RESUMO
SCOPE: In the attempt to develop new therapeutic treatments for colitis, fractions containing phenolic compound isolate (Phi) and phenolic reduced-flaxseed protein hydrolysate (phr-FPH) from flaxseed are evaluated for their effects on the in vitro production of pro-inflammatory mediators and on the course of experimental colitis. METHODS AND RESULTS: The anti-inflammatory effects of Phi and phr-FPH from flaxseeds are studied in RAW264.7 cells and in trinitrobenzene sulphonic acid (TNBS) colitis model. It is observed that the incubation with Phi or phr-FPH result in lower levels of tumor necrosis factor α and nitric oxide in macrophages stimulated with bacterial lipopolysaccharide + interferon-γ. Prophylactic and therapeutic treatments with Phi and phr-FPH, respectively, greatly contribute to the prevention of weight loss and colon inflammation in colitic BALB/c mice. T cell proliferation, expansion of TH1 and TH17 cells, and pro-inflammatory cytokines are lower, whereas Treg cells are higher in spleen cell cultures from Phi-treated mice. In addition, therapeutic phr-FPH treatment is able to reduce the expansion of TH17 in splenic cell cultures. CONCLUSION: The consumption of phenolic and protein compounds extracted from flaxseeds has a protective effect on TNBS-induced colitis, and may be useful in the control of other inflammatory disorders.
Assuntos
Colite/tratamento farmacológico , Linho/química , Hidrolisados de Proteína/farmacologia , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Colite/induzido quimicamente , Colite/etiologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Óxido Nítrico/metabolismo , Fenóis/farmacologia , Células RAW 264.7 , Células Th17/efeitos dos fármacos , Ácido Trinitrobenzenossulfônico/toxicidadeRESUMO
INTRODUCTION: In addition to conventional therapies, several new strategies have been proposed for modulating autoimmune diseases, including the adoptive transfer of immunological cells. In this context, dendritic cells (DCs) appear to be one of the most promising treatments for autoimmune disorders. The present study aimed to evaluate the effects of adoptive transfer of DCs obtained from both naïve and ovalbumin (OVA)-tolerant mice on the severity of TNBS induced colitis and analyze the eventual protective mechanisms. METHODS AND RESULTS: To induce oral tolerance, BALB/c mice were fed 4mg/mL OVA solution for seven consecutive days. Spleen DCs were isolated from tolerant (tDC) and naïve (nDC) mice, and then adoptively transferred to syngeneic mice. Three days later, colitis was induced in DC treated mice by intrarectal instillation of 100µg2,4,6-trinitrobenzenesulfonic acid (TNBS) dissolved in 50% ethanol. Control subjects received only intrarectal instillation of either TNBS solution or a vehicle. Five days later, mice from all groups were euthanized and examined for physiological and immunological parameters. Regarding the phenotype, we observed that the frequencies of CD11+ MHC II+ and CD11+ MHCII+ CD86+ cells were significantly lower in DCs isolated from tolerant mice than in those from naive mice. However, pretreatment with both types of DCs was able to significantly reduce clinical signs of colitis such as diarrhea, rectal prolapse, bleeding, and cachexia, although only treatment with tDCs was able to prevent weight loss from instillation of TNBS. In vitro proliferation of spleen cells from mice treated with either type of DCs was significantly lower than that observed in splenic cell cultures of naïve mice. Although no significant difference was observed in the frequencies of Treg cells in the experimental groups, the frequency of Th17+CD4+cellsand the secretion of IL-17 were more reduced in the cultures of spleen cells from mice treated with either type of DCs. The levels of IL-9 and IFN-γ were lower in supernatants of cells from mice treated with nDCs. CONCLUSION: The results allow us to conclude that the adoptive transfer of cells expressing CD11c is able to reduce the clinical and immunological signs of drug-induced colitis. Adoptive transfer of CD11c+DC isolated from both naive and tolerant mice altered the proliferative and T cell responses. To the best of our knowledge, there is no previously published data showing the protective effects of DCs from naïve or tolerant mice in the treatment of colitis.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Colite/terapia , Células Dendríticas/transplante , Tolerância Imunológica , Transferência Adotiva/métodos , Animais , Antígeno B7-2/imunologia , Antígeno CD11c/imunologia , Colite/induzido quimicamente , Colite/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Humanos , Camundongos , Ovalbumina/efeitos adversos , Ovalbumina/imunologia , Baço/citologia , Baço/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Ácido Trinitrobenzenossulfônico/toxicidadeRESUMO
