Helminth-induced alterations of the gut microbiota exacerbate bacterial colitis.

Infection with the intestinal helminth parasite Heligmosomoides polygyrus exacerbates the colitis caused by the bacterial enteropathogen Citrobacter rodentium. To clarify the underlying mechanism, we analyzed fecal microbiota composition of control and helminth-infected mice and evaluated the functional role of compositional differences by microbiota transplantation experiments. Our results showed that infection of Balb/c mice with H. polygyrus resulted in significant changes in the composition of the gut microbiota, characterized by a marked increase in the abundance of Bacteroidetes and decreases in Firmicutes and Lactobacillales. Recipients of the gut microbiota from helminth-infected wide-type, but not STAT6-deficient, Balb/c donors had increased fecal pathogen shedding and significant worsening of Citrobacter-induced colitis compared to recipients of microbiota from control donors. Recipients of helminth-altered microbiota also displayed increased regulatory T cells and IL-10 expression. Depletion of CD4+CD25+ T cells and neutralization of IL-10 in recipients of helminth-altered microbiota led to reduced stool C. rodentium numbers and attenuated colitis. These results indicate that alteration of the gut microbiota is a significant contributor to the H. polygyrus-induced exacerbation of C. rodentium colitis. The helminth-induced alteration of the microbiota is Th2-dependent and acts by promoting regulatory T cells that suppress protective responses to bacterial enteropathogens.

Influence of adjuvant formulation on inducing immune response in mice immunized with a recombinant serpin from Trichinella spiralis.

Nematodes of the genus Trichinella are one of the most widespread zoonotic pathogens on the world, and they can still cause major public health problems in many parts of the world. Vaccination against the helminth nematode Trichinella could be a good strategy to reduce the risk of human and animal infection. It was our aim to evaluate three adjuvants, which could be used as an efficient vaccine for animals in combination with rTs-Serpin antigen. In this study, BALB/c mice were vaccinated by an intramuscular route with rTs-Serpin antigen from the parasite Trichinella spiralis in combination with three different adjuvant formulations: Montanide ISA201, Montanide IMS 1313 N PR VG and Freund's complete adjuvant/Freund's incomplete adjuvant (FCA/FIA). The dynamics of IgG, IgM, IgE and cytokine production from spleen cells and worm reduction rate of the vaccinated mice were analysed. The results showed that rTs-serpin can induce partial protection against Trichinella larvae challenge in mice, when compared to the FCA-/FIA-formulated vaccination, the IMS1313 plus rTs-serpin mixture showed higher humoral immunity and similar levels of cellular immunity and worm reduction rate. The study suggested that Montanide IMS nanoparticles 1313 are as effective as FCA but less toxic; thus, Montanide IMS nanoparticles 1313 can be used as a good candidate of adjuvant for developing vaccine against Trichinella spiralis.

Developmental profile of select immune cells in mice infected with Trichinella spiralis during the intestinal phase.

Trichinella spiralis can cause immunosuppression during the intestinal phase of early infection. However, changes in the peripheral blood during T. spiralis early infection remain unclear. Here, select immune cells in mice infected with 500 muscle larvae (ML) of T. spiralis during the intestinal phase of infection were studied. First, the recovery rates of the intestinal worms and female fecundity were determined, and the results showed that the intestinal worms were completely eliminated at 17 days post-infection (dpi) and that large numbers of new-born larvae (NBL) were generated from 5 to 9dpi. Using flow cytometry, it was shown that the number of CD4+ T cells and CD8+ T cells increased over the entire intestinal phase, except on 7dpi when CD4+ T cells decreased significantly compared to the control groups. Although both CD4+ and CD8+ T cells increased, CD8+ T cells increased more than CD4+ T cells, leading to a lower CD4+/CD8+ ratio compared to the control group. Subsequently, a depression of the proliferative response of T cells to concanavalin A (Con A) was noticed at 7 and 11dpi. Although the proliferative response of B cells to LPS was enhanced, the number of B cells from mouse peripheral blood stimulated by T. spiralis antigens showed no differences with the control group prior to 11dpi. The expression of CD14 on monocyte-macrophages decreased during the same period, which meant that the antigen-presenting response was reduced in the immune system of the infected mice. Moreover, the alternatively activated macrophages were induced in T. spiralis early infection. These data provide a better understanding of the development of the intestinal immune response in mice infected with T. spiralis.

The roles of supernatant of macrophage treated by excretory-secretory products from muscle larvae of Trichinella spiralis on the differentiation of C2C12 myoblasts.

