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1.
J Cell Sci ; 136(2)2023 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-36620952

RESUMO

SART3 is a multifunctional protein that acts in several steps of gene expression, including assembly and recycling of the spliceosomal U4/U6 small nuclear ribonucleoprotein particle (snRNP). In this work, we provide evidence that SART3 associates via its N-terminal HAT domain with the 12S U2 snRNP. Further analysis showed that SART3 associates with the post-splicing complex containing U2 and U5 snRNP components. In addition, we observed an interaction between SART3 and the RNA helicase DHX15, which disassembles post-splicing complexes. Based on our data, we propose a model that SART3 associates via its N-terminal HAT domain with the post-splicing complex, where it interacts with U6 snRNA to protect it and to initiate U6 snRNA recycling before a next round of splicing.


Assuntos
Splicing de RNA , Spliceossomos , Splicing de RNA/genética , Spliceossomos/genética , Spliceossomos/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6/genética , Ribonucleoproteína Nuclear Pequena U4-U6/metabolismo , Ribonucleoproteína Nuclear Pequena U5/genética , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Ribonucleoproteínas Nucleares Pequenas/genética , Ribonucleoproteínas Nucleares Pequenas/metabolismo
2.
Nucleic Acids Res ; 46(7): 3774-3790, 2018 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-29415178

RESUMO

Cajal bodies (CBs) are nuclear non-membrane bound organelles where small nuclear ribonucleoprotein particles (snRNPs) undergo their final maturation and quality control before they are released to the nucleoplasm. However, the molecular mechanism how immature snRNPs are targeted and retained in CBs has yet to be described. Here, we microinjected and expressed various snRNA deletion mutants as well as chimeric 7SK, Alu or bacterial SRP non-coding RNAs and provide evidence that Sm and SMN binding sites are necessary and sufficient for CB localization of snRNAs. We further show that Sm proteins, and specifically their GR-rich domains, are important for accumulating snRNPs in CBs. Accordingly, core snRNPs containing the Sm proteins, but not naked snRNAs, restore the formation of CBs after their depletion. Finally, we show that immature but not fully assembled snRNPs are able to induce CB formation and that microinjection of an excess of U2 snRNP-specific proteins, which promotes U2 snRNP maturation, chases U2 snRNA from CBs. We propose that the accessibility of the Sm ring represents the molecular basis for the quality control of the final maturation of snRNPs and the sequestration of immature particles in CBs.


Assuntos
Núcleo Celular/genética , RNA Nuclear Pequeno/genética , Ribonucleoproteína Nuclear Pequena U2/genética , Spliceossomos/genética , Corpos Enovelados/genética , Corpos Enovelados/metabolismo , Regulação da Expressão Gênica/genética , Células HeLa , Humanos
3.
Gastroenterology ; 144(2): 381-391, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23142137

RESUMO

BACKGROUND & AIMS: The Wnt signaling pathway is required for maintenance of the intestinal epithelia; blocking this pathway reduces the proliferative capacity of the intestinal stem cells. However, aberrant Wnt signaling leads to intestinal cancer. We investigated the roles of the Wnt pathway in homeostasis of the intestinal epithelium and during malignant transformation in human cells and mice. METHODS: We performed chromatin immunoprecipitation (ChIP) with DNA microarray analysis (ChIP-on-chip) to identify genes regulated by Wnt signaling in human colorectal cancer cells Colo320, DLD1, LS174T, and SW480. Formation of intestinal tumor was induced in C57BL/6J mice using azoxymethane and dextran sulfate. Intestinal tissues from these mice, as well as Apc(+/Min) and Apc(CKO/CKO)/Lgr5-EGFP-IRES-CreERT2 mice, were analyzed by immunohistochemistry and in situ hybridization. RESULTS: We identified promoter regions of 960 genes that interacted with the Wnt pathway nuclear effector T-cell factor 4 in 4 different human colorectal cancer-derived cell lines; 18 of these promoters were present in all chromatin precipitates. Wnt signaling up-regulated a member of the tumor necrosis factor receptor superfamily called TROY. Levels of TROY messenger RNA were increased in human cells with deficiencies in the adenomatous polyposis coli (APC) gene and in cells stimulated with the Wnt3a ligand. Expression of Troy was significantly up-regulated in neoplastic tissues from mice during intestinal tumorigenesis. Lineage tracing experiments revealed that Troy is produced specifically by fast-cycling intestinal stem cells. TROY associated with a unique marker of these cells, leucine-rich repeat-containing G-protein coupled receptor (LGR) 5. In organoids established from the intestinal crypts, Troy suppressed signaling mediated by R-spondin, a Wnt agonist. CONCLUSIONS: TROY is up-regulated in human colorectal cancer cell lines and in intestinal tumors in mice. It functions as a negative modulator of the Wnt pathway in LGR5-positive stem cells.