INTRODUCTION: Literature data have shown that the consumption of dietary proteins may cause modulatory effects on the host immune system, process denominated oral tolerance by bystander suppression. It has been shown that the bystander suppression induced by dietary proteins can improve inflammatory diseases such as experimental arthritis. Here, we evaluated the effects of oral tolerance induced by ingestion of ovalbumin (OVA) on TNBS-induced colitis in mice, an experimental model for human Crohn's disease. METHODS AND RESULTS: Colitis was induced in BALB/c mice by instilling a single dose of TNBS (100 mg/kg) in ethanol into the colon. Tolerized mice received OVA (4mg/mL) dissolved in the drinking water for seven consecutive days, prior to or concomitantly with the intrarectal instillation. Control groups received protein-free water and ethanol by intrarectal route. We observed that either the prior or concomitant induction of oral tolerance were able to reduce the severity of colitis as noted by recovery of body weight gain, improvement of clinical signs and reduction of histological abnormalities. The in vitro proliferation of spleen cells from tolerant colitic mice was lower than that of control mice, the same as the frequencies of CD4+ T cells secreting IL-17 and IFN-γ. The frequencies of regulatory T cells and T cells secreting IL-10 have increased significantly in mice orally treated with OVA. The levels of inflammatory cytokines (IL-17A, TNF-α, IL-6 and IFN-γ) were lower in supernatants of cells from tolerant colitic mice, whereas IL-10 levels were higher. CONCLUSION: Our data show that the modulation of immune response induced by oral tolerance reduces the severity of experimental colitis. Such modulation may be partially attributed to the increase of Treg cells and reduction of pro-inflammatory cytokines in peripheral lymphoid organs of tolerant mice by bystander suppression.
Assuntos
Efeito Espectador/imunologia , Colite/imunologia , Tolerância Imunológica/imunologia , Ovalbumina/imunologia , Linfócitos T Reguladores/imunologia , Animais , Colite/induzido quimicamente , Feminino , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-17/imunologia , Interleucina-17/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Ovalbumina/farmacologia , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
High-fat diets are used to induce adverse alterations in the intestinal microbiota, or dysbiosis, generalized inflammation and metabolic stress, which ultimately may lead to obesity. The influence of dietary whey proteins, whether intact or hydrolyzed, has been reported to improve glucose homeostasis and reduce stress. Therefore, the purpose of this work was to test if dietary milk-whey proteins, both in the intact form and hydrolyzed, could have an effect on the compositional changes of the cecal microbiota that can be induced in mice when receiving a high-fat diet in combination with the standard casein. Male C57BL/6 mice were fed a control casein diet (AIN 93-G); high-fat-casein (HFCAS); high-fat-whey protein concentrate (HFWPC) and high-fat whey-protein hydrolysate (HFWPH) for 9weeks. The intestinal microbiota composition was analyzed by 16S-rRNA of the invariant (V1-V3) gene, potentially endotoxemic lipopolysaccharide (LPS) release was determined colorimetrically, and liver fat infiltration assessed by light microscopy. The high-fat diet proved to induce dysbiosis in the animals by inverting the dominance of the phylum Firmicutes over Bacteroidetes, promoted the increase of LPS and resulted in liver fat infiltration. The whey proteins, whether intact or hydrolyzed, resisted the installation of dysbiosis, prevented the surge of circulating LPS and prevented fat infiltration in the liver. It is concluded that dietary whey proteins exert metabolic actions that tend to preserve the normal microbiota profile, while mitigating liver fat deposition in mice consuming a high-fat diet for nine weeks. Such beneficial effects were not seen when casein was the dietary protein. The hydrolyzed whey protein still differed from the normal whey protein by selectively protecting the Bacteroidetes phylum.
