RESUMO
IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response against helminth infections, where it plays an important role in the expulsion of the parasite. In a murine model of Nippostrongylus brasiliensis infection, IL-9 is produced mainly by CD4+ T lymphocytes and innate lymphoid cells found in the lung, small intestine, and draining lymph nodes. Given the technical difficulties involved in the intracellular staining of IL-9, as well as the complexity of isolating hematopoietic cells from the small intestine upon infection, there is a pressing need for a comprehensive but straightforward protocol to analyze the expression of IL-9 in different lymphoid and non-lymphoid tissues in this model. The protocol described here outlines the kinetics of IL-9 produced by CD4+ T cells and innate lymphoid cells in the lung and small intestine, the main organs targeted by N. brasiliensis, as well as in the mediastinal and mesenteric lymph nodes, throughout the infection. In addition, it details the number of larvae needed for infection, depending on the cell type and organ of interest. This protocol aims to assist in the standardization of assays to save time and resources by offering the opportunity to focus on the specific cells, organs, and disease stages of interest in the N. brasiliensis infection model.
Assuntos
Interleucina-9 , Nippostrongylus , Camundongos , Animais , Nippostrongylus/fisiologia , Interleucina-9/metabolismo , Imunidade Inata , Citocinas/metabolismo , Linfócitos T CD4-PositivosRESUMO
Molybdate (Mo+) supplements can suppress or enhance nematode infections in ruminants, depending on exposure level, but there have been no investigations in non-ruminants. Three groups of 16 mature rats were each fed a commercial diet and given Mo+ (10 mg Mo/l), tungstate (a molybdenum [Mo] antagonist) (MoO4, 350 mg W/l) or no supplement (C) via drinking water for 40 days before acute infection with 3,600 Nippostrongylus brasiliensis larvae. Group Mo- also received allopurinol (1 g/l), a molybdenoenzyme inhibitor, from 4 days post infection (dpi). Subgroups of four rats from each group were killed at 7-14 dpi. A group of six rats was left untreated and uninfected and subgroups killed 10 or 12 dpi. Infection reduced intakes of food and water but impacts were greatest in group Mo-. Median worm counts in groups C, Mo- and Mo+ were 900, 941 and 510, respectively, at 7 dpi and 9, 40 and 0 (P = 0.05) at 10 dpi. Median faecal egg counts were consistently lowest in group Mo+. Worm weight was reduced (P <0.05), worm tissue protease increased and superoxide dismutase activities increased in worm (P < 0.01) and host duodenal homogenates (P < 0.01) from group Mo+. In group Mo-, liver Mo concentration decreased, duodenal xanthine oxidoreductase activity (DXOR) became totally inhibited and plasma uric acid was barely detectable at 10 dpi. Plasma mast cell protease activity and duodenal malonyldialdehyde concentrations, markers of inflammation, were increased by nematode infection (P <0.001) but unaffected by water treatments. Liver Mo, liver copper (Cu) and plasma Cu concentrations were increased in group Mo+ and plasma Cu concentration was increased in group Mo- suggesting systemic exposure to partially thiolated MoO4 and WO4. Supplementary MoO4 impaired larval establishment and changed parasite biochemistry without affecting the inflammatory response to infection but may have required partial thiolation to do so. Rats did not rely on DXOR activity to expel N. brasiliensis.
Assuntos
Infecções por Nematoides , Doenças dos Roedores , Animais , Mastócitos , Molibdênio , Infecções por Nematoides/veterinária , Nippostrongylus/fisiologia , Peptídeo Hidrolases , RatosRESUMO
Hookworm infection is endemic in developing countries, leading to poor cognitive function-among other disruptions. In this study, the effects of Nippostrongylus brasiliensis infection (a murine model of Necator Americanus) on cognitive function were investigated. Though impaired cognition has been extensively reported, the exact domain of cognition affected is still unknown, hence requiring investigation. The objective of this study was to identify possible cognitive changes during Nippostrongylus brasiliensis infection in mice, using the Morris water maze. Here, we show for the first time that mice infected with Nippostrongylus brasiliensis were able to learn the Morris water maze task, but demonstrated impaired reference memory. Anxiety measured by thigmotaxis in the maze, did not play a role for the observed cognitive impairment. Of further interest, an increase in the number of hippocampal macrophages and microglia with training and/or infection suggested a significant role of these cell types during spatial learning. Together, these experimental mouse studies suggest that helminth infections do have an impact on cognition. Further experimental animal studies on cognition and infection might open new approaches for a better understanding and impact of pathogen infections.