Assuntos
Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Mucosa Intestinal/metabolismo , Células-Tronco Neoplásicas/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Receptores do Fator de Necrose Tumoral/fisiologia , Via de Sinalização Wnt/fisiologia , Animais , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Humanos , Imuno-Histoquímica , Hibridização In Situ , Mucosa Intestinal/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias Experimentais , Células-Tronco Neoplásicas/patologia , Análise de Sequência com Séries de Oligonucleotídeos , RNA Neoplásico/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
4.
J Clin Immunol ; 32(6): 1372-80, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22777159

RESUMO

PURPOSE: Human colostrum and milk provide a newborn with immunomodulatory components, ensuring protection and proper development of the immune system. Secretory IgA antibodies in colostrum represent the first line of defence against harmful substances, but their potential spectra of reactivity with autoantigens remains unclear. Here, we characterised the repertoire of natural sectretory IgA autoantibodies in colostrum of healthy mothers. METHODS: The human colostrum samples from 39 healthy mothers were analyzed for autoantibodies by indirect immunofluorescence, dot blots, immunoblots and ELISA. RESULTS: We found that there is high diversity in reactivities of colostral IgA antibodies to autoantigens among individual samples. Using tissue sections and biochips commonly used for autoimmunity testing, we found that most samples reacted with monkey ovary (79.3%), monkey pancreatic tissue (78.6%), human HEp-2 cells (69%) and monkey adrenal gland (69.0%), fewer samples reacted with monkey liver tissue (47.2%), rat stomach (42.9%), monkey testicular tissue (41.4%), monkey salivary gland (39.3%), rat kidney (32.1%) and monkey cerebellar tissue (17.9%). At the protein level, we detected reactivity of IgA with 21 out of 25 (auto) antigens. The majority of the samples reacted with the pyruvate dehydrogenase complex, E3 ubiquitin ligase, cytosolic liver antigen, promyelocytic leukemia protein and nuclear pore glycoprotein-210. Using ELISA, we found reactivity of colostral IgA antibodies against examined extractable nuclear antigens, double stranded DNA, phospholipids and neutrophil cytoplasm. CONCLUSIONS: The broad spectrum of polyreactive natural autoantibodies present in human colostrum may contribute to proper development of mucosal immune system of the breastfed infant.


Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Colostro/imunologia , Imunoglobulina A Secretora/imunologia , Adolescente , Adulto , Animais , Especificidade de Anticorpos , Autoanticorpos/metabolismo , Autoantígenos/metabolismo , Aleitamento Materno , Colostro/química , Ensaio de Imunoadsorção Enzimática , Feminino , Haplorrinos , Humanos , Imunoglobulina A Secretora/biossíntese , Imuno-Histoquímica , Lactente , Lactação/imunologia , Mães , Gravidez , Ligação Proteica , Proteínas/imunologia , Proteínas/metabolismo , Ratos
5.
BMC Gastroenterol ; 11: 47, 2011 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-21545711

RESUMO

BACKGROUND: Oral thiopurines are effective and widely used in treatment of inflammatory bowel disease (IBD) in humans, although their use is limited due the development of adverse events. Here, we examine the efficacy and toxicity of oral treatment with 6-tioguanine (6-TG) and azathioprine (AZA) in a murine model of IBD. METHODS: We induced acute or chronic colitis in BALB/c mice by one or four cycles of 3% dextran sulphate sodium (DSS), respectively. Mice were treated by daily gavages of various dosages of 6-tioguanine, azathioprine, or by phosphate buffered saline (PBS) starting the first day of DSS or after two cycles of DSS, respectively. We monitored the efficacy and toxicity by measuring the weight change and serum alanine aminotransferase (ALT) activity and by disease severity and histology, at the end of the experiment. Moreover, we measured cytokine production after colon fragment cultivation by enzyme-linked immunoabsorbent assay and numbers of apoptotic cells in the spleen by flow cytometry. RESULTS: 6-TG is effective in the treatment of acute DSS-induced colitis in a dose-dependent manner and 40 µg of 6-TG is significantly more effective in the treatment of acute colitis than both AZA and PBS. This effect is accompanied by decrease of IL-6 and IFN-γ production in colon. We did not observe histological abnormalities in liver samples from control (PBS) or 6-TG treated mice. However, liver samples from most mice treated with AZA showed mild, yet distinct signs of hepatotoxicity. In chronic colitis, all thiopurine derivatives improved colitis, 20 µg of 6-TG per dose was superior. High doses of 6-TG led to significant weight loss at the end of the therapy, but none of the thiopurine derivatives increased levels of serum ALT. Both thiopurine derivatives reduced the proportion of apoptotic T helper cells, but a high production of both IL-6 and TGF-ß was observed only in colon of AZA-treated mice. CONCLUSIONS: Use of 6-TG in the treatment of experimental colitis in mice appears superior to AZA administration and placebo. In contrast to 6-TG, the use of AZA resulted in histological liver abnormalities.