RESUMO
uNK cells differ from cNK cells, as they produce angiogenic molecules critical for normal implantation site development. We evaluated heterogeneity among DBA(+)uNK cells for Prf, Gzma, and Vegfa. Ctsd and Srgn expression was used to assign intracellular sorting of these molecules on gd7, -9, and -14. Vegfa was present in small, granule-free DBA(+)uNK cells at gd7 and in large, granule-rich DBA(+)uNK cells at gd9 and -14. Prf and Gzma were only found in granulated DBA(+)uNK cells (gd9 and -14). All granule-rich Prf(+)DBA(+)uNK cells appeared to coexpress Vegfa. Thus, all DBA(+)uNK cells were Vegfa-producing cells. PC analysis and immunogold ultrastructure confirmed colocalization of Prf/Ctsd in secretory-lysosome granules (PC>0.5). Surprisingly, Gzma and Prf(+)Ctsd(+) were not colocalized (PC<0.5). Rather, Gzma colocalized with Srgn (PC>0.5) in small granules in cells with Vegfa expression (PC<0.5). NK1.1(+)sNK cells and DBA(+)uNK cells expressed genes regulating vesicular traffic (rab11, rab27a, snap23, vamp7), but uNK cells also expressed rab34 and vamp8, molecules associated with constitutive secretion. SEE activated the regulated secretory pathway of DBA(+)uNK cells in vivo, mobilizing Prf and Gzma but not Vegfa. Thus, DBA(+)uNK cells display constitutive and regulated secretion. Further, these results demonstrate that granule-free DBA(+)uNK cells are not quiescent immature cells, but they are cells with potentially significant angiogenic roles before and in addition to their initiation of spiral arterial remodeling.
Assuntos
Biomarcadores/metabolismo , Grânulos Citoplasmáticos , Células Matadoras Naturais/classificação , Células Matadoras Naturais/citologia , Útero/citologia , Animais , Feminino , Técnicas Imunoenzimáticas , Células Matadoras Naturais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Lectinas de Plantas , Gravidez , Útero/metabolismoRESUMO
Remodeling and relaxation of the mouse pubic symphysis (PS) are central events in parturition. The involvement of endogenous proteins such as matrix metalloproteinases (MMPs), tissue inhibitors of matrix metalloproteinases (TIMPs), and cathepsins in these phenomena remains unclear. In this work, we used a combination of immunolocalization, protein expression/activity, and relative messenger RNA (mRNA) expression to examine the changes in selected MMPs (-2, -9, and -8), TIMPs (-1 and -2), and cathepsins (B and K) during pregnancy and postpartum in mice. Immunohistochemistry revealed the presence of all of these proteins in the cytoplasm of chondrocytes, fibrochondrocytes, and fibroblast-like cells in the interpubic tissues. Zymography showed increases in the active forms of MMP-2 and -9 primarily on days 15 to 19 of pregnancy. Western blotting showed enhanced expression of MMP-8 on days 12 to 15 of pregnancy, with no changes in cathepsins B and K. Matrix metalloproteinases 2, TIMP-1 and -2, and cathepsin B had significant relative gene expression throughout pregnancy. These findings indicate that during pregnancy and postpartum there are variations in the expression and activity of proteins that may have an important role in remodeling the pubic symphysis during these events.
Assuntos
Catepsinas/metabolismo , Metaloproteinases da Matriz/metabolismo , Período Pós-Parto/metabolismo , Prenhez/metabolismo , Sínfise Pubiana/metabolismo , Inibidores Teciduais de Metaloproteinases/metabolismo , Animais , Catepsinas/genética , DNA/química , DNA/genética , Feminino , Metaloproteinases da Matriz/genética , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Reação em Cadeia da Polimerase , Gravidez , Sínfise Pubiana/enzimologia , Sínfise Pubiana/ultraestrutura , Inibidores Teciduais de Metaloproteinases/genéticaRESUMO
Os aspectos morfofuncionais da oogênese do Poecilia vivipara foram estudados nesse trabalho. Esse estudo contribuiu para o aprimoramento das informações sobre a biologia reprodutiva e do desenvolvimento de espécies com fecundação interna, em especial aquelas pertencentes à família Poecilidae. Para tanto, caracterizou-se os estágios de maturação gonadal e desenvolvimento folicular através de análises mesoscópicas, histológicas, histoquímicas e citoquímicas com lectinas. O estudo mesoscópico permitiu a classificação do desenvolvimento ovariano em seis estágios: imaturo, em maturação I, em maturação II, maturo I, maturo II e pós-desova. O exame microscópico dos ovários permitiu a identificação dos oócitos pré-vitelogênicos (OPVt) e vitelogênicos (OVt). Na fase pré-vitelogênese, verificou-se os oócitos tipo I (OI) e tipo II (OII), e na fase vitelogênese, foram encontrados oócitos tipo III (OIII), tipo IV (OIV) e os pós-fertilização (OV). Com o avanço da oogênese, o citosol dos oócitos aumentaram em volume, com crescente acúmulo de grânulos citoplasmáticos, caracterizando a vitelogênese. Além disso, a zona radiata (ZR) aumentou de espessura e alterou suas características histocitoquímicas, e o epitélio folicular (EF), inicialmente delgado e constituído por células pavimentosas, nos FOs na fase III tornaram-se cúbico simples. As análises histoquímicas e citoquímicas permitiram diagnosticar alterações na composição molecular das estruturas que formam os FOs ao longo do desenvolvimento gonadal. O presente estudo indica diferenças no sistema reprodutor feminino entre espécies de peixes com fecundação interna e aquelas com fecundação externa e sugere que o P. vivipara pode ser usado como modelo experimental em testes de toxicidade ambiental.
