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1.
Clin Gastroenterol Hepatol ; 22(1): 22-33.e6, 2024 01.
Artigo em Inglês | MEDLINE | ID: mdl-37716619

RESUMO

BACKGROUND & AIMS: Withdrawal of immunomodulators (IMMs) or tumor necrosis factor (TNF) antagonists in patients with inflammatory bowel diseases (IBDs) in remission on combination therapy is attractive. We evaluated the efficacy and safety of (1) IMM, or (2) TNF antagonist withdrawal in patients with IBD in sustained remission on combination therapy. METHODS: Through a systematic review till March 31, 2023, we identified randomized controlled trials (RCTs) that compared the efficacy and safety of IMM or TNF antagonist withdrawal vs continued combination therapy, in patients with IBD in sustained corticosteroid-free clinical remission for >6 months on combination therapy. Primary outcome was risk of relapse and serious adverse events at 12 months. We conducted meta-analysis to calculate relative risk (RR) and 95% confidence interval (CI) and used Grading of Recommendations Assessment, Development and Evaluation (GRADE) to appraise certainty of evidence. RESULTS: We identified 8 RCTs with 733 patients (77% with Crohn's disease, 91% on infliximab-based combination therapy). On meta-analysis of 5 RCTs, there was no difference in the risk of relapse between patients with IMM withdrawal (continued TNF antagonist monotherapy) vs continued combination therapy (16.8% vs 14.9%; RR, 1.15; 95% CI, 0.75-1.76) without heterogeneity (low certainty of evidence). TNF antagonist withdrawal (continued IMM monotherapy) was associated with 2.4-times higher risk of relapse compared with continuing combination therapy