Assuntos
Memória , Células Mieloides/citologia , Nippostrongylus/fisiologia , Infecções por Strongylida/imunologia , Infecções por Strongylida/fisiopatologia , Animais , Cognição , Macrófagos/citologia , Aprendizagem em Labirinto , Camundongos , Microglia/patologia , Infecções por Strongylida/patologiaRESUMO
Hookworm infections (Necator americanus or Ancylostoma duodenale) represent a major neglected tropical disease, affecting approximately 700 million people worldwide, and can cause severe morbidity due to the need for these worms to feed on host blood. N. brasiliensis and H. polygrus, both rodent parasites, are the two most commonly employed laboratory models of experimental hookworm infection. Both parasites evoke type 2 immune responses, and their use has been instrumental in generating fundamental insight into the molecular mechanisms of type-2 immunity and for understanding how the immune response can control parasite numbers. Here we provide a complete set of methods by which to investigate the natural progression of infection and the host immunological responses in the lung and intestine of H. polygyrus- and N. brasiliensis-infected mice. Detailed information is included about the most important parasitological and immunological measurements to perform at each time point. © 2017 by John Wiley & Sons, Inc.
Assuntos
Modelos Animais de Doenças , Imunidade Humoral , Camundongos , Nematospiroides dubius/fisiologia , Nippostrongylus/fisiologia , Infecções por Strongylida/imunologia , Animais , Progressão da DoençaRESUMO
Mucin is a major component of mucus in gastrointestinal mucosa. Increase of specific sialomucins having Sda blood group antigen, NeuAcα2-3(GalNAcß1-4)Galß1-4GlcNAcß-, is considered to be associated with expulsion of the parasitic intestinal nematode Nippostrongylus brasiliensis. In this study, we examined the relationship between interleukin (IL)-13 pathway and expression of Sda-sialomucins in small intestinal mucosa with N. brasiliensis infection. Nematode infection induced marked increases in small intestinal mucins that reacted with anti-Sda antibody in wild type (wt) mice. However, this increase due to infection was supressed in IL-4 receptor α deficient (IL-4Rα-/-) mice, which lack both IL-4 and IL-13 signaling via IL-4R, and severe combined immunodeficient (SCID) mice, which have defects in B- and T-lymphocytes. Analysis using tandem mass spectroscopy showed that Sda-glycans were not expressed in small intestinal mucins in IL-4Rα-/- and SCID mice after infection despite the appearance of Sda-glycans in the infected wt mice. Inoculation of recombinant IL-13 into the infected SCID mice restored expression of Sda-glycan. Our results suggest that the IL-13/IL-4R axis is important for the production of Sda-sialomucins in the host intestinal mucosa with parasitic nematode infection.
Assuntos
Imunidade Adaptativa , Enteropatias Parasitárias/imunologia , Mucosa Intestinal/imunologia , Receptores Tipo II de Interleucina-4/genética , Sialomucinas/metabolismo , Infecções por Strongylida/imunologia , Animais , Enteropatias Parasitárias/parasitologia , Intestino Delgado/imunologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Nippostrongylus/fisiologia , Receptores Tipo II de Interleucina-4/metabolismo , Transdução de Sinais , Infecções por Strongylida/parasitologiaRESUMO
The present study investigates the in vitro efficacy of derivatives of the cyclooctadepsipeptides and the aminophenylamidines, which are promising candidates for the evaluation of the treatment of human soil-transmitted helminthiases. The effects of emodepside and PF1022A as well as of amidantel, deacylated amidantel and tribendimidine were evaluated in a concentration range between 0.01 and 100 µg/ml against third-stage larvae (L3) and adult worms of Nippostrongylus brasiliensis and first-stage larvae (L1) of Trichinella spiralis. Furthermore, drug combinations of PF1022A plus deacylated amidantel or tribendimidine and of tribendimidine plus levamisole were tested for any potential additive or even synergistic interactions. Emodepside had a significantly lower EC(50) value than PF1022A in the T. spiralis (0.02788 vs. 0.05862 µg/ml) and the N. brasiliensis (0.06188 vs. 0.1485 µg/ml) motility assays but not in the acetylcholine esterase secretion assay with adult N. brasiliensis (0.05650 vs. 0.06886 µg/ml). While amidantel showed only minimal or at best partial inhibition of nematode motility and acetylcholine esterase secretion, tribendimidine was nearly as potent as deacylated amidantel. Whereas deacylated amidantel had a significantly lower EC(50) than tribendimidine in the N. brasiliensis L3 motility assay (0.05492 vs. 0.2080 µg/ml), differences were not significant in the T. spiralis L1 motility assay (0.7766 vs. 1.145 µg/ml). Surprisingly, none of the combinations showed improved efficacy when compared to the individual drugs including levamisole/tribendimidine, which have previously been reported to act synergistically against Ancylostoma ceylanicum.