Assuntos
Azatioprina/toxicidade , Azatioprina/uso terapêutico , Colite/tratamento farmacológico , Colo/patologia , Tioguanina/toxicidade , Tioguanina/uso terapêutico , Doença Aguda , Alanina Transaminase/sangue , Análise de Variância , Animais , Apoptose , Doença Crônica , Colite/sangue , Colite/induzido quimicamente , Colite/patologia , Colo/metabolismo , Sulfato de Dextrana , Feminino , Interferon gama/metabolismo , Interleucina-6/metabolismo , Fígado/efeitos dos fármacos , Fígado/patologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Animais , Linfócitos T Auxiliares-Indutores , Fator de Crescimento Transformador beta/metabolismo , Redução de Peso/efeitos dos fármacos
6.
Nat Commun ; 12(1): 3646, 2021 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-34131137

RESUMO

U5 snRNP is a complex particle essential for RNA splicing. U5 snRNPs undergo intricate biogenesis that ensures that only a fully mature particle assembles into a splicing competent U4/U6•U5 tri-snRNP and enters the splicing reaction. During splicing, U5 snRNP is substantially rearranged and leaves as a U5/PRPF19 post-splicing particle, which requires re-generation before the next round of splicing. Here, we show that a previously uncharacterized protein TSSC4 is a component of U5 snRNP that promotes tri-snRNP formation. We provide evidence that TSSC4 associates with U5 snRNP chaperones, U5 snRNP and the U5/PRPF19 particle. Specifically, TSSC4 interacts with U5-specific proteins PRPF8, EFTUD2 and SNRNP200. We also identified TSSC4 domains critical for the interaction with U5 snRNP and the PRPF19 complex, as well as for TSSC4 function in tri-snRNP assembly. TSSC4 emerges as a specific chaperone that acts in U5 snRNP de novo biogenesis as well as post-splicing recycling.


Assuntos
Ribonucleoproteína Nuclear Pequena U5/química , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Spliceossomos/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Regulação para Baixo , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Fatores de Alongamento de Peptídeos , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Splicing de RNA , Fatores de Processamento de RNA/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão , Ribonucleoproteínas Nucleares Pequenas/química , Fatores de Transcrição , Proteínas Supressoras de Tumor/genética
7.
Inflamm Res ; 58(8): 503-12, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19271150

RESUMO

OBJECTIVE: It is an open question whether multifunctional galectin-3 can be a serum marker in inflammatory bowel disease. METHODS: Western blots and commercial ELISA detected and quantitated the lectin immunocytochemistry using double labeling localized it in tissue sections. RESULTS: Serum concentrations were significantly increased in specimen of patients with active and remission-stage ulcerative colitis and Crohn's disease, associated with emerging positivity of CD14(+) cells. CONCLUSION: Enhanced concentration of galectin-3 in serum reflects presence of disease and points to its involvement in the pathogenesis.


Assuntos
Galectina 3/sangue , Doenças Inflamatórias Intestinais/sangue , Animais , Biomarcadores , Western Blotting , Colite/induzido quimicamente , Colite Ulcerativa/sangue , Colo/metabolismo , Doença de Crohn/sangue , Sulfato de Dextrana , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Feminino , Fluoresceína-5-Isotiocianato , Corantes Fluorescentes , Humanos , Imuno-Histoquímica , Doenças Inflamatórias Intestinais/induzido quimicamente , Doenças Inflamatórias Intestinais/diagnóstico , Lectinas/metabolismo , Receptores de Lipopolissacarídeos/análise , Receptores de Lipopolissacarídeos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C
8.
J Histochem Cytochem ; 56(3): 267-74, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18040078