The morphofunctional aspects of oogenesis of Poecilia vivipara were studied aiming to understand the reproductive biology and development of species with internal fertilization, particularly those belonging to the family Poeciliidae. The stages of gonadal maturation and follicular development were characterized using mesoscopic, histological, histochemical, and lectin cytochemical analyses. Through mesoscopic evaluation the ovarian development was classified in six phases of development: immature, in maturation I, in maturation II, mature I, mature II, and post-spawn. Based on microscopic examination of the ovaries, we identified the presence of oocytes types I and II during the previtellogenic phase and types III, IV, and V during the vitellogenic phase. As oogenesis proceeded the oocyte cytosol increased in volume and presented increased cytoplasmic granule accumulation, characterizing vitellogenesis. The zona radiata (ZR) increased in thickness and complexity, and the follicular epithelium, which was initially thin and consisting of pavimentous cells, in type III oocytes exhibited cubic simple cells. The histochemical and cytochemical analyses revealed alterations in the composition of the molecular structures that form the ovarian follicle throughout the gonadal development. Our study demonstrated differences in the female reproductive system among fish species with internal and external fertilization and we suggest P. vivipara can be used as experimental model to test environmental toxicity.
Assuntos
Animais , Glicoproteínas/análise , Poecilia/classificação , Fertilização/fisiologia , Reprodução/fisiologiaRESUMO
Uterine natural killer (uNK) cells expand rapidly during endometrial decidualization and account for 70% of leukocytes in early gestational uteri of humans and rodents. These cells make unique contributions to pregnancy, contributing to the success of embryo implantation and maintenance of decidual tissue that supports placental and fetal development. We postulated that uNK cells express molecules that are not shared by circulating NK (cNK) cells or other leukocytes and, therefore, would be immunogenic for male mice. We isolated viable uNK cells from gestation day 9 pregnant mice and inoculated them into syngeneic males. This induced antibodies reactive with mouse uNK cells but not with cNK cells or other lymphocytes. The antibodies reacted identically with uNK cells in tissue sections from five different mice strains from gestational day 7-12 and in pregnant rat uterus, suggesting that the recognized antigen should be a specific marker of uNK cell. Spleen cells from inoculated males were used subsequently to produce a monoclonal antibody reactive to a uNK cell surface antigen. These experiments confirm that uNK cells are a pregnancy-specific subset of NK cells expressing distinct surface antigen from those found in other tissues.