Assuntos
Anti-Helmínticos/farmacologia , Depsipeptídeos/farmacologia , Nippostrongylus/efeitos dos fármacos , Fenilenodiaminas/farmacologia , Trichinella spiralis/efeitos dos fármacos , Animais , Sinergismo Farmacológico , Larva/efeitos dos fármacos , Larva/fisiologia , Locomoção/efeitos dos fármacos , Nippostrongylus/fisiologia , Trichinella spiralis/fisiologiaRESUMO
Infections with the parasitic helminth, Nippostrongylus brasiliensis, cause changes in rat small intestinal goblet cell mucin, particularly in the peripheral sugar residues of oligosaccharide. These changes may correlate with expulsion. In this study, we examined changes in mucin oligosaccharides caused by primary infection and reinfection with N. brasiliensis, using two monoclonal antibodies, HCM31 and PGM34, that react with sialomucin and sulfomucin, respectively. Enzyme-linked immunosorbent assay of jejunal mucins showed that the relative reactivity of mucins with HCM31, but not PGM34, increased up to 16 days after primary infection and 6 days after reinfection, the times when the worms were expelled from the rats. Immunohistochemical studies confirmed that goblet cells stained with HCM31 greatly increased at the time of worm expulsion. These results indicate that the marked increase observed in HCM31-reactive sialomucins may be related to expulsion of the worms.
Assuntos
Enteropatias Parasitárias/metabolismo , Jejuno/metabolismo , Nippostrongylus/fisiologia , Sialomucinas/metabolismo , Infecções por Strongylida/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Ensaio de Imunoadsorção Enzimática , Fezes/parasitologia , Células Caliciformes/metabolismo , Imunidade nas Mucosas , Imuno-Histoquímica , Enteropatias Parasitárias/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/parasitologia , Jejuno/parasitologia , Cinética , Lectinas , Masculino , Nippostrongylus/imunologia , Contagem de Ovos de Parasitas , Ratos , Ratos Wistar , Sialomucinas/imunologia , Infecções por Strongylida/imunologiaRESUMO
Expulsion of adult Nippostrongylus brasiliensis worms from the small intestine is profoundly impaired in signal transducer and activator of transcription (STAT)6-deficient mice. IL-5 transgenic (Tg) mice with constitutive eosinophilia show profound early resistance in the skin and/or later pre-lung phase of primary infections with N. brasiliensis. This study was designed to assess the importance of the eosinophil chemokine eotaxin and the STAT6/interleukin (IL)-4/IL-13 signalling pathway in early resistance to N. brasiliensis. Eosinophil recruitment into the skin following injection of N. brasiliensis larvae was reduced in STAT6- or eotaxin-deficient/IL-5 Tg double mutant mice. While ablation of eotaxin did not impair resistance in the pre-lung phase of N. brasiliensis infections in IL-5 Tg mice, elimination of STAT6 caused a modest reduction in resistance in both primary and secondary infections on this genetic background. STAT6(-/-)-, IL-13(-/-)- and IL-4Ralpha(-/-)-deficient single mutant and IL-13(-/-)/IL-4Ralpha(-/-) double mutant mice were more susceptible than WT mice during the pre-lung phase of secondary N. brasiliensis infections. In contrast, primary or secondary resistance were unaffected at either the pre-lung or gut stages of infection in eotaxin(-/-) single mutant mice. STAT6(-/-) and eotaxin(-/-) mice with or without the IL-5 transgene, were no more susceptible than WT or IL-5 Tg mice to protracted primary infections with Heligmosomoides bakeri, a parasitic nematode that is restricted to the gut. Our data suggest that parasitic nematodes that transit through the skin and lungs en route to the gut may be susceptible to early (pre-lung) innate and adaptive immune mechanisms that are dependent on the STAT6/IL-4/IL-13 signalling pathway, and this may be important for the development of effective therapies and vaccines.
