atg16l1 regulates autophagy in intestinal epithelial cells and is required for bacterial clearance. It also is required to prevent systemic infection of mice with enteric bacteria., (Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Scope: This study investigated the effects of supplementing different ratios of omega-6 and omega-3 fatty acids (O6H = 10:1, O3O6 = 4:1, and O3H = 1:4) to western-style diets on cow β-lactoglobulin (BLG) induced allergic reactions in Balb/c mice., Methods and Results: Three-week-old mice were randomly assigned to three diet groups (n = 20/group). At 9 wk of age, half of the mice from each dietary treatment (n = 10) were intraperitoneally (i.p.) sensitized with three weekly doses of BLG and alum while the remaining half from each group was sham sensitized (controls). One week after the final sensitization, all mice were orally challenged with BLG. Elevated BLG-specific serum Igs were observed in all sensitized and challenged mice. IFN-γ, MCP-1, and IL-12p40 concentrations from lymphocytes of mesenteric lymph nodes were highest in O3H mice, compared to O3O6 and O6H mice. O6H mice had the highest IL-4 concentrations from splenic lymphocytes and a significantly lower rectal temperature after the challenge in comparison to O3O6 and O3H mice., Conclusions: Our results suggest that the ω-3 PUFA rich diets alleviated the severity of allergic reactions, and may modulate immune response toward T helper cell (Th)1-favoured immune response while the ω-6 PUFA rich diet exhibited no allergy alleviation with a stronger Th2 polarized immune response., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background & Aims: Caspase recruitment domain 9 (CARD9) is an adaptor protein that integrates signals downstream of pattern recognition receptors. CARD9 has been associated with autoinflammatory disorders, and loss-of-function mutations have been associated with chronic mucocutaneous candidiasis, but the role of CARD9 in intestinal inflammation is unknown. We characterized the role of Card9 in mucosal immune responses to intestinal epithelial injury and infection., Methods: We induced intestinal inflammation in Card9-null mice by administration of dextran sulfate sodium (DSS) or Citrobacter rodentium. We analyzed body weight, assessed inflammation by histology, and measured levels of cytokines and chemokines using quantitative reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Cell populations were compared between wild-type and Card9-null mice by flow cytometry analysis., Results: Colon tissues and mesenteric lymph nodes of Card9-null mice had reduced levels of interleukin (IL)-6, interferon-γ, and T-helper (Th)17 cytokines after administration of DSS, compared with wild-type mice. IL-17A and IL-22 expression were reduced in the recovery phase after DSS administration, coincident with decreased expression of antimicrobial peptides and the chemokine (C-C motif) ligand 20 (Ccl20). Although Card9-null mice had more intestinal fungi based on 18S analysis, their Th17 responses remained defective even when an antifungal agent was administered throughout DSS exposure. Moreover, Card9-null mice had impaired immune responses to C rodentium, characterized by decreased levels of colonic IL-6, IL-17A, IL-22, and regenerating islet-derived 3 gamma (RegIIIγ), as well as fewer IL-22-producing innate lymphoid cells (ILCs) in colon lamina propria., Conclusions: The adaptor protein CARD9 coordinates Th17- and innate lymphoid cell-mediated intestinal immune responses after epithelial injury in mice., (Copyright © 2013 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Infection of mice with Trichinella spiralis redirects the mucosal immune system from a Th1 to a protective Th2 response with a reduction in the severity of trinitrobenzesulfonic acid-induced colonic damage. T. spiralis infection induced IL-10 production in a dose-dependent manner in oxazolone (OXZ)-induced colitis. This phenomenon may be responsible for the lack of efficacy of T. spiralis in the treatment of OXZ-induced colitis. These results indicate that if the source of increased IL-10 production is identified and addressed, T. spiralis may alter the Th2 response., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Trichinella spiralis infection confers effective resistance to tumor cell expansion. In this study, a T7 phage cDNA display library was constructed to express genes encoded by T. spiralis. Organic phase multi-cell screening was used to sort through candidate proteins in a transfected human chronic myeloid leukemia cell line (K562) and a human hepatoma cell line (H7402) using the display library. The protein encoded by the A200711 gene was identified and analyzed using protein analysis software. To test the antitumor effects of A200711, variations in cell proliferation and apoptosis were monitored after recombinant pEGFP-N1-A200711 was transfected into H7402 cells. The results show that the expressed target gene successfully induced apoptosis in H7402 cells as measured by Hoechst-PI staining, MTT assay (p<0.05). This study warrants further investigation into the therapeutic use of A200711 for anti-hepatocellular carcinomas., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Parasite-induced and parasite-regulated larval capsule formation and host immunosuppression are two major characteristics that are unique in Trichinella spp. infections, but the molecule(s) and mechanism(s) that mediate these processes remain largely unknown. Trichinella pseudospiralis and Trichinella spiralis, are obviously different with respect to these two characteristics. A comparative study of these two species, in particular their antigen expression profiles at different developmental stages (the main molecules involved in the cross-talk or interaction between each parasite and its host), may help us better understand the parasite molecules and mechanisms involved. Here, we constructed cDNA libraries from T. pseudospiralis adults (Ad), newborn larvae (NBL) and muscle larvae (ML) mRNA and screened them with pig anti-T. pseudospiralis serum collected 26, 32 and 60 days post-infection (p.i.). The most abundant antigens were found to vary among life-cycle stages. Pyroglutamy peptidase 1-like and 6-phosphogluconolactonase-like genes predominated in the Ad stage and a serine protease (SS2-1-like gene) predominated in NBL similar to that observed in T. spiralis. Muscle larvae expressed proteasome activator complex subunit 3-like and 21 kDa excretory/secretory protein-like genes. This study indicated that parasites of two species may utilise different molecules and mechanisms for larvae capsule formation and host immunosuppression during their infections. Proteins of antigenic genes identified in this study may be also good candidates for diagnosis, treatment or vaccination for T. pseudospiralis infection, and also for the differential diagnosis of two species' infections., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Autophagy is a conserved homeostatic process in which cytoplasmic contents are degraded and recycled. Two ubiquitin-like conjugation pathways are required for the generation of autophagosomes, and ATG5 is necessary for both of these processes. Studies of mice deficient in ATG5 reveal a key role for autophagy in T lymphocyte function, as well as in B cell development and B-1a B cell maintenance. However, the role of autophagy genes in B cell function and antibody production has not been described. Using mice in which Atg5 is conditionally deleted in B lymphocytes, we showed here that this autophagy gene is essential for plasma cell homeostasis. In the absence of B cell ATG5 expression, antibody responses were significantly diminished during antigen-specific immunization, parasitic infection and mucosal inflammation. Atg5-deficient B cells maintained the ability to produce immunoglobulin and undergo class-switch recombination, yet had impaired SDC1 expression, significantly decreased antibody secretion in response to toll-like receptor ligands, and an inability to upregulate plasma cell transcription factors. These results build upon previous data demonstrating a role for ATG5 in early B cell development, illustrating its importance in late B cell activation and subsequent plasma cell differentiation.