RESUMO

Dysregulation of innate and adaptive intestinal immune responses to bacterial microbiota is supposed to be involved in pathogenetic mechanisms of inflammatory bowel diseases (IBDs). We investigated expression of Toll-like receptor 2 (TLR2), TLR4, and their transmembrane coreceptor CD14 in biopsy samples from patients with IBD and in non-inflamed gut mucosa from controls. Small intestine and colon samples were obtained by colonoscopy from patients with Crohn's disease (CD), ulcerative colitis (UC), and controls. Immunohistochemical analysis of cryostat sections using polyclonal and monoclonal antibodies specific for TLR2, TLR4, and CD14 showed a significant increase in TLR2 expression in the terminal ileum of patients with inactive and active UC against controls. Significant upregulation of TLR4 expression relative to controls was found in the terminal ileum and rectum of UC patients in remission and in the terminal ileum of CD patients with active disease. CD14 expression was upregulated in the terminal ileum of CD patients in remission and with active disease, in the cecum of UC patients in remission and with active disease, and in rectum of UC patients with active disease. Hence, dysregulation of TLR2, TLR4, and CD14 expression in different parts of the intestinal mucosa may be crucial in IBD pathogenesis.


Assuntos
Colite Ulcerativa/metabolismo , Doença de Crohn/metabolismo , Receptores de Lipopolissacarídeos/biossíntese , Receptor 2 Toll-Like/biossíntese , Receptor 4 Toll-Like/biossíntese , Biópsia , Ceco/metabolismo , Ceco/patologia , Colite Ulcerativa/patologia , Doença de Crohn/patologia , Humanos , Íleo/metabolismo , Íleo/patologia , Imuno-Histoquímica , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Reto/metabolismo , Reto/patologia
9.
Front Microbiol ; 9: 774, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29731748

RESUMO

Host's physiology is significantly influenced by microbiota colonizing the epithelial surfaces. Complex microbial communities contribute to proper mucosal barrier function, immune response, and prevention of pathogen invasion and have many other crucial functions. The oral cavity and large intestine are distant parts of the digestive tract, both heavily colonized by commensal microbiota. Nevertheless, they feature different proportions of major bacterial and fungal phyla, mostly due to distinct epithelial layers organization and different oxygen levels. A few obligate anaerobic strains inhabiting the oral cavity are involved in the pathogenesis of oral diseases. Interestingly, these microbiota components are also enriched in gut inflammatory and tumor tissue. An altered microbiota composition - dysbiosis - and formation of polymicrobial biofilms seem to play important roles in the development of oral diseases and colorectal cancer. In this review, we describe the differences in composition of commensal microbiota in the oral cavity and large intestine and the mechanisms by which microbiota affect the inflammatory and carcinogenic response of the host.

10.
PLoS One ; 11(7): e0159539, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27434104

RESUMO

Psoriasis is a chronic inflammatory skin disease in which Th17 cells play a crucial role. Since indigenous gut microbiota influences the development and reactivity of immune cells, we analyzed the link among microbiota, T cells and the formation of psoriatic lesions in the imiquimod-induced murine model of psoriasis. To explore the role of microbiota, we induced skin inflammation in germ-free (GF), broad-spectrum antibiotic (ATB)-treated or conventional (CV) BALB/c and C57BL/6 mice. We found that both mice reared in GF conditions for several generations and CV mice treated with ATB were more resistant to imiquimod-induced skin inflammation than CV mice. The ATB treatment dramatically changed the diversity of gut bacteria, which remained stable after subsequent imiquimod application; ATB treatment resulted in a substantial increase in the order Lactobacillales and a significant decrease in Coriobacteriales and Clostridiales. Moreover, as compared to CV mice, imiquimod induced a lower degree of local and systemic Th17 activation in both GF and ATB-treated mice. These findings suggest that gut microbiota control imiquimod-induced skin inflammation by altering the T cell response.