Assuntos
Quimiocina CCL11/fisiologia , Eosinófilos/metabolismo , Heligmosomatoidea/fisiologia , Nippostrongylus/fisiologia , Fator de Transcrição STAT6/fisiologia , Transdução de Sinais/fisiologia , Animais , Quimiocina CCL11/deficiência , Quimiocina CCL11/genética , Eosinófilos/citologia , Eosinófilos/parasitologia , Fezes/parasitologia , Feminino , Interações Hospedeiro-Parasita , Imunidade Inata , Interleucina-5/genética , Interleucina-5/metabolismo , Interleucina-5/fisiologia , Mucosa Intestinal/metabolismo , Intestinos/parasitologia , Larva/fisiologia , Pulmão/metabolismo , Pulmão/parasitologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Camundongos Knockout , Camundongos Transgênicos , Contagem de Ovos de Parasitas , Receptores de Superfície Celular/deficiência , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/fisiologia , Fator de Transcrição STAT6/deficiência , Fator de Transcrição STAT6/genética , Pele/metabolismo , Pele/parasitologiaRESUMO
Plasma butyrylcholinesterase (BChE) hydrolyzes ester-containing compounds such as succinylcholine, as well as acting as a scavenger against neurotoxic organophosphates (OPs). We previously found that Nippostrongylus brasiliensis infection makes rats more susceptible to OP toxicity by decreasing serum paraoxonase-1 (PON1) activity. In the present study, we examined the effects of N.brasiliensis infection on acetylcholinesterase (AChE) activity in plasma, red blood cells (RBCs), brain and diaphragm, as well as serum PON1 activity, in rats at day 7 after infection. N.brasiliensis infection significantly decreased plasma BChE and PON1 activities without significantly altering AChE activity in RBCs, brain and diaphragm. These results provide further insight into the unusual deleterious effects of intestinal nematode infections on body homeostasis.
Assuntos
Butirilcolinesterase/sangue , Nippostrongylus/fisiologia , Infecções por Strongylida/enzimologia , Acetilcolinesterase/análise , Acetilcolinesterase/sangue , Animais , Arildialquilfosfatase/sangue , Encéfalo/enzimologia , Hidrolases de Éster Carboxílico/metabolismo , Diafragma/enzimologia , Eritrócitos/enzimologia , Fezes/parasitologia , Masculino , Contagem de Ovos de Parasitas , Distribuição Aleatória , Ratos , Ratos Wistar , Infecções por Strongylida/sangueRESUMO
Nippostrongylus brasiliensis larvae are particularly susceptible to immunological attack during the pre-lung stage of primary and secondary infections in mice. Whilst most of the common laboratory strains of mice are permissive hosts for the parasite, in this study we report for the first time, the strong resistance of naive FVB/N mice to N. brasiliensis. Damage to larvae is evident within the first 24 h of infection and this may be critical to later larval development and reproductive success. Inflammatory responses in the skin, and larval escape from this tissue were comparable in susceptible CBA/Ca and resistant FVB/N mice, with most larvae exiting within 4 h of a primary infection. Lung larval burdens were also similar between strains, but larvae recovered from FVB/N mice were smaller and less motile. In FVB/N mice, larval colonization of the gut was impaired and worms produced very few eggs. However FVB/N mice did not show enhanced resistance to Heligmosomoides bakeri (also known as Heligmosomoides polygyrus), a nematode largely restricted to the gut. Damage done in the pre-lung or lung stages of infection with N. brasiliensis is likely to contribute to ongoing developmental and functional abnormalities, which are profoundly evident in the gut phase of infection.