Assuntos
Células Matadoras Naturais/imunologia , Útero/imunologia , Animais , Anticorpos/análise , Antígeno CD56/análise , Feminino , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos , Gravidez , Ratos , Ratos WistarRESUMO
The hypoxia-inducible factor-1alpha (HIF-1alpha) is expressed in response to hypoxia and has been recently demonstrated in a variety of cells such as tumor cells and tumor-associated macrophages. Several characteristics of leishmanial lesions in humans and in animal models, such as microcirculation impairment, metabolic demand for leukocyte infiltration into infected tissue, parasite proliferation, and secondary bacterial infection, are strong indications of a hypoxic microenvironment in the lesions. We evaluated HIF-1alpha expression in the cutaneous lesions of BALB/c mice during Leishmania amazonensis infection. Immunohistochemical analyses of the lesions demonstrated, only in the later stages of infection when the lesion size is maximal and parasite burden is enormous and massive numbers of recruited macrophages and ulcers are observed, positive HIF-1alpha-infected cells throughout the lesions. HIF-1alpha is expressed mainly in the cytoplasm and around parasites inside the parasitophorous vacuoles of macrophages. This is the first evidence that macrophages in the microenvironment of lesions caused by a parasite produce a hypoxia-inducible factor.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Leishmania braziliensis/fisiologia , Leishmaniose Cutânea/metabolismo , Proteínas Nucleares/metabolismo , Pele/metabolismo , Fatores de Transcrição/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Pé/parasitologia , Pé/patologia , Membro Posterior , Fator 1 Induzível por Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Técnicas Imunoenzimáticas , Leishmania braziliensis/patogenicidade , Leishmaniose Cutânea/parasitologia , Leishmaniose Cutânea/patologia , Camundongos , Camundongos Endogâmicos BALB C , Pele/parasitologia , Pele/patologiaRESUMO
Spermatozoa in testicular fluid are known to have weak forward motility and cannot fertilize eggs. The epididymis is known to participate in sperm maturation leading fertilization, but little is known about the specific epididymal molecules involved in the modification of sperm. In this study, we characterized the new pattern of expression of an antigen previously identified in testicular germ cells by monoclonal antibody (mAb) TRA 54. This antigen is expressed in epididymal and vas deferens epithelial cells in mice older than 24 days but not during younger developmental stages. Evaluation by immunohistochemistry shows that antigen expression is limited to the cytoplasm of a specific cell population of epithelia along the epididymal regions and vas deferens of adult mice. The molecules synthesized and released by epididymal and vas deferens epithelia into their lumen seem to bind on spermatozoa moving down through the ducts. Immunoblot analysis showed that the molecules recognized by mAb TRA 54 in testis and epididymis were similar and share a common epitope involving carbohydrate domains. Interestingly, the antigens identified in epididymal and vas deferens epithelial cells were expressed independently of testicular germ cells and are produced in an androgen-dependent manner. Finally, the molecules recognized by mAb TRA 54 seem to play an important role in spermatogenesis, as well as in epididymal function related to spermatozoa maturation and ability to fertilize.
Assuntos
Anticorpos Monoclonais , Antígenos/metabolismo , Epididimo/imunologia , Ducto Deferente/imunologia , Androgênios/fisiologia , Animais , Western Blotting , Criptorquidismo/imunologia , Epididimo/efeitos dos fármacos , Imuno-Histoquímica/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Orquiectomia , Coloração e Rotulagem , Testosterona/farmacologia , Ducto Deferente/efeitos dos fármacosRESUMO
In the present work, in vitro assays were used to investigate the toxicity of Serratia marcescens cytotoxin in cultured Chinese hamster ovary (CHO) cells. The time necessary to detect cellular alterations such as the onset of apoptosis, the perturbation of mitochondrial function, and cytoskeletal changes was assessed. The internalization of the cytotoxin by CHO cells was also examined. Within 10-15 min of exposure to cytotoxin, CHO cells became round, the nucleus shrank, the chromatin became more compact, and cytoplasmic blebs appeared on the cell surface. TUNEL (TdT-mediated dUTP nick end labeling) and propidium iodide staining identified some nuclei with fragmented DNA, and electrophoresis of CHO cell DNA obtained after 30-min exposure to S. marcescens toxin showed a pattern of DNA fragments typically associated with apoptosis. The cells also lost their characteristic actin organization within 10 min of exposure to cytotoxin. Lactate dehydrogenase leakage was detected after 20-min exposure to the cytotoxin and increased with time thereafter. Concomitantly, there was a time-dependent reduction in mitochondrial activity. Fluorescein-labeled S. marcescens cytotoxin was detected only on the surface of CHO cells, even after 30-min exposure to the toxin. These results show that there was no internalization of the toxin by CHO cells, and that, once bound to the cell surface, the toxin was able to induce changes in intracellular metabolism and to trigger cell death by apoptosis.