Autophagy is an important mechanism used by macrophages to kill intracellular pathogens. The results reported in this study demonstrate that autophagy is also involved in the macrophage killing of the extracellular enteropathogen Citrobacter rodentium after phagocytosis. The process was significantly impaired in macrophages isolated from mice chronically infected with the helminth parasite Heligmosomoides polygyrus. The H. polygyrus-mediated inhibition of autophagy was Th2 dependent because it was not observed in macrophages isolated from helminth-infected STAT6-deficient mice. Moreover, autophagy of Citrobacter was inhibited by treating macrophages with IL-4 and IL-13. The effect of H. polygyrus on autophagy was associated with decreased expression and processing of L chain protein 3 (LC3), a key component of the autophagic machinery. The helminth-induced inhibition of LC3 expression and processing was STAT6 dependent and could be recapitulated by treatment of macrophages with IL-4 and IL-13. Knockdown of LC3 significantly inhibited autophagic killing of Citrobacter, attesting to the functional importance of the H. polygyrus-mediated downregulation of this process. These observations reveal a new aspect of the immunosuppressive effects of helminth infection and provide mechanistic insights into our earlier finding that H. polygyrus significantly worsens the in vivo course of Citrobacter infection.

Immaturity of gut-associated immunity may contribute to pediatric mortality associated with enteric infections. A murine model to parallel infantile enteric disease was used to determine the effects of probiotic, Lactobacillus acidophilus (La), prebiotic, inulin, or both (synbiotic, syn) on pathogen-induced inflammatory responses, NF-κB, and Smad 7 signaling. Newborn mice were inoculated bi-weekly for 4 weeks with La, inulin, or syn and challenged with Citrobacter rodentium (Cr) at 5 weeks. Mouse intestinal epithelial cells (CMT93) were exposed to Cr to determine temporal alterations in NF-Kappa B and Smad 7 levels. Mice with pretreatment of La, inulin, and syn show reduced intestinal inflammation following Cr infection compared with controls, which is associated with significantly reduced bacterial colonization in La, inulin, and syn animals. Our results further show that host defense against Cr infection correlated with enhanced colonic IL-10 and transforming growth factor-β expression and inhibition of NF-κB in syn-treated mice, whereas mice pretreated with syn, La, or inulin had attenuation of Cr-induced Smad 7 expression. There was a temporal Smad 7 and NF-κB intracellular accumulation post-Cr infection and post-tumor necrosis factor stimulation in CMT93 cells. These results, therefore, suggest that probiotic, La, prebiotic inulin, or synbiotic may promote host-protective immunity and attenuate Cr-induced intestinal inflammation through mechanisms affecting NF-κB and Smad 7 signaling., (© 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Modulation of the cellular response by the administration of probiotic bacteria may be an effective strategy for preventing or inhibiting tumour growth. We orally pre-inoculated mice with probiotics Lactobacillus acidophilus NCFM (La) for 14 d. Subcutaneous dorsal-flank tumours and segmental orthotopic colon cancers were implanted into mice using CT-26 murine colon adenocarcinoma cells. On day 28 after tumour initiation, the lamina propria of the colon, mesenteric lymph nodes (MLN) and spleen were harvested and purified for flow cytometry and mRNA analyses. We demonstrated that La pre-inoculation reduced tumour volume growth by 50·3 %, compared with untreated mice at 28 d after tumour implants (2465·5 (SEM 1290·4) v. 4950·9 (SEM 1689·3) mm³, P<0·001). Inoculation with La reduced the severity of colonic carcinogenesis caused by CT-26 cells, such as level of colonic involvement and structural abnormality of epithelial/crypt damage. Moreover, La enhanced apoptosis of CT-26 cells both in dorsal-flank tumour and segmental orthotopic colon cancer, and the mean counts of apoptotic body were higher in mice pre-inoculated with La (P<0·05) compared with untreated mice. La pre-inoculation down-regulated the CXCR4 mRNA expressions in the colon, MLN and extra-intestinal tissue, compared with untreated mice (P<0·05). In addition, La pre-inoculation reduced the mean fluorescence index of MHC class I (H-2Dd, -Kd and -Ld) in flow cytometry analysis. Taken together, these findings suggest that probiotics La may play a role in attenuating tumour growth during CT-26 cell carcinogenesis. The down-regulated expression of CXCR4 mRNA and MHC class I, as well as increasing apoptosis in tumour tissue, indicated that La may be associated with modulating the cellular response triggered by colon carcinogenesis.

Objective: There are approximately 8.5 million Alzheimer disease (AD) patients who need anesthesia and surgery care every year. The inhalation anesthetic isoflurane, but not desflurane, has been shown to induce caspase activation and apoptosis, which are part of AD neuropathogenesis, through the mitochondria-dependent apoptosis pathway. However, the in vivo relevance, underlying mechanisms, and functional consequences of these findings remain largely to be determined., Methods: We therefore set out to assess the effects of isoflurane and desflurane on mitochondrial function, cytotoxicity, learning, and memory using flow cytometry, confocal microscopy, Western blot analysis, immunocytochemistry, and the fear conditioning test., Results: Here we show that isoflurane, but not desflurane, induces opening of mitochondrial permeability transition pore (mPTP), increase in levels of reactive oxygen species, reduction in levels of mitochondrial membrane potential and adenosine-5'-triphosphate, activation of caspase 3, and impairment of learning and memory in cultured cells, mouse hippocampus neurons, mouse hippocampus, and mice. Moreover, cyclosporine A, a blocker of mPTP opening, attenuates isoflurane-induced mPTP opening, caspase 3 activation, and impairment of learning and memory. Finally, isoflurane may induce the opening of mPTP via increasing levels of reactive oxygen species., Interpretation: These findings suggest that desflurane could be a safer anesthetic for AD patients as compared to isoflurane, and elucidate the potential mitochondria-associated underlying mechanisms, and therefore have implications for use of anesthetics in AD patients, pending human study confirmation., (Copyright © 2012 American Neurological Association.)