Assuntos
Imunidade Inata/genética , Nippostrongylus/fisiologia , Infecções por Strongylida/genética , Infecções por Strongylida/imunologia , Animais , Proteínas de Ligação a Ácido Graxo/genética , Feminino , Imunidade Celular , Imunidade Inata/imunologia , Intestinos/parasitologia , Larva/fisiologia , Leucócitos/imunologia , Pulmão/parasitologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos CBA , Camundongos Endogâmicos , Camundongos Transgênicos , Nematospiroides/fisiologia , Contagem de Ovos de ParasitasRESUMO
Protease-activated receptor 2 (PAR(2)) is a cell surface receptor that detects trypsin and trypsin-like enzymes. Although the precise pathophysiological roles of PAR(2) are yet to be determined, the receptor has been broadly implicated in inflammation and allergy. However, no studies have investigated the possible roles of PAR(2) in hosts infected by parasitic helminths. Therefore, in this preliminary investigation, we compared the infectivity of the nematode Nippostrongylus brasiliensis in mice lacking the PAR(2) gene (PAR2-/- ) and in their 'background-strain' controls (129SV). PAR2-/- mice displayed elevated fecal egg counts and decreased levels of total serum IgE, after a subcutaneous infection with 900 infective third-stage N. brasiliensis larvae compared with 129SV mice that were not susceptible to infection. In addition, in a separate study in BALB/c mice, two immunological hallmarks of parasite infection, IgE- and IL-10-expressing lymphocytes, were shown to be augmented after the coadministration of the classic antigen ovalbumin with the PAR(2)-activating peptide SLIGRL (single letter amino acid sequence) but not the inactive reverse peptide LRGILS. These findings provide initial support for the proposal that PAR(2) is a recognition receptor for nematode-derived proteases.
Assuntos
Proteínas de Helminto/imunologia , Nippostrongylus/fisiologia , Infecções por Strongylida/imunologia , Animais , Fezes/parasitologia , Deleção de Genes , Proteínas de Helminto/metabolismo , Imunoglobulina E/sangue , Interleucina-10/sangue , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Contagem de Ovos de ParasitasRESUMO
Female and male worms of Nippostrongylus brasiliensis exhibited sexual dimorphism based on the number of coelomocytes present. A surprising multiplicity of diverse morphological types of coelomocytes developed in both female and male worms during the parasitic cycle. Cytoplasmic processes began to appear on the surface membrane of coelomocytes in the late third-stage larvae (L3s) in the lungs, and they increased greatly in type, size, and morphology during the fourth and fifth stages. These structures were characterized primarily as complex filopodia, pseudopodia, and cytoplasmic pearls, which resulted in the formation of highly pleomorphic cells. Pearls, starting as small protuberances, progressively increased in size and number with larval growth and development. In the adult worms, a novel process of autocannibalism was initiated in many of the very large coelomocytes. The pearls grew enormously in size at the expense of the cytoplasm, forming a peripheral garland in 1 plane surrounding a residual, small, flat, cytoplasmic core containing the nucleus. The underlying "strategy" was to increase the surface-to-volume ratio of these huge cells to overcome the restriction imposed by eutely; the coelomocytes do not undergo cell division. This morphological innovation makes possible a more efficient uptake of nutrients and exocytosis of waste matter. Vesicles (presumably lysosomes) in the coelomocytes of the infective L3 store an extraordinarily high concentration of vitamin B12 (cobalamin, Cbl). At present, the only physiological function that can be assigned to coelomocytes of N. brasiliensis is the uptake, concentration, and storage of Cbl in the free-living stages, with the subsequent release of the molecule from the vesicles in the early phase of parasitism. Thus, stored Cbl in the infective L3 is made available for biochemical processes during the critical period of larval growth and differentiation initiated in the lung. A model of a hypothetical coelomocyte is presented relative to the processing and use of Cbl. Based on many criteria, it is possible that functional differences exist between different morphological types of coelomocytes in the parasitic stages of N. brasiliensis and that future studies will have to address this matter.
Assuntos
Estágios do Ciclo de Vida , Nippostrongylus/citologia , Nippostrongylus/crescimento & desenvolvimento , Infecções por Strongylida/parasitologia , Animais , Feminino , Intestinos/parasitologia , Larva/citologia , Larva/fisiologia , Estágios do Ciclo de Vida/fisiologia , Pulmão/parasitologia , Masculino , Nippostrongylus/fisiologia , Ratos , Ratos Sprague-Dawley , Caracteres Sexuais , Maturidade Sexual , Fatores de TempoRESUMO
Complement activation and C3 deposition on the surface of parasitic helminths may be important for recruitment of leukocytes and for damage to the target organism via cell-mediated mechanisms. Inhibition of complement activation would therefore be advantageous to parasites, minimizing damage and enhancing migration through tissues. The aim of this study was to determine ex vivo if complement activation by, and leukocyte adherence to, the nematode Nippostrongylus brasiliensis change as the parasite matures and migrates through the murine host. Pathways of activation of complement and the mechanism of adherence of leukocytes were also defined using sera from mice genetically deficient in either C1q, factor B, C1q and factor B, C3, or C4. Substantive deposition of C3 and adherence of eosinophil-rich leukocytes were seen with infective-stage (L3) but not with lung-stage (L4) larvae. Adult intestinal worms had low to intermediate levels of both C3 and leukocyte binding. For L3 and adult worms, complement deposition was principally dependent on the alternative pathway. For lung-stage larvae, the small amount of C3 detected was dependent to similar degrees on both the lectin and alternative pathways. The classical pathway was not involved for any of the life stages of the parasite. These results suggest that in primary infections, the infective stage of N. brasiliensis is vulnerable to complement-dependent attack by leukocytes. However, within the first 24 h of infection, N. brasiliensis acquires the ability to largely avoid complement-dependent immune responses.