Assuntos
Apoptose , Toxinas Bacterianas/toxicidade , Tamanho Celular , Citotoxinas/toxicidade , Serratia marcescens/metabolismo , Animais , Toxinas Bacterianas/metabolismo , Células CHO , Linhagem Celular , Núcleo Celular/ultraestrutura , Cromatina/ultraestrutura , Cricetinae , Citoplasma/ultraestrutura , Citoesqueleto/ultraestrutura , Citotoxinas/metabolismo , Fragmentação do DNA , Formazans/metabolismo , Marcação In Situ das Extremidades Cortadas , L-Lactato Desidrogenase/metabolismo , Mitocôndrias/metabolismo , Propídio/metabolismo , Ligação Proteica , Sais de Tetrazólio/metabolismoRESUMO
Intrauterine and early postnatal malnutrition has profound consequences on fetal and postnatal development in both humans and animals. In addition, low birth weight has been reported to be associated with impaired insulin secretion, insulin resistance and diminished area of pancreatic islets. Because the transcription factor pancreatic and duodenal homeobox 1 (PDX-1) is important for the maintenance of B-cell physiology, PDX-1 expression and islet area were assessed in neonatal rats of dams fed low (6%) or normal (17%) protein diets during pregnancy. PDX-1 protein and mRNA levels, as well as insulin secretion and islet area, were measured after 28 d of life in normal, low protein and recovered rats whose dams consumed a normal protein diet after delivery. Insulin secretion by isolated islets in response to 2.8 and 16.7 mmol glucose/L was reduced in 28-d-old low protein rats compared with the control (P < 0.05). At birth and after 28 d of life, the islet area and PDX-1 protein expression were also reduced (P < 0.05). In contrast, PDX-1 mRNA levels in islets from 28-d-old low protein rats were not different from control rats. PDX-1 protein expression in pancreatic islets, the area of islets and insulin secretion were restored in recovered rats, whereas PDX-1 mRNA levels were higher than in normal rats (P < 0.05). These results suggest a link among diminished PDX-1 protein expression, a reduction in islet area and impaired insulin secretion in low protein rats. The reintroduction of a normal diet early in life restored islet area and cell physiology.
Assuntos
Dieta com Restrição de Proteínas , Proteínas de Homeodomínio , Ilhotas Pancreáticas/metabolismo , Efeitos Tardios da Exposição Pré-Natal , Deficiência de Proteína/metabolismo , Transativadores/genética , Animais , Animais Recém-Nascidos , Proteínas Alimentares/administração & dosagem , Feminino , Feto/metabolismo , Imuno-Histoquímica , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/fisiologia , Lactação , Gravidez , Distribuição Aleatória , Ratos , Ratos Wistar , Transativadores/metabolismoRESUMO
A cell-associated mannose-resistant hemagglutinating factor (HAF) was extracted from enteroinvasive Escherichia coli (EIEC) serotype O124:H- by sonication. Ultrastructural analysis of EIEC and immunocytochemical assays with the cell-free HAF and EIEC bacterial cells on HeLa cells, suggested that the HAF is a non-fimbrial putative adhesive factor that mediates in vivo adherence of EIEC to human epithelial cells.
Assuntos
Aderência Bacteriana/fisiologia , Escherichia coli/fisiologia , Hemaglutininas/fisiologia , Anticorpos Antibacterianos/metabolismo , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Infecções por Escherichia coli/microbiologia , Células HeLa , Testes de Hemaglutinação , Hemaglutininas/metabolismo , Hemaglutininas/ultraestrutura , Humanos , Microscopia Eletrônica , Microscopia de FluorescênciaRESUMO
One strain (S32) of Clostridium perfringens type A was isolated from a case of catarrhal enteritis of piglets. This strain was able to adhere to HeLa cells showing an adherence index (AI) of 25.15ñ1.26(mean ñ 1 standard error of the mean). Treatment of the bacterial cells with trypsin (025mg/ml) decreased in 70 (per cent)-80 (per cent) the AI and metaperiodate (10mg/ml) abolished completely the adherence, suggesting that the structure responsible for this phenomenon was probably a glycoprotein. Heating of bacterial suspensions (100§C/5 min) before carrying out the adhesion test decreased the AI rendering it equal to the negative controls. Rabbit homologous S32 antiserum inhibited the adherence up to dilutions of 1:640, at least. The piglet ileal loop assay carried out with strains S32 and Jab-1 (negative control) demonstrated that the strain S32 was able to adhere to the intestinal epithelial cells when examined after Gram staining. Transmission electron microscopy (TEM) demonstrated that S32 strain displayed a loose fibrillar material not seen with Jab-1. Stabilization of the bacterial cells with homologous antiserum of strain S32, followed by staining with rhutenium red, revealed loose long fibrillar material on the outer surface of the cells, that sometimes could be seen spreading out from the cells and linking bacterial cells. The question whether this structure might be an adhesin for this strain of Cl. perfringes type A, perhaps playing a role in the pathogenesis of the catarrhal enteritis of piglets, is dependent on further studies.