The excretory-secretory products (ESPs) released by the muscle-larvae (ML) stage of Trichinella spiralis have been suggested to be involved in nurse cell formation. However, the molecular mechanisms by which ML-ESPs modulate nurse cell formation remain unclear. Macrophages exert either beneficial or deleterious effects on tissue repair, depending on their activation/polarization state. They are crucial for skeletal muscle repair, notably, via their actions on myogenic precursor cells. However, these interactions during T. spiralis infection have not been characterized. In the present study, the ability of conditioned medium (CM) from J774A.1 macrophages treated with ML-ESPs to influence the differentiation of murine myoblasts, and the mechanisms of this influence, were investigated in vitro. The results showed that the expression of Myogenic Regulatory Factors (MRFs) MyoD and myogenin, myosin heavy chain (MyHC), and the p21 cyclin-dependent kinase inhibitor were reduced in CM treated cells compared to their expression in the control group. These findings indicated that CM inhibited myoblast differentiation. Conversely, CM promoted myoblast proliferation and increased cyclin D1 levels. Taken together, results of our study suggested that CM can indirectly influence myoblast differentiation and proliferation, which provides a new method for the elucidation of the complex mechanisms involved in cell-parasite and cell-cell interactions during T. spiralis infection.

Genetic diversity and taxonomic status of Gymnocypris chilianensis based on the mitochondrial DNA cytochrome b gene.

In order to study the genetic diversity and taxonomic status of Gymnocypris chilianensis on a molecular level, the mitochondrial DNA cytochrome b gene was sequenced for 74 individuals of G. chilianensis from two locations (Heihe River and Shule River) and 42 individuals of its affinis species Gymnocypris przewalskii. Analyses of genetic diversity and sequence differences were conducted for these samples, combined with the analysis of 30 homologous sequences of another affinis species Gymnocypris eckloni, which were downloaded from GenBank. The results showed that both the haplotype diversity (h = 0.9820) and nucleotide diversity (π= 0.0039) of the Shule River G. chilianensis were lower than the other populations, thus, the Shule River G. chilianensis should be prioritized for protection because of its lower genetic diversity level. The results of sequence analysis showed that the genetic distance between the Heihe River G. chilianensis population and the Shule River G. chilianensis population was 0.0064, and the genetic distance between these two populations and the G. przewalskii population was 0.0838 and 0.0810, respectively. The genetic distance between the two G. chilianensis populations and the G. eckloni population was 0.0805 and 0.0778, respectively. Analysis of sequence differences indicates that G. chilianensis is sufficiently diverged from G. przewalskii and G. eckloni to the extent that it has reached species level, thus, G. chilianensis can be considered an independent species of Gymnocypris.

Short Communication: Effect of heat stress on heat-shock protein (Hsp60) mRNA expression in rainbow trout Oncorhynchus mykiss.

The enhanced expression of heat shock proteins (hsps) in organisms can be detected in response to many kinds of stressor. For fish, high temperature is an important stressor, and hsp expression is associated with differences in environmental temperature. In this study, rainbow trout (Oncorhynchus mykiss) that were accustomed to an aquatic temperature of 18°C were exposed to an elevated temperature (25°C), and hsp60 expression in the gill, liver, spleen, heart, and head kidney was quantified using real-time polymerase chain reaction in unstressed and heat-stressed animals. The fish responded to heat stress in a time- and tissue-specific manner. Cardiac hsp60 mRNA levels were largely unchanged, and the greatest induction of hsp60 in heat-stressed animals was recorded in the liver, suggesting that protein damage and the consequent requirement for the Hsp60 protein are probably greater in hepatic tissue. Therefore, fish must be provided with optimal temperature conditions in order to realize their potential growth and maximize fish farm profits.

An anti-tumor protein produced by Trichinella spiralis induces apoptosis in human hepatoma H7402 cells.

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.

Unique antigenic gene expression at different developmental stages of Trichinella pseudospiralis.

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.

Modulation of inflammatory bowel disease in a mouse model following infection with Trichinella spiralis.

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.

Peripheral nonresponsiveness to orally administered soluble protein antigens.

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.

Enteric infection acts as an adjuvant for the response to a model food 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.

Concurrent enteric helminth infection modulates inflammation and gastric immune responses and reduces helicobacter-induced gastric atrophy.

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.

Orally induced peripheral nonresponsiveness is maintained in the absence of functional Th1 or Th2 cells.

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.

Mucosal T-cell responses to enteric infection.

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.

A helminth-induced mucosal Th2 response alters nonresponsiveness to oral administration of a soluble antigen.

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.

Energy restriction and zinc deficiency impair the functions of murine T cells and antigen-presenting cells during gastrointestinal nematode 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.

[Expression of c-erb B-2 concoprotein in salivary adenoid cystic carcinoma]

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.

Zinc deficiency and energy restriction modify immune responses in mice during both primary and challenge infection with Heligmosomoides polygyrus (Nematoda).

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 effect of FN(fibronectin) on invasion and metastasis of human acinic cell carcinoma cell strain of the salivary gland]

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.

Energy restriction and severe zinc deficiency influence growth, survival and reproduction of Heligmosomoides polygyrus (Nematoda) during primary and challenge infections in mice.

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.