Assuntos
Ativação do Complemento/imunologia , Leucócitos/citologia , Leucócitos/fisiologia , Nippostrongylus/fisiologia , Infecções por Strongylida/imunologia , Infecções por Strongylida/parasitologia , Animais , Adesão Celular , Complemento C1q/imunologia , Complemento C3/imunologia , Complemento C4/imunologia , Fator B do Complemento/fisiologia , Camundongos , Mutação , Nippostrongylus/imunologiaRESUMO
The resistance of cotton rats, Sigmodon hispidus to Nippostrongylus brasiliensis infection was examined and compared the response to that of the susceptible Indian soft-furred rat, Millardia meltada. After a primary infection with infective third-stage N. brasiliensis larvae (L3), the number of eggs in feces and adult worm recovery rates from the small intestine of cotton rats were significantly lower than in the controls. To determine whether cotton rat resistance was observed during the migratory phase or the intestinal phase, cotton rats and control animals were challenged subcutaneously with L3 or intraduodenally with adult worms, and larval recovery from lungs and adult worm burden were evaluated. The recovery rate of larvae from the lungs of cotton rats was about five-fold lower than from controls. Adult worm recovery from the small intestine of cotton rats was also lower than that from the controls, but the difference (two-fold lower) was smaller than that observed for lung recovery. Carbon treatment at a dose of 250-500 mg/kg effectively increased larval worm recovery from the lungs of cotton rats. However, this treatment had no effect on worm recovery from the intestine after intraduodenal implantation of adult N. brasiliensis. These results suggest that macrophage function have important role in the expression of strong resistance during the migratory phase of N. brasiliensis infection in cotton rats.
Assuntos
Suscetibilidade a Doenças , Nippostrongylus/fisiologia , Doenças dos Roedores/parasitologia , Sigmodontinae/parasitologia , Animais , Carbono/farmacologia , Fezes/parasitologia , Intestino Delgado/parasitologia , Larva/efeitos dos fármacos , Larva/parasitologia , Pulmão/parasitologia , Macrófagos/imunologia , Masculino , Nippostrongylus/efeitos dos fármacos , Nippostrongylus/imunologia , Contagem de Ovos de Parasitas , Ratos , Doenças dos Roedores/imunologia , Sigmodontinae/imunologia , Infecções por Strongylida/imunologia , Infecções por Strongylida/parasitologiaRESUMO
Infection of mice with the nematode parasite Nippostrongylus brasiliensis results in a well characterized intestinal mastocytosis with intraepithelial migration of mucosal mast cells (MMC). The molecules mediating this response are unknown. We examined expression of several putative mast cell chemoattractants in intestinal epithelium following N. brasiliensis infection. Expression of the chemokines monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1 alpha (MIP-1alpha), RANTES (regulated on activation normal T-cell expressed and secreted), fractalkine, and thymocyte expressed chemokine (TECK); and the cytokines stem cell factor (SCF) and transforming growth factor beta1 (TGFbeta1), was constitutive and no alteration was detected following infection. MCP-1 expression was also constitutive but at much lower levels and increased expression was detected on days 7 and 14 postinfection. Expression of MCP-1 in whole jejunum was at much higher levels than in epithelium. Constitutive expression of MCP-1, MIP-1alpha and TGFbeta1 was also detected in cultured bone marrow-derived homologues of MMC. In an intestinal epithelial cell line (CMT-93), there was constitutive expression of SCF, TGFalpha1, fractalkine and MCP-1. The results show that, in vivo, epithelium is a potentially important source of mast cell chemoattractants.