Assuntos
Microbioma Gastrointestinal/fisiologia , Psoríase/imunologia , Psoríase/microbiologia , Pele/imunologia , Células Th17/imunologia , Actinobacteria/efeitos dos fármacos , Actinobacteria/fisiologia , Aminoquinolinas/farmacologia , Animais , Antibacterianos/farmacologia , Clostridiales/efeitos dos fármacos , Clostridiales/fisiologia , Modelos Animais de Doenças , Feminino , Microbioma Gastrointestinal/efeitos dos fármacos , Expressão Gênica , Vida Livre de Germes , Humanos , Imiquimode , Interleucina-17/genética , Interleucina-17/imunologia , Lactobacillales/efeitos dos fármacos , Lactobacillales/fisiologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/imunologia , Psoríase/induzido quimicamente , Psoríase/patologia , Receptores de Antígenos de Linfócitos T gama-delta/genética , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Pele/efeitos dos fármacos , Pele/microbiologia , Pele/patologia , Especificidade da Espécie , Células Th17/efeitos dos fármacos , Células Th17/microbiologia
11.
Urolithiasis ; 43(2): 107-17, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25269440

RESUMO

Hyperoxaluria significantly increases the risk of calcium oxalate kidney stone formation. Since several bacteria have been shown to metabolize oxalate in vitro, including probiotic bifidobacteria, we focused on the efficiency and possible mechanisms by which bifidobacteria can influence oxalate handling in vivo, especially in the intestines, and compared these results with the reported effects of Oxalobacter formigenes. Bifidobacterium animalis subsp. lactis DSM 10140 and B. adolescentis ATCC 15703 were administered to wild-type (WT) mice and to mice deficient in the hepatic enzyme alanine-glyoxylate aminotransferase (Agxt(-/-), a mouse model of Primary Hyperoxaluria) that were fed an oxalate-supplemented diet. The administration of B. animalis subsp. lactis led to a significant decrease in urinary oxalate excretion in WT and Agxt(-/-) mice when compared to treatment with B. adolescentis. Detection of B. animalis subsp. lactis in feces revealed that 3 weeks after oral gavage with the bacteria 64% of WT mice, but only 37% of Agxt(-/-) mice were colonized. Examining intestinal oxalate fluxes showed there were no significant changes to net oxalate secretion in colonized animals and were therefore not associated with the changes in urinary oxalate excretion. These results indicate that colonization with B. animalis subsp. lactis decreased urinary oxalate excretion by degrading dietary oxalate thus limiting its absorption across the intestine but it did not promote enteric oxalate excretion as reported for O. formigenes. Preventive or therapeutic administration of B. animalis subsp. lactis appears to have some potential to beneficially influence dietary hyperoxaluria in mice.


Assuntos
Bifidobacterium , Suplementos Nutricionais , Hiperoxalúria Primária/dietoterapia , Hiperoxalúria Primária/urina , Oxalatos/urina , Oxalobacter formigenes , Animais , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL
12.
Cell Rep ; 10(3): 429-440, 2015 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-25600876

RESUMO

Cajal bodies (CBs) are evolutionarily conserved nuclear structures involved in the metabolism of spliceosomal small nuclear ribonucleoprotein particles (snRNPs). CBs are not present in all cell types, and the trigger for their formation is not yet known. Here, we depleted cells of factors required for the final steps of snRNP assembly and assayed for the presence of stalled intermediates in CBs. We show that depletion induces formation of CBs in cells that normally lack these nuclear compartments, suggesting that CB nucleation is triggered by an imbalance in snRNP assembly. Accumulation of stalled intermediates in CBs depends on the di-snRNP assembly factor SART3. SART3 is required for both the induction of CB formation as well as the tethering of incomplete snRNPs to coilin, the CB scaffolding protein. We propose a model wherein SART3 monitors tri-snRNP assembly and sequesters incomplete particles in CBs, thereby allowing cells to maintain a homeostatic balance of mature snRNPs in the nucleoplasm.

13.
Cancer J ; 20(3): 217-24, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24855011

RESUMO

The mammalian microbiota plays a crucial role in the pathogenesis of many diseases. Thanks to recent advances in metagenomics, proteomics, and metabolomics, microbiome composition and metabolic activity can now be studied in detail. Results obtained by such fascinating and provocative studies would be meaningless without considering the perspective of the whole organism. Our work using gnotobiology as the major tool to unravel the mechanisms of host-microbe interaction has demonstrated the crucial role of microbiota in the initiation and progression of inflammation-associated colorectal neoplasia. Carcinogenesis in the gut is driven by the presence of potentially harmful microbes or by lack of protective ones, by the production of carcinogens generated by microbes, and by the induction of inflammation and modulation of the immune system. Here, we review these mechanisms with special emphasis on those where gnotobiology has yielded important insights.