Background & Aims: T helper (Th) 17 cells produce the effector cytokine interleukin (IL)-17, along with IL-22, which stimulates colonic epithelial cells to produce a membrane-bound mucin, Muc1. Muc1 is a component of the colonic mucus, which functions as a lubricant and a physiologic barrier between luminal contents and mucosal surface. The gene MUC1 has been associated with susceptibility to inflammatory bowel disease; we investigated the role of Muc1 in development of colitis in mice., Methods: Muc1 and RAG1 were disrupted in mice (Muc/RAG double knockout mice); Th1-mediated colitis was induced by intravenous injection of CD4(+)CD45RB(high) T cells. We also studied Th2-mediated colitis using mice with disruptions in Muc1 and T-cell receptor α chain (Muc/TCR double knockout mice)., Results: Muc1 deficiency led to the development of more severe forms of Th1- and Th2-induced colitis than controls. Loss of Muc1 increased colonic permeability and the Th17-cell, but not Th2 or Th1 cell, response in the inflamed colon. Loss of Muc1 also promoted expansion of an innate lymphoid cell population (Lin(-) ckit(-) Thy1(+) Sca1(+)) that produces IL-17. The expansion of Th17 adaptive immune cells and innate lymphoid cells required the commensal microbiota., Conclusions: Muc1, which is up-regulated by Th17 signaling, functions in a negative feedback pathway that prevents an excessive Th17 cell response in inflamed colons of mice. Disruption of this negative feedback pathway, perhaps by variants in Muc1, might contribute to inflammatory bowel disease in patients., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Chronic infection with intestinal helminth parasites is a major public health problem, particularly in the developing world, and can have significant effects on host physiology and the immune response to other enteric infections and antigens. The mechanisms underlying these effects are not well understood. In the current study, we investigated the impact of infection with the murine nematode parasite Heligmosomoides polygyrus, which resides in the duodenum, on epithelial barrier function in the colon. We found that H. polygyrus infection produced a significant increase in colonic epithelial permeability, as evidenced by detection of elevated serum levels of the tracer horseradish peroxidase following rectal administration. This loss of normal barrier function was associated with clear ultrastructural changes in the tight junctions of colonic epithelial cells and an alteration in the expression and distribution of the junctional protein E-cadherin. These parasite-induced abnormalities were not observed in SCID mice but did occur in SCID mice that were adoptively transferred with wild-type T cells, indicating a requirement for adaptive immunity. Furthermore, the helminth-induced increase in gut permeability was not seen in STAT6 knockout (KO) mice. Taken together, the results demonstrate that one of the mechanisms by which helminths exert their effects involves the lymphocyte- and STAT6-dependent breakdown of the intestinal epithelial barrier. This increase in epithelial permeability may facilitate the movement of lumenal contents across the mucosa, thus helping to explain how helminth infection can alter the immune response to enteric antigens.

The role of microorganisms in the gastrointestinal tract has undergone significant modification in the past few decades with new observations from clinical, epidemiologic, and basic science research. We now know that the perception of these gut microbes as pathogens or even as commensals is somewhat outdated. It is becoming increasingly clear that the gut microbiome plays an important role in a host of activities including digestion, protection from potentially pathogenic organisms, and the regulation and development of the host immune system. The complex interactions between microbes and host combined with recent clinical observations and epidemiologic trends may point to the convergence of two well-supported (though imperfect) hypotheses: the "hygiene hypothesis" and the "fetal programming hypothesis." We are beginning to understand that exposure to microbes before conception, during gestation, and in the neonatal period have profound effects on the developing immune system. Recent observations from a variety of fields help support the expansion of the "fetal programming hypothesis" to a host-microbe corollary that microbe-host interactions at critical windows influence the future immune phenotype, the maintenance of immune health, and the development of immune-mediated disease.

Mice deficient in the hemochromatosis gene, Hfe, have attenuated inflammatory responses to Salmonella infection associated with decreased macrophage TNF-alpha and IL-6 biosynthesis after exposure to LPS. In this study, we show that the abnormal cytokine production is related to impaired TLR4 signaling. Despite their abnormal response to LPS, Hfe KO macrophages produced amounts of TNF-alpha similar to those in WT cells after TLR2 stimulation. Consistent with this finding, LPS-induced activation of Mal/MyD88-dependent events was normal in the mutant macrophages. However, LPS-induced IFN-beta expression, a TRAM/TRIF-dependent response activated by TLR4, was reduced by Hfe deficiency. This reduction could be replicated in WT macrophages with the use of iron chelators. In contrast, TLR3-activated expression of IFN-beta, a TRIF-dependent response, was normal in Hfe KO macrophages and was unaffected by iron chelation. Our data suggest that low intracellular iron selectively impairs signaling via the TLR4/TRAM/TRIF pathway proximal to TRIF and results in reduced LPS-induced cytokine expression. Furthermore, by mimicking the altered iron metabolism associated with Hfe deficiency, we found that 3 different inhibitors of hepcidin attenuated Salmonella-induced and noninfectious enterocolitis. Thus, manipulation of iron homeostasis could represent a new therapeutic approach to controlling inflammation.

Modulation of the intestinal immune response early in life by administration of probiotic bacteria may be an effective strategy for preventing or attenuating infectious diarrhea. We preinoculated the mice early in life with the probiotic bacteria Lactobacillus acidophilus NCFM (La) at age 2 wk. Dendritic cells (DCs) were collected and purified from mesenteric lymph nodes (MLN) and spleens of the BalbC/ByJ mice. DC isolation and adoptive transfer was used to examine the function of probiotics. We demonstrated that when mice were adoptively transferred with La-primed DCs (t-LaDC) instead of oral consumption with La, there was a similar effect on fecal bacteria counts, IgA levels, and colonic histopathology, as well as cytokine levels in MLN when there was intestinal bacterial infection. The above findings suggest that DCs play a key role in probiotics attenuating Citrobacter rodentium (Cr) colitis. Moreover, the location of La-primed DC hints that there is interaction of DCs and T cells in the digestive system of the host. Up-regulated expression of a surface marker on DCs indicated that inoculation with probiotics will stimulate the function of DCs, thereby further increasing immune response triggered by DC.

We show that the enzymatic acetylation and deacetylation of a cell surface carbohydrate controls B cell development, signaling, and immunological tolerance. Mice with a mutation in sialate:O-acetyl esterase, an enzyme that specifically removes acetyl moieties from the 9-OH position of alpha2-6-linked sialic acid, exhibit enhanced B cell receptor (BCR) activation, defects in peripheral B cell development, and spontaneously develop antichromatin autoantibodies and glomerular immune complex deposits. The 9-O-acetylation state of sialic acid regulates the function of CD22, a Siglec that functions in vivo as an inhibitor of BCR signaling. These results describe a novel catalytic regulator of B cell signaling and underscore the crucial role of inhibitory signaling in the maintenance of immunological tolerance in the B lineage.

Neutrophil transmigration across mucosal surfaces contributes to dysfunction of epithelial barrier properties, a characteristic underlying many mucosal inflammatory diseases. Thus, insight into the directional movement of neutrophils across epithelial barriers will provide important information relating to the mechanisms of such inflammatory disorders. The eicosanoid hepoxilin A(3), an endogenous product of 12-lipoxygenase activity, is secreted from the apical surface of the epithelial barrier and establishes a chemotactic gradient to guide neutrophils from the submucosa across epithelia to the luminal site of an inflammatory stimulus, the final step in neutrophil recruitment. Currently, little is known regarding how hepoxilin A(3) is secreted from the intestinal epithelium during an inflammatory insult. In this study, we reveal that hepoxilin A(3) is a substrate for the apical efflux ATP-binding protein transporter multidrug resistance-associated protein 2 (MRP2). Moreover, using multiple in vitro and in vivo models, we show that induction of intestinal inflammation profoundly up-regulates apical expression of MRP2, and that interfering with hepoxilin A(3) synthesis and/or inhibition of MRP2 function results in a marked reduction in inflammation and severity of disease. Lastly, examination of inflamed intestinal epithelia in human biopsies revealed up-regulation of MRP2. Thus, blocking hepoxilin A(3) synthesis and/or inhibiting MRP2 may lead to the development of new therapeutic strategies for the treatment of epithelial-associated inflammatory conditions.