Assuntos
Quimiocinas/metabolismo , Citocinas/metabolismo , Mucosa Intestinal/imunologia , Nippostrongylus/fisiologia , Infecções por Strongylida/imunologia , Animais , Linhagem Celular , Células Cultivadas , Mucosa Intestinal/parasitologia , Mucosa Intestinal/ultraestrutura , Jejuno/imunologia , Jejuno/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Contagem de Ovos de Parasitas , Homologia de Sequência , Infecções por Strongylida/parasitologia , Infecções por Strongylida/patologiaRESUMO
Neutral mucin oligosaccharides from the small intestine of control rats and rats infected with the parasite Nippostrongylus brasiliensis were released and analyzed by gas chromatography-mass spectrometry. Infected animals expressed seven blood group A-like structures that were all absent in the control animals. The blood group A nature of these epitopes was confirmed by blood group A reactivity of the prepared mucins, of which Muc2 was one. Transferase assays and Northern blotting on small intestines from infected animals showed that an alpha-N-acetylgalactosaminyltransferase similar to the human blood group A glycosyltransferase had been induced. The expression was a transient event, with a maximum at day 6 of the 13-day-long infection. The rat blood group A glycosyltransferase was cloned, revealing two forms with an amino acid similarity of 95%. Both types had blood group A transferase activity and were probably allelic because none of 12 analyzed inbred strains carried both types. The second type was found in outbred rats and in one inbred strain. First generation offspring of inbred rats of each type were heterozygous, further supporting the allelic hypothesis. The transient induction and the large allelic variation could suggest that glycosyltransferases are part of a dynamic system altering mucins and other glycoconjugates as a protecting mechanism against microbial challenges.
Assuntos
Sistema ABO de Grupos Sanguíneos/genética , Alelos , Glicosiltransferases/genética , Nippostrongylus/fisiologia , Animais , Sequência de Bases , Sequência de Carboidratos , DNA Complementar , Cromatografia Gasosa-Espectrometria de Massas , Glicosiltransferases/química , Dados de Sequência Molecular , Nippostrongylus/enzimologia , Nippostrongylus/genética , Ratos , Ratos Sprague-DawleyRESUMO
Migration of L3 larvae of Nippostrongylus brasiliensis through the lungs of the rat, during primary infection, was studied at 24 h, 72 h and 8 days. At 24 h p.i., there was evidence of damage to lung epithelial cells and microvasculature, with increased protein and gamma-glutamyl transpeptidase in the bronchoalveolar lavage (BAL) fluid. However, there was little evidence of inflammatory cell recruitment. At 24 h p.i., there was a significant reduction in the inflammatory cytokine tumour necrosis factor alpha. Superoxide (O2-*) production was also reduced, accompanied by an increase in superoxide dismutase activity. Lipid peroxidation was reduced at 24 h p.i. and L3 larvae were shown to possess high levels of glutathione compared to host lung tissue. Nitric oxide, detected as nitrite, was produced in BAL fluid, and inducible nitric oxide synthase protein was increased by 72 h p.i. There was evidence of peroxynitrite production throughout the infection period with specific protein bands nitrosylated at 75, 30 and 25 kDa. It appears that despite early evidence of lung damage, the inflammation was reduced in response to L3 larvae of N. brasiliensis.