Assuntos
Neoplasias Colorretais/microbiologia , Modelos Animais de Doenças , Vida Livre de Germes , Microbiota , Animais , Humanos , Inflamação/microbiologia , Microambiente Tumoral
14.
Inflamm Bowel Dis ; 19(6): 1266-77, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23567778

RESUMO

BACKGROUND: Microbial sensing by Toll-like receptors (TLR) and its negative regulation have an important role in the pathogenesis of inflammation-related cancer. In this study, we investigated the role of negative regulation of Toll-like receptors signaling and gut microbiota in the development of colitis-associated cancer in mouse model. METHODS: Colitis-associated cancer was induced by azoxymethane and dextran sodium sulfate in wild-type and in interleukin-1 receptor-associated kinase M (IRAK-M)-deficient mice with or without antibiotic (ATB) treatment. Local cytokine production was analyzed by multiplex cytokine assay or enzyme-linked immunosorbent assay, and regulatory T cells were analyzed by flow cytometry. Changes in microbiota composition during tumorigenesis were analyzed by pyrosequencing, and ß-glucuronidase activity was measured in intestinal content by fluorescence assay. RESULTS: ATB treatment of wild-type mice reduced the incidence and severity of tumors. Compared with nontreated mice, ATB-treated mice had significantly lower numbers of regulatory T cells in colon, altered gut microbiota composition, and decreased ß-glucuronidase activity. However, the ß-glucuronidase activity was not as low as in germ-free mice. IRAK-M-deficient mice not only developed invasive tumors, but ATB-induced decrease in ß-glucuronidase activity did not rescue them from severe carcinogenesis phenotype. Furthermore, IRAK-M-deficient mice had significantly increased levels of proinflammatory cytokines in the tumor tissue. CONCLUSIONS: We conclude that gut microbiota promotes tumorigenesis by increasing the exposure of gut epithelium to carcinogens and that IRAK-M-negative regulation is essential for colon cancer resistance even in conditions of altered microbiota. Therefore, gut microbiota and its metabolic activity could be potential targets for colitis-associated cancer therapy.


Assuntos
Colite/complicações , Neoplasias do Colo/etiologia , Trato Gastrointestinal/microbiologia , Quinases Associadas a Receptores de Interleucina-1/fisiologia , Metagenoma , Animais , Azoximetano/toxicidade , Western Blotting , Carcinógenos/toxicidade , Colite/induzido quimicamente , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Citocinas/genética , Citocinas/metabolismo , Sulfato de Dextrana/toxicidade , Feminino , Citometria de Fluxo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores de Interleucina-1/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/patologia , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo
15.
PLoS One ; 7(3): e34043, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22479515

RESUMO

The most important trigger for immune system development is the exposure to microbial components immediately after birth. Moreover, targeted manipulation of the microbiota can be used to change host susceptibility to immune-mediated diseases. Our aim was to analyze how differences in early gut colonization patterns change the composition of the resident microbiota and future immune system reactivity. Germ-free (GF) mice were either inoculated by single oral gavage of caecal content or let colonized by co-housing with specific pathogen-free (SPF) mice at different time points in the postnatal period. The microbiota composition was analyzed by denaturing gradient gel electrophoresis for 16S rRNA gene followed by principal component analysis. Furthermore, immune functions and cytokine concentrations were analyzed using flow cytometry, ELISA or multiplex bead assay. We found that a single oral inoculation of GF mice at three weeks of age permanently changed the gut microbiota composition, which was not possible to achieve at one week of age. Interestingly, the ex-GF mice inoculated at three weeks of age were also the only mice with an increased pro-inflammatory immune response. In contrast, the composition of the gut microbiota of ex-GF mice that were co-housed with SPF mice at different time points was similar to the gut microbiota in the barrier maintained SPF mice. The existence of a short GF postnatal period permanently changed levels of systemic regulatory T cells, NK and NKT cells, and cytokine production. In conclusion, a time window exists that enables the artificial colonization of GF mice by a single oral dose of caecal content, which may modify the future immune phenotype of the host. Moreover, delayed microbial colonization of the gut causes permanent changes in the immune system.


Assuntos
Trato Gastrointestinal/microbiologia , Vida Livre de Germes/imunologia , Administração Oral , Animais , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Regulação da Expressão Gênica , Sistema Imunitário , Leucócitos Mononucleares/citologia , Linfócitos/citologia , Linfócitos/microbiologia , Masculino , Metagenoma , Camundongos , Camundongos Endogâmicos BALB C , RNA Ribossômico 16S/genética , Fatores de Tempo
16.
PLoS One ; 7(5): e37156, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22629361

RESUMO

BACKGROUND AND AIMS: The egg protein ovalbumin (OVA) belongs to six most frequent food allergens. We investigated how thermal processing influences its ability to induce allergic symptoms and immune responses in mouse model of food allergy. METHODOLOGY/PRINCIPAL FINDINGS: Effect of increased temperature (70°C and 95°C) on OVA secondary structure was characterized by circular dichroism and by the kinetics of pepsin digestion with subsequent HPLC. BALB/c mice were sensitized intraperitoneally and challenged with repeated gavages of OVA or OVA heated to 70°C (h-OVA). Levels of allergen-specific serum antibodies were determined by ELISA (IgA and IgGs) or by ß-hexosaminidase release test (IgE). Specific activities of digestive enzymes were determined in brush border membrane vesicles of jejunal enterocytes. Cytokine production and changes in regulatory T cells in mesenteric lymph nodes and spleen were assessed by ELISA and FACS. Heating of OVA to 70°C caused mild irreversible changes in secondary structure compared to boiling to 95°C (b-OVA), but both OVA treatments led to markedly different digestion kinetics and Tregs induction ability in vitro, compared to native OVA. Heating of OVA significantly decreased clinical symptoms (allergic diarrhea) and immune allergic response on the level of IgE, IL-4, IL-5, IL-13. Furthermore, h-OVA induced lower activities of serum mast cell protease-1 and enterocyte brush border membrane alkaline phosphatase as compared to native OVA. On the other hand h-OVA stimulated higher IgG2a in sera and IFN-γ secretion by splenocytes. CONCLUSIONS: Minor irreversible changes in OVA secondary structure caused by thermal processing changes both its digestion and antigenic epitopes formation, which leads to activation of different T cell subpopulations, induces shift towards Th1 response and ultimately reduces its allergenicity.


Assuntos
Apresentação de Antígeno/imunologia , Hipersensibilidade Alimentar/imunologia , Ovalbumina/química , Linfócitos T Reguladores/imunologia , Animais , Citocinas/sangue , Citocinas/imunologia , Modelos Animais de Doenças , Feminino , Hipersensibilidade Alimentar/sangue , Temperatura Alta , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Camundongos , Ovalbumina/imunologia , Estrutura Secundária de Proteína
17.
PLoS One ; 6(11): e27961, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22132181

RESUMO

BACKGROUND: Probiotic bacteria can be used for the prevention and treatment of human inflammatory diseases including inflammatory bowel diseases (IBD). However, the nature of active components and exact mechanisms of this beneficial effects have not been fully elucidated. Our aim was to investigate if lysate of probiotic bacterium L. casei DN-114 001 (Lc) could decrease the severity of intestinal inflammation in a murine model of IBD. METHODOLOGY/PRINCIPAL FINDINGS: The preventive effect of oral administration of Lc significantly reduces the severity of acute dextran sulfate sodium (DSS) colitis in BALB/c but not in SCID mice. In order to analyze how this beneficial effect interferes with well-known phases of intestinal inflammation pathogenesis in vivo and in vitro, we evaluated intestinal permeability using the FITC-labeled dextran method and analysed tight junction proteins expression by immunofluorescence and PCR. We also measured CD4(+)FoxP3(+) regulatory T cells proportion by FACS analysis, microbiota composition by pyrosequencing, and local cytokine production by ELISA. Lc leads to a significant protection against increased intestinal permeability and barrier dysfunction shown by preserved ZO-1 expression. We found that the Lc treatment increases the numbers of CD4(+)FoxP3(+) regulatory T cells in mesenteric lymph nodes (MLN), decreases production of pro-inflammatory cytokines TNF-α and IFN-γ, and anti-inflammatory IL-10 in Peyer's patches and large intestine, and changes the gut microbiota composition. Moreover, Lc treatment prevents lipopolysaccharide-induced TNF-α expression in RAW 264.7 cell line by down-regulating the NF-κB signaling pathway. CONCLUSION/SIGNIFICANCE: Our study provided evidence that even non-living probiotic bacteria can prevent the development of severe forms of intestinal inflammation by strengthening the integrity of intestinal barrier and modulation of gut microenvironment.


Assuntos
Colite/microbiologia , Colite/prevenção & controle , Sistema Digestório/microbiologia , Lacticaseibacillus casei/metabolismo , Probióticos/farmacologia , Doença Aguda , Administração Oral , Animais , Colite/patologia , Colite/fisiopatologia , Sistema Digestório/efeitos dos fármacos , Sistema Digestório/fisiopatologia , Regulação para Baixo/efeitos dos fármacos , Feminino , Humanos , Imunidade/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Lipopolissacarídeos/farmacologia , Contagem de Linfócitos , Ativação de Macrófagos/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Metagenoma/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , NF-kappa B/metabolismo , Permeabilidade/efeitos dos fármacos , Fosfoproteínas/metabolismo , Probióticos/administração & dosagem , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/efeitos dos fármacos , Fator de Necrose Tumoral alfa/biossíntese , Proteína da Zônula de Oclusão-1
18.
Cell Mol Immunol ; 8(2): 110-20, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21278760

RESUMO

Metagenomic approaches are currently being used to decipher the genome of the microbiota (microbiome), and, in parallel, functional studies are being performed to analyze the effects of the microbiota on the host. Gnotobiological methods are an indispensable tool for studying the consequences of bacterial colonization. Animals used as models of human diseases can be maintained in sterile conditions (isolators used for germ-free rearing) and specifically colonized with defined microbes (including non-cultivable commensal bacteria). The effects of the germ-free state or the effects of colonization on disease initiation and maintenance can be observed in these models. Using this approach we demonstrated direct involvement of components of the microbiota in chronic intestinal inflammation and development of colonic neoplasia (i.e., using models of human inflammatory bowel disease and colorectal carcinoma). In contrast, a protective effect of microbiota colonization was demonstrated for the development of autoimmune diabetes in non-obese diabetic (NOD) mice. Interestingly, the development of atherosclerosis in germ-free apolipoprotein E (ApoE)-deficient mice fed by a standard low-cholesterol diet is accelerated compared with conventionally reared animals. Mucosal induction of tolerance to allergen Bet v1 was not influenced by the presence or absence of microbiota. Identification of components of the microbiota and elucidation of the molecular mechanisms of their action in inducing pathological changes or exerting beneficial, disease-protective activities could aid in our ability to influence the composition of the microbiota and to find bacterial strains and components (e.g., probiotics and prebiotics) whose administration may aid in disease prevention and treatment.


Assuntos
Doenças Autoimunes/etiologia , Trato Gastrointestinal/microbiologia , Vida Livre de Germes , Inflamação/etiologia , Metagenoma/imunologia , Mucosa/imunologia , Neoplasias/etiologia , Animais , Doenças Autoimunes/microbiologia , Modelos Animais de Doenças , Humanos , Imunidade , Inflamação/microbiologia , Neoplasias/microbiologia
19.
J Immunotoxicol ; 6(4): 217-26, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19908940

RESUMO

The intestinal environment is considered to play an important role both in colorectal tumor development and in the evolution and modulation of mucosal immunity. Studies in animals reared in germ-free (GF, without any intestinal microflora) versus conventional (CV, with regular microflora in bowel) conditions can aid in clarifying the influence of bacteria on carcinogenesis and anti-cancer immune responses in situ. The lower incidence of colon cancers and better immunological parameters in GF animals versus CV ones after chemically-induced carcinogenesis raises questions about specific characteristics of the immunological networks in each respective condition. Different levels of tolerance/regulatory mechanisms in the GF versus CV animals may influence the development of immune responses not only at the level of mucosal, but also at the systemic, immunity. We hypothesize that GF animals can better recognize and respond to evolving neoplasias in the bowel as a consequence of their less-tolerogenic immunity (i.e., due to their more limited exposure to antigens to become tolerated against at the intestinal level). In this paper, we review the role of bacteria in modulating gut environment and mucosal immunity, their importance in cancer development, and aspects of immune regulation (both at local and systemic level) that can be modified by bacterial microflora. Lastly, the use of GF animals in comparison with conventionally-raised animals is proposed as a suitable and potent model for understanding the inflammatory network and its effect on cancer immunity especially during colorectal cancer development.


Assuntos
Adenocarcinoma/imunologia , Colo/imunologia , Neoplasias Colorretais/imunologia , Vida Livre de Germes/imunologia , Imunidade Inata , Adenocarcinoma/microbiologia , Animais , Colo/microbiologia , Neoplasias Colorretais/microbiologia , Modelos Animais de Doenças , Vida Livre de Germes/efeitos dos fármacos , Humanos , Tolerância Imunológica , Imunidade nas Mucosas/imunologia , Células Matadoras Naturais/imunologia , Camundongos , Ratos , Receptor Cross-Talk/imunologia
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