Evidence from epidemiological studies indicates an inverse correlation between the incidence of certain immune-mediated diseases, including inflammatory bowel diseases (IBD), and exposure to helminths. Helminth parasites are the classic inducers of Th2 responses. The Th2-polarized T cell response driven by helminth infection has been linked to the attenuation of some damaging Th1 driven inflammatory responses, preventing some Th1-mediated autoimmune diseases in the host, including experimentally induced colitis. Helminth parasites (the porcine whipworm, Trichuris suis) have been tested for treating IBD patients, resulting in clinical amelioration of the disease. As a result, there is a great deal of interest in the research community in exploring the therapeutic use of helminth parasites for the control of immune-mediated diseases, including IBD. However, recent studies have provided evidence indicating the exacerbating effects of helminths on bacterial as well as non-infectious colitis in animal models. Therefore, a better understanding of mechanisms by which helminths modulate host immune responses in the gut may reveal novel, more effective and safer approaches to helminth-based therapy of IBD.

Disturbances of iron homeostasis are associated with altered susceptibility to infectious disease, but the underlying molecular mechanisms are poorly understood. To study this phenomenon, we examined innate immunity to oral Salmonella infection in Hfe knockout (Hfe(-/-)) mice, a model of the human inherited disorder of iron metabolism type I hemochromatosis. Salmonella- and LPS-induced inflammatory responses were attenuated in the mutant animals, with less severe enterocolitis observed in vivo and reduced macrophage TNF-alpha and IL-6 secretion measured in vitro. The macrophage iron exporter ferroportin (FPN) was up-regulated in the Hfe(-/-) mice, and correspondingly, intramacrophage iron levels were lowered. Consistent with the functional importance of these changes, the abnormal cytokine production of the mutant macrophages could be reproduced in wild-type cells by iron chelation, and in a macrophage cell line by overexpression of FPN. The results of analyzing specific steps in the biosynthesis of TNF-alpha and IL-6, including intracellular concentrations, posttranslational stability and transcript levels, were consistent with reduced translation of cytokine mRNAs in Hfe(-/-) macrophages. Polyribosome profile analysis confirmed that elevated macrophage FPN expression and low intracellular iron impaired the translation of specific inflammatory cytokine transcripts. Our results provide molecular insight into immune function in type I hemochromatosis and other disorders of iron homeostasis, and reveal a novel role for iron in the regulation of the inflammatory response.

Indoleamine 2,3-dioxygenase (IDO) is a negative regulator of lymphocyte responses that is expressed predominantly in macrophages and dendritic cells. We detected it at high levels in the small intestine and mesenteric lymph node of young adult mice, suggesting a role in intestinal immunity. Consistent with this idea, we found that IDO-deficient mice had elevated baseline levels of immunoglobulin A (IgA) and IgG in the serum and increased IgA in intestinal secretions. These abnormalities were corrected by a course of broad-spectrum oral antibiotics started at weaning, indicating that they were dependent on the intestinal microbiota. Kynurenine and picolinic acid, two IDO-generated metabolites of tryptophan, were able to inhibit lipopolysaccharide-induced antibody production by splenocytes in vitro, and kynurenine also induced B-cell apoptosis, findings that provide an explanation for the elevated Ig levels in animals lacking IDO. The intestinal secretions of IDO-deficient mice had elevated levels of IgA antibodies that cross-reacted with the gram-negative enteric bacterial pathogen Citrobacter rodentium. In keeping with the functional importance of this natural secretory IgA, the mutant animals were more resistant to intestinal colonization by Citrobacter, developed lower levels of serum Citrobacter-specific IgM and IgG antibodies following oral infection, and had significantly attenuated Citrobacter-induced colitis. Our observations point to an important role for IDO in the regulation of immunity to the gut commensal microbiota that has a significant impact on the response to intestinal pathogens.

Over 80% of the body's activated B cells are located in mucosal sites, including the intestine. The intestine contains IgM(+) B cells, but these cells have not been characterized phenotypically or in terms of their developmental origins. We describe a previously unidentified and unique subset of immunoglobulin M(+) B cells that present with an AA4.1(-)CD21(-)CD23(-) major histocompatibility complex class II(bright) surface phenotype and are characterized by a low frequency of somatic hypermutation and the potential ability to produce interleukin-12p70. This B cell subset resides within the normal mucosa of the large intestine and expands in response to inflammation. Some of these intestinal B cells originate from the AA4.1(+) immature B2 cell pool in the steady state and are also recruited from the recirculating naive B cell pool in the context of intestinal inflammation. They develop in an antigen-independent and BAFF-dependent manner in the absence of T cell help. Expansion of these cells can be induced in the absence of the spleen and gut-associated lymphoid tissues. These results describe the existence of an alternative pathway of B cell maturation in the periphery that gives rise to a tissue-specific B cell subset.

Acute gastroenteritis caused by Salmonella infection is a significant public health problem. Using a mouse model of this condition, the authors demonstrated previously that the cytokine gamma interferon (IFN-gamma) is required for a normal intestinal inflammatory response to the pathogen. In the present study, these experiments are extended to show that natural killer (NK) cells constitute an early source of intestinal IFN-gamma during Salmonella infection, and that these cells have a significant impact on intestinal inflammation. It was found that infection of mice with Salmonella increased both intestinal IFN-gamma production and the numbers of NK cells in the intestine and mesenteric lymph nodes. NK cells, along with other types of lymphocytes, produced IFN-gamma in response to the bacteria in vitro, while antibody-mediated depletion of NK cells in vivo resulted in a significant reduction in Salmonella-induced intestinal IFN-gamma expression. In a mouse strain lacking NK cells and T and B lymphocytes, intestinal production of IFN-gamma and Salmonella-induced intestinal inflammation were both significantly decreased compared with a strain deficient only in T and B cells. The authors' observations point to an important function for NK cells and NK-derived IFN-gamma in regulating the intestinal inflammatory response to Salmonella.

The distribution of several pathogenic helminth infections coincides geographically with many devastating microbial diseases, including enteric bacterial infections. To dissect the mechanisms by which helminths modulate the host's response to enteric bacteria and bacteria-mediated intestinal inflammation, we have recently established a coinfection model and shown that coinfection with the helminth Heligmosomoides polygyrus exacerbates colitis induced by infection with the gram-negative bacterial pathogen Citrobacter rodentium. The disease severity of the coinfected mice was correlated with high Citrobacter loads in the gut, translocation of the bacteria into mucosal and systemic immune compartments, delayed bacterial clearance, and a significantly enhanced colonic TNF-alpha response. In the present study, using our in vivo coinfection model as well as in vitro approaches, we test the hypothesis that the phenotypic and functional alterations in macrophages induced by the helminth-driven T cell response may contribute to the observed alterations in the response to C. rodentium. We show that via a STAT6-dependent mechanism H. polygyrus coinfection results in a marked infiltration into the colonic lamina propria of F4/80+ cells that have the phenotype of alternatively activated macrophages. Functional analysis of these macrophages further shows that they are impaired in their killing of internalized bacteria. Yet, these cells produce an enhanced amount of TNF-alpha in response to C. rodentium infection. These results demonstrate that helminth infection can impair host protection against concurrent enteric bacterial infection and promote bacteria-induced intestinal injury through a mechanism that involves the induction of alternatively activated macrophages.

In human intestinal disease induced by Salmonella enterica serotype Typhimurium (S. typhimurium) transepithelial migration of polymorphonuclear leukocytes (PMNs) rapidly follows attachment of the bacteria to the epithelial apical membrane. Previously, we have shown that the S. typhimurium effector protein, SipA, plays a pivotal role in signalling epithelial cell responses that lead to the transepithelial migration of PMNs. Thus, the objective of this study was to determine the functional domain of SipA that regulates this signalling event. SipA was divided into two fragments: the SipAb C-terminal fragment(426-684) (259 AA), which binds actin, and the SipAa fragment(2-425) (424 AA), which a role has yet to be described. In both in vitro and in vivo models of S. typhimurium-induced intestinal inflammation the SipAa fragment exhibited a profound ability to induce PMN transmigration, whereas the SipAb actin-binding domain failed to induce PMN transmigration. Subsequent mapping of the SipAa domain identified a 131-amino-acid region (SipAa3(294-424)) responsible for modulating PMN transepithelial migration. Interestingly, neither intracellular translocation nor actin association of SipA was necessary for its ability to induce PMN transepithelial migration. As these results indicate SipA has at least two separate functional domains, we speculate that during infection S. typhimurium requires delivery of SipA to both extracellular and intracellular spaces to maximize pro-inflammatory responses and mechanisms of bacterial invasion.

Disparate models for the development of peripheral B cells may reflect significant heterogeneity in recirculating long-lived B cells that have not been previously accounted for. We show in this study that the murine recirculating B cell pool contains two distinct, long-lived, posttransitional, follicular B cell populations. Follicular Type I IgM(low) B cells require Ag-derived and Btk-dependent signals for their development and make up the majority of cells in the recirculating follicular B cell pool. Follicular type II B cells do not require Btk- or Notch-2-derived signals, make up about a third of the long-lived recirculating B cell pool, and can develop in the absence of Ag. These two follicular populations exhibit differences in basal tyrosine phosphorylation and in BCR-induced proliferation, suggesting that they may represent functionally distinct populations of long-lived recirculating B cells.

Protein kinase C-associated kinase (PKK; DIK/RIP4) is an ankyrin-repeat containing serine/threonine receptor-interacting protein (RIP)-family kinase that can activate NFkappaB, and is required for keratinocyte development. In earlier studies, the expression of a catalytically inactive mutant of PKK in the B cell lineage resulted in a marked decrease in peripheral B cells in the spleen and a severe reduction of B-1 B cells. Here we explore the consequences of a null mutation in PKK with respect to the generation of peripheral B cell lineages and the activation of NFkappaB. We show that PKK is not required for the production of B cells in the bone marrow or for the development and maintenance of all mature B lymphocyte populations. We also show that PKK is not required for the activation of NFkappaB downstream of the BCR, CD40, or TLR-4 in B cells. Taken together, these data demonstrate that the loss of this RIP-family kinase does not compromise B lymphocyte development and maintenance, but leaves open the possibility that PKK may have a redundant role in these processes.

Intestinal epithelial cells (IEC) are constantly exposed to bacterial components, such as LPS, without triggering proinflammatory immune responses. This study demonstrates that chronic exposure of human-derived IEC to LPS induces tolerance to an endogenous inflammatory cytokine (IL-1beta) activated IL-8 response that occurs independently of TLR-4/MD-2 signaling. IL-8 production in response to activation by unrelated TNF-alpha and PMA signaling pathways is also inhibited, indicating a broad-spanning tolerance. Quantitative rtPCR and IL-8 promoter-luciferase assays demonstrate that tolerance is regulated at the transcriptional level and occurs independently of IEC cytodifferentiation. By contrast, LPS does not significantly alter other proinflammatory signaling cascades in IEC that function independently of IL-8 production, e.g., IL-6 secretion and PEEC (Hepoxilin A3)-induced neutrophil transepithelial migration in response to invasive Salmonella typhimurium. Human IEC have therefore developed LPS-induced signaling cascades that promote an IL-8 hyporesponsiveness to proinflammatory cytokines while LPS exposure does not compromise the ability of IEC to mount other proinflammatory immune responses to invasive enteropathogens.

To examine whether intestinal helminth infection may be a risk factor for enteric bacterial infection, a murine model was established using the intestinal helminth Heligomosomoides polygyrus and a murine pathogen Citrobacter rodentium, which causes infectious colitis. Using this model we recently have shown that coinfection with the Th2-inducing H. polygyrus and C. rodentium promotes bacterial-associated disease and colitis. In this study, we expand our previous observations and examine the hypothesis that dendritic cells (DC) stimulated by helminth infection may play an important role in the regulation of the intestinal immune response to concurrent C. rodentium infection as well as in the modulation of the bacterial pathogenesis. We show that H. polygyrus infection induces DC activation and IL-10 expression, and that adoptive transfer of parasite-primed DC significantly impairs host protection to C. rodentium infection, resulting in an enhanced bacterial infection and in the development of a more severe colonic injury. Furthermore, we demonstrate that adoptive transfer of parasite-primed IL-10-deficient DCs fails to result in the development of a significantly enhanced C. rodentium-mediated colitis. Similarly, when the DC IL-10 response was neutralized by anti-IL-10 mAb treatment in mice that received parasite-primed DC, no deleterious effect of the parasite-primed DC on the host intestinal response to C. rodentium was detected. Thus, our results provide evidence to indicate that the H. polygyrus-dependent modulation of the host response to concurrent C. rodentium infection involves IL-10-producing DCs.

Enteropathogenic Escherichia coli (EPEC) is a common pathogen in infantile diarrhea, causing a characteristic histopathologic attaching and effacing (A/E) lesion in the intestinal mucosa. The mouse pathogen Citrobacter rodentium causes a similar A/E lesion in the murine intestine. Like EPEC, C. rodentium infection results in colonic crypt hyperplasia, goblet cell depletion, epithelial proliferation, and mucosal disruption. Using this murine model, we tested the hypothesis that preinoculation of murine gut with Lactobacillus acidophilus early in life can enhance host defense against enteric bacterial infection and attenuate bacteria-mediated colitis. Two-week old BALB/c mice were inoculated with L. acidophilus twice per week for 4 weeks before C. rodentium infection or concomitantly with the exposure to C. rodentium at 6-8 weeks of age. The probiotics were administered twice weekly thereafter. We observed that L. acidophilus inoculation in mice inhibits C. rodentium-induced colitis, which is associated with a decrease in C. rodentium colonization and translocation, an increase in its clearance, and a suppression of colonic myeloperoxidase (MPO) activity. Probiotic treatment also stimulates regulatory cytokine expression in the colon [transforming growth factor beta (TGF-beta), interleukin (IL)-10]. Preinoculation with L. acidophilus is more effective than concomitant use of probiotics in the induction of intestinal IgA secretion and in the downregulation of proinflammatory cytokine expression [tumor necrosis factor alpha (TNF-alpha), IL-6, and IL-12]. These observations suggest that inoculation with probiotics can effectively prevent bacteria-induced colitis by limiting enteric bacteria infection and promoting mucosal protective regulatory immune responses. This study may have ramifications for prevention of infectious diarrhea in human infants and children, particularly in developing countries.

Mature recirculating B cells are generally assumed to exist in follicular niches in secondary lymphoid organs, and these cells mediate T-dependent humoral immune responses. We show here that a large proportion of mature B lymphocytes occupy an anatomically and functionally distinct perisinusoidal niche in the bone marrow. Perisinusoidal B cells circulate freely, as revealed by parabiosis studies. However, unlike their counterparts in the follicular niche, these cells are capable of being activated in situ by blood-borne microbes in a T-independent manner to generate specific IgM antibodies. The bone marrow represents a unique type of secondary lymphoid organ in which mature B cells are strategically positioned in the path of circulating microbes.

Infections with intestinal helminth and bacterial pathogens, such as enteropathogenic Escherichia coli, continue to be a major global health threat for children. To test the hypothesis that intestinal helminth infection may be a risk factor for enteric bacterial infection, a murine model was established by using the intestinal helminth Heligomosomoides polygyrus. To analyze the modulatory effect of a Th2-inducing helminth on the outcome of enteric bacterium Citrobacter rodentium infection, BALB/c and STAT 6 knockout (KO) mice were infected with H. polygyrus, C. rodentium, or both. We found that only BALB/c mice coinfected with H. polygyrus and C. rodentium displayed a marked morbidity and mortality. The enhanced susceptibility to C. rodentium and intestinal injury of coinfected BALB/c mice were shown to be associated with a significant increase in helminth-driven Th2 responses, mucosally and systemically, and correlated with a significant downregulation of protective gamma interferon and with a dramatic upregulation of the proinflammatory tumor necrosis factor alpha response. In addition, C. rodentium-associated colonic pathology in coinfected BALB/c mice was significantly enhanced, whereas bacterial burden was increased and clearance was delayed. In contrast, coinfection in STAT 6 KO mice failed to promote C. rodentium infection or to induce a more severe intestinal inflammation and tissue injury, demonstrating a mechanism by which helminth influences the development of host protective immunity and susceptibility to bacterial infections. We conclude that H. polygyrus coinfection can promote C. rodentium-associated disease and colitis through a STAT 6-mediated immune mechanism.

In humans, intestinal defences develop during gestation and, at full term, have the capacity to respond in an appropriate manner to infectious agents and foreign antigens. Before an active protective response can occur, however, the gut must first be exposed to colonizing bacteria. Colonization with diverse intestinal microbes is necessary for the development of important gut defenses such as the synthesis and secretion of polymeric immunoglobulin A and the generation of a balanced T helper (Th) cell response. Insights into normal immune physiological development of the gut have been made by studying the germ-free animal and intestinal defenses. These studies have provided insights into the physiology of immune responses. Two important immunological functions are the secretion of polymeric immunoglobulin A to protect the intestinal surface against harmful stimuli and inhibition of the systemic response to commensal bacteria and food proteins (eg, oral tolerance) to prevent chronic inflammation. Neither function exists in the germ-free state, but rapidly develops after conventionalization (colonization) of the germ-free animal. In the present review, the importance of bacterial colonization on the appearance of normal mucosal immune function and to the clinical consequences of inadequate colonization to the development of disease will be discussed. For example, excessive Th2 activity can lead to atopy, whereas Th1 predominance is found in conditions such as Helicobacter pylori gastritis and Crohn's disease. With the eradication of infectious diseases in developed countries in the past three decades, the incidence of atopic and autoimmune diseases has increased. This epidemiological observation has been explained by the 'hygiene hypothesis', which suggests that a reduction in microbial burden by public health measures has contributed to an immunological imbalance in the intestine. A family of pattern recognition receptors (Toll-like receptors) on gut lymphoid and epithelial cells mediates innate immune responses to bacterial molecular patterns and, thereby, orchestrates acquired immunity. As the role of bacterial communication within the gut (bacterial-epithelial cross-talk) is clarified, physicians should be able to modulate gut immune responses, for example, by the use of probiotics.

The mechanisms by which signaling by the innate immune system controls susceptibility to allergy are poorly understood. In this report, we show that intragastric administration of a food allergen with a mucosal adjuvant induces allergen-specific IgE, elevated plasma histamine levels, and anaphylactic symptoms in three different strains of mice lacking a functional receptor for bacterial LPS (Toll-like receptor 4 (TLR4)), but not in MHC-matched or congenic controls. Susceptibility to allergy correlates with a Th2-biased cytokine response in both the mucosal (mesenteric lymph node and Peyer's patch) and systemic (spleen) tissues of TLR4-mutant or -deficient mice. TLR4-mutant mice are not inherently impaired in their ability to regulate Th1 cytokine production because they respond to stimulation via TLR9. Coadministration of CpG oligodeoxynucleotides during sensitization of TLR4-mutant mice with allergen plus CT abrogates anaphylactic symptoms and Ag-specific IgE, and results in a Th1-polarized cytokine response. When the composition of the bacterial flora is reduced and altered by antibiotic administration (beginning at 2 wk of age), TLR4 wild-type mice become as susceptible to the induction of allergy as their TLR4-mutant counterparts. Both allergen-specific IgE and Th2 cytokine responses are reduced in antibiotic-treated mice in which the flora has been allowed to repopulate. Taken together, our results suggest that TLR4-dependent signals provided by the intestinal commensal flora inhibit the development of allergic responses to food Ags.

The functionally different CD4 T helper cell subsets known as T helper 1 (Th1) and T helper 2 (Th2) display a unique and different cytokine profile. Abnormal skewing toward Th1 or Th2 cells has been suggested to play an important role in the pathogenesis of autoimmune disorders and in inflammatory and allergic diseases. The Th1/Th2 paradigm continues to serve as a model to understand the pathogenesis of several pathologic conditions and provides the rationale for the development of new strategies for treating and preventing these diseases. Over the past year, efforts have continued to increase our understanding of the mechanisms underlying the development and regulation of Th cells and their pathogenic role and therapeutic potential in the induction and treatment of clinic diseases. Several teams of researchers have further examined the association of Th1/Th2 inducing factors and allergic disease and intestinal inflammatory diseases. The protective effect of helminth infection on allergy and the role of regulatory T cells in both Th1- and Th2-mediated diseases have been further examined.

Although helminths induce a polarized Th2 response they have been shown, in clinical studies, to confer protection against allergies. To elucidate the basis for this paradox, we have examined the influence of an enteric helminth infection on a model of food allergy. Upon Ag challenge, mice fed peanut (PN) extract plus the mucosal adjuvant cholera toxin (CT) produced PN-specific IgE that correlated with systemic anaphylactic symptoms and elevated plasma histamine. PN-specific IgE was not induced in helminth-infected mice fed PN without CT. Moreover, when PN plus CT was fed to helminth-infected mice, both PN-specific IgE and anaphylactic symptoms were greatly diminished. The down-regulation of PN-specific IgE was associated with a marked reduction in the secretion of IL-13 by PN-specific T cells. When helminth-infected PN plus CT-sensitized mice were treated with neutralizing Abs to IL-10, the PN-specific IgE response and anaphylactic symptoms were similar to, or greater than, those seen in mice that receive PN and CT alone. Taken together, these results suggest that helminth-dependent protection against allergic disease involves immunoregulatory mechanisms that block production of allergen-specific IgE.

The presentation of soluble model food antigens to the intestinal immune system typically induces antigen-specific systemic nonresponsiveness. Yet, the gut-associated lymphoid tissue (GALT) must launch an effective attack against potentially invasive pathogens even as it avoids mounting a response to innocuous food antigens. Although the mechanism by which the GALT is able to recognize and respond to these different forms of antigen is not clear, recent studies have shown that, initially, both tolerogenic and immunogenic forms of orally administered antigen elicit transient T-cell activation and proliferation. The unique microenvironment of the GALT plays a central role in determining whether functional T-cell anergy or adaptive immunity is the ultimate response. Administration of model food proteins with adjuvants (microbial products that activate the innate immune system) induces a productive immune response to this normally tolerogenic form of antigen. Recent work from our laboratory has shown that an ongoing enteric infection can itself act as an adjuvant and prime for a response to an orally administered soluble protein antigen.

Oral administration of soluble protein Ags typically induces Ag-specific systemic nonresponsiveness. However, we have found that feeding a model food protein, OVA, to helminth-infected mice primes for a systemic OVA-specific Th2 response. In this report we show that, in addition to creating a Th2-priming cytokine environment, helminth infection up-regulates costimulatory molecule expression on mucosal, but not peripheral, APCs. To examine the consequences of mucosal infection for the T cell response to orally administered Ag, we adoptively transferred transgenic, OVA-specific, T cells into normal mice. We found that helminth infection enhances the expansion and survival of transgenic T cells induced by Ag feeding. Transfer of 5,6-carboxyfluorescein diacetate succinimidyl ester-labeled donor cells showed that T cell proliferation in response to Ag feeding takes place primarily in the mesenteric lymph nodes. Upon subsequent peripheral exposure to Ag in adjuvant, the proliferative capacity of the transferred transgenic T cells was reduced in noninfected mice that had been fed OVA. Helminth infection abrogated this reduction in proliferative capacity. Our data suggests that enteric infection can act as an adjuvant for the response to dietary Ags and has implications for allergic responses to food and the efficacy of oral vaccination.

Helicobacter pylori is causally associated with gastritis and gastric cancer. Some developing countries with a high prevalence of infection have high gastric cancer rates, whereas in others, these rates are low. The progression of helicobacter-induced gastritis and gastric atrophy mediated by type 1 T-helper cells may be modulated by concurrent parasitic infection. Here, in mice with concurrent helminth infection, helicobacter-associated gastric atrophy was reduced considerably despite chronic inflammation and high helicobacter colonization. This correlated with a substantial reduction in mRNA for cytokines and chemokines associated with a gastric inflammatory response of type 1 T-helper cells. Thus, concurrent enteric helminth infection can attenuate gastric atrophy, a premalignant lesion.

Although immunologists typically examine immune responses in peripheral lymphoid tissues, mucosal surfaces are the first sites at which most antigens are encountered. The role of lymphocytes in the gut-associated lymphoid tissue (GALT) in the production of secretory IgA has been well characterized. Although T cells of the GALT are located in areas likely to have a key role in cell-mediated immunity at mucosal surfaces, the ways in which these cells help defend against mucosal infection are only beginning to be understood. This review examines mucosal T-cell responses to enteric infection with bacteria, viruses, and parasites.

Intragastric administration of soluble protein Ags results in peripheral tolerance to the fed Ag. To examine the role of cytokine regulation in the induction of oral tolerance, we fed OVA to mice deficient in Th1 (Stat 4-/-) and Th2 (Stat 6-/-) cells and compared their response to that of normal BALB/c controls. We found that, in spite of these deficiencies, OVA-specific peripheral cell-mediated and humoral nonresponsiveness was maintained in both Stat 4-/- and Stat 6-/- mice. In the mucosa, both Peyer's patch T cell proliferative responses and OVA-specific fecal IgA were reduced in Stat 4-/- and Stat 6-/- mice fed OVA but not in normal BALB/c controls. Mucosal, but not peripheral, nonresponsiveness was abrogated by the inclusion of a neutralizing Ab to TGF-beta in the culture medium. Our results show that, in the periphery, tolerance to oral Ag can be induced in both a Th1- or Th2-deficient environment. In the mucosa, however, the absence of Th1 and Th2 cytokines can markedly affect this response, perhaps by regulation of TGF-beta-secreting cells.

OBJECTIVE:In order to analyze the correlation between immunohistochemical expressin for c-erb B-2 oncoprotein and salivary adenoid cystic carcinoma (ACC). METHODS: 25 cases of ACC and two ACC cell strain was studied immunohistochemically using a monoclone antibody against c-erb B-2 oncoprotein. RESULTS: No positive results were obtained in any cell strain,but the invasive breast cancer expressed strong positive. CONCLUSION: It demonstrated that c-erb B-2 expression can not be used as a predictive marker for prognosis of ACC.

A fascinating feature of the intestinal mucosal immune system is its ability to guard against invasion by pathogens while avoiding a response to the many potential Ags present in food. The phenomenon of systemic tolerance after oral administration of protein Ags is well documented, but the cellular and molecular basis for the observed nonresponsiveness is not fully understood. To gain insight into the role of the mucosal microenvironment in the induction of orally induced nonresponsiveness, we attempted to induce tolerance to OVA in mice primed for a Th2-biased mucosal immune response by infection with the nematode parasite Heligmosomoides polygyrus. We found that oral tolerance for Th1-type responses to OVA is maintained when OVA is fed during the peak of the mucosal immune response to H. polygyrus. Tolerance for Th2 cytokine responses or a Th2-dependent isotype of IgG is not induced in this Th2-biased mucosal environment. Treatment of infected mice with rIL-12 to reverse the Th2 polarity of the parasite-specific immune response restores tolerance of both Th1 and Th2 responses to OVA. We conclude that the polarized Th2 response induced by this enteric infection plays a central role in determining whether or not systemic tolerance is induced. Our results imply that attempts to use oral administration of Ag to suppress systemic immune responses will be influenced strongly by the presence of mucosal infection.

This study examined whether the impaired immune responses in zinc deficient- and/or energy-restricted mice exposed to a challenge infection of Heligmosomoides polygyrus might be associated with reduced numbers of spleen cells, altered proportions of spleen cell subpopulations and/or altered function of the T cells or antigen-presenting cells (APC). Female BALB/c mice were given free access to either a zinc-sufficient (60 mg zinc/kg diet, Zn+) or a zinc-deficient diet (0.75 mg zinc/kg diet, Zn-) or were pair-fed (PF) the zinc-sufficient diet. Significant differences in parasite burdens were observed. Worm numbers were lowest in Zn+ mice, intermediate in the PF mice and highest in the Zn- mice, showing that both zinc deficiency and energy restriction reduced protective immunity against the gastrointestinal nematode H. polygyrus. Although the absolute numbers of spleen cells were reduced in both Zn- and energy-restricted (PF) mice, neither deficiency altered the phenotypic distribution of the subpopulations of positive marker cells in the spleen. In vitro functional assays using a 1:1 ratio of APC:T cells showed that T-cell proliferation in response to parasite antigen (Ag) was impaired by a dietary effect of zinc deficiency on T cells and of energy restriction and zinc deficiency on APC function. Consequences of the nutritional deficiencies on cytokine production in response to parasite antigen were more complex: zinc deficiency reduced T-cell function [interleukin-4 and interleukin-5 (IL-4 and IL-5) production], and both nutritional deficits depressed APC functions [IL-4, IL-5, and interferon-gamma (IFN-gamma) production] and T-cell function (IFN-gamma production). Thus, this study showed that zinc deficiency and energy restriction played identifiably distinct roles in regulating host immune responses against the gastrointestinal nematode H. polygyrus.

FN distribution in ACC-2 and high lung-metastatic ACC-M cells was studied with immunohistochemical staining.There were less FN found in ACC-2,but much more FN were observed in the highly metastatic clone Acc-M.In vitro motility of two cell lines was measured by Boyden chamber.With the presence of exgenous FN,Acc-2 became activated with a higher migration rate.The cell adhesion test showed that Acc-2 had stronger ability of adhesion than Acc-M with the presence of FN.The result suggested FN may play a vital role in the invasion and metastasis of ACC.

This study characterized the consequences of zinc-sufficient (Zn+, 60 mg zinc/kg diet, ad libitum), zinc-deficient (Zn-075 mg zinc/kg diet, ad libitum) and energy-restricted (ER, 60 mg zinc/kg diet which was restricted to match food intake of Zn- mice) diets on the in vivo and in vitro immune response of BALB/c mice during both primary and challenge infection with Heligmosomoides polygyrus. In Zn+ mice, both primary and challenge infection with H. polygyrus induced not only a strong Th2 response (IgE, IgG1, eosinophilia, IL-4, IL-5, IL-10), but also elements of a TH1 response (IgG3, IFN-gamma). Zinc deficiency significantly depressed Th2-dependent antibody production during both primary and challenge infection, and reduced mitogen and antigen-induced T cell proliferation during the challenge infection. Th2 cytokine production was reduced by zinc deficiency (IL-4), energy restriction (IL-5) and by zinc deficiency possibly in combination with energy restriction (IL-10) during the primary infection whereas TH1 cytokine production (IFN-gamma) was depressed during the challenge infection by zinc deficiency, possibly together with energy restriction. Both zinc deficiency and energy restriction reduced eosinophilia with the more profound effect being exerted by zinc deficiency. Thus, both zinc deficiency and its concurrent energy restriction modify immune responses in the mice during primary and challenge infection with H. polygyrus.

The objectives of this study were (1) to determine the impact of severe zinc deficiency on the establishment, growth, survival and reproduction of Heligmosomoides polygyrus in the laboratory mouse, during both primary and challenge infection protocols, and (2) to determine whether the observed effects resulted from zinc deficiency per se, or from the accompanying energy restriction. Three diet groups were used: zinc-sufficient (Zn+:60 mg zinc/kg diet), zinc-deficient (Zn-:0.75 mg zinc/kg diet) and energy restricted (ER:60 mg zinc/kg diet pair fed to Zn- mice). Neither Zn- nor ER influenced the establishment of the parasite during a primary infection. However, both significantly influenced the early development of the parasite. The proportion of adult worms recovered 9 days post-infection (p.i.) was highest in Zn- mice, intermediate in ER mice and lowest in Zn+ mice. Worms were also distributed more distally in the intestine of the Zn- mice and worm survival was highest in Zn- mice, intermediate in ER mice and lowest in Zn+ mice at both 4 and 5 weeks p.i. Although the length of female worms was reduced in Zn- mice, neither per capita fecundity nor egg viability was affected by zinc deficiency. Energy restriction, on the other hand, significantly reduced worm fecundity at 5 weeks post-primary infection, but had no effect on egg viability. Zinc concentration of adult H. polygyrus was similar among dietary groups. The effects of zinc deficiency and energy restriction were also investigated 4 and 5 weeks after a challenge infection. Whereas strong host resistance was evident in Zn+ and ER mice, based on comparison of worm numbers between challenged mice and primary infection controls, no evidence of resistance was detected in Zn- mice. As in the primary infection, female worms were shorter in Zn- mice than in ER and Zn+ mice, and energy restriction but not zinc deficiency significantly affected per capita fecundity. However, in contrast to the primary infection, ER mice had elevated rather than reduced fecundity. This study demonstrates a complex interaction between H. polygyrus and zinc and energy restriction, and highlights the importance of controlling for reduced food intake in nutrition-infection studies.

This study was designed to determine whether severe zinc deficiency would prolong the course of a primary Heligmosomoides polygyrus infection in mice, and whether this could be related to impaired T cell function. Female BALB/c mice were fed a zinc-sufficient (Zn+; 60 mg/kg), a zinc-deficient (Zn-; 0.75 mg/kg) or an energy restricted (PF; 60 mg zinc/kg) diet. After four weeks, some mice in each dietary group were given a primary infection with 100 larvae; nutritional, parasitological and immunological parameters were assayed over the following five weeks. Liver zinc concentrations were significantly reduced in Zn- mice compared with Zn+ mice. In certain cases, PF mice also had reduced liver zinc concentrations, showing the negative effects of restricted food intake on zinc status. Zinc deficiency prolonged the course of a primary infection, with the effects being most evident five weeks post-infection when Zn+ mice had only 40% as many worms as Zn- mice. Parasite infection induced strong immunological responses in Zn+ mice in contrast to Zn- mice. The reduced production of IL-4 and IFN-gamma, the reduced peripheral eosinophilia and reduced serum levels of IgE and IgG1 in Zn- mice were attributed to the zinc deficiency, whereas the reduced delayed type hypersensitivity response to parasite antigen and reduced production of IL-5 were in certain instances attributed to reduced energy intake rather than zinc deficiency. These results show that zinc deficiency significantly impairs functions normally attributed to both Th1 and Th2 cell populations, and that these alterations are associated with elevated worm numbers in zinc-deficient mice.