Assuntos
Nippostrongylus/patogenicidade , Óxido Nítrico/metabolismo , Estresse Oxidativo , Pneumonia/fisiopatologia , Infecções por Strongylida/fisiopatologia , Animais , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Feminino , Pulmão/parasitologia , Pulmão/patologia , Masculino , Nippostrongylus/crescimento & desenvolvimento , Nippostrongylus/fisiologia , Pneumonia/imunologia , Pneumonia/parasitologia , Pneumonia/patologia , Ratos , Ratos Wistar , Infecções por Strongylida/imunologia , Infecções por Strongylida/parasitologia , Infecções por Strongylida/patologiaRESUMO
The glycosylation alterations of mouse small intestinal mucins during a 12-day infectious cycle caused by the parasite Nippostrongylus brasiliensis have been studied. The guanidinium chloride insoluble mucins were isolated at day 0 to 12 from the small intestine of infected and non-infected C57BL/6 mice. The O-linked oligosaccharides were released by reductive beta-elimination from the mucins and separated into neutral, sialylated and sulfated fractions. All fractions were analyzed by monosaccharide composition analysis and the neutral oligosaccharides were structurally characterized by gas chromatography/mass spectrometry. Two oligosaccharides containing blood group H-type epitopes (Fucalpha1-2Gal-) were transiently expressed with a maximum at day 6. Additional oligosaccharides with the common structure HexNAc-Gal-3GalNAcol were transiently induced with a maximum at day 10. Northern blot analysis on total RNA showed a transient expression at day 4-6 of the Fut2 gene encoding a Fucalpha1-2 fucosyltransferase, probably responsible for the detected blood group H-type epitopes. Comparisons with the corresponding infection in rat studied previously, revealed structurally different alterations, although occurring as transient events in both species. Both showed an induced blood group-type transferase halfway through the infection (a blood group A transferase in rat) and an induced transferase adding a terminal GalNAc (to a sialic acid- containing epitope in rat) towards the end of the infection. These differences between closely related species suggest rapid evolutionary alterations in glycosyltransferase expression.
Assuntos
Mucosa Intestinal/metabolismo , Intestinos/parasitologia , Mucinas/metabolismo , Nippostrongylus/fisiologia , Infecções por Strongylida/metabolismo , Animais , Sequência de Carboidratos , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Cromatografia Gasosa-Espectrometria de Massas , Glicosilação , Camundongos , Dados de Sequência Molecular , Mucinas/química , Ácido N-Acetilneuramínico/metabolismo , Oligossacarídeos/química , Oligossacarídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Infecções por Strongylida/parasitologia , Sulfatos/metabolismoRESUMO
The gastrointestinal nematode Nippostrongylus brasiliensis is thought to feed on host ingesta, and it is generally thought that the presence of ingesta determines the distribution of this parasite within the host intestine. However, these assertions have not been supported by direct evidence. The purpose of this study was to test the hypothesis that N. brasiliensis worms are preferentially found in regions of the host small intestine containing ingesta. The relationship between worm and ingesta distribution was investigated using mice infected with N. brasiliensis and killed on day 8 postinfection at 0130, 0730, 1330, or 1930 hr. There was an inverse relationship between worm and ingesta distributions, and the worms were distributed significantly more anteriad in the intestine than host ingesta, at all times during the 24 hr. To determine what the worms fed on, host ingesta, tissue, and blood were differentially labeled with the fluorescent dyes rhodamine B and Fluoresbrite. The results of this study suggest that N. brasiliensis feeds on the host's intestinal wall, and that habitat distribution of this parasite within the small intestine is not directly related to the presence of luminal ingesta.
Assuntos
Conteúdo Gastrointestinal/parasitologia , Intestino Delgado/parasitologia , Nippostrongylus/fisiologia , Animais , Ingestão de Alimentos , Masculino , Camundongos , Nippostrongylus/isolamento & purificaçãoRESUMO
We and others have previously shown that nematodes or nematode products can stimulate or inhibit the generation of lymphocyte responses, suggesting that nematodes exert diverse effects on the developing immune responses of their host. In this study we examined the immunomodulatory effect of a soluble extract of Nippostrongylus brasiliensis (adult worm homogenate [AWH]) on B-cell responsiveness. We found that the extract inhibited the proliferation of B cells to lipopolysaccharide (LPS) stimulation in a dose-dependent manner. This effect was specific to B cells, since the extract did not inhibit T-cell proliferation to concanavalin A or anti-CD3 stimulation. The data presented here confirm that the extract is not toxic to B cells. We present evidence that the active factor is proteinaceous in nature and that the inhibitory activity is restricted to the adult stage of Nb. The extract does not appear to interfere with early activation events since it can be added up to 48 h after LPS stimulation, and it inhibited responses to phorbol myristate acetate and ionomycin. Furthermore, the proliferation of B cells to other activators was also inhibited by AWH. This observation shows that the inhibitory activity of AWH is not restricted to LPS-mediated B-cell proliferation. We present evidence that, in the absence of accessory cells, the inhibitory effect of the extract was ablated. This observation shows that the activity of AWH is not mediated directly on B cells but is mediated via the production of negative signals from accessory cells (macrophages), which affect a downstream pathway required by all B-cell activators tested. These effects on B-cell and accessory cell function are likely to have a significant effect on the outcome of infections experienced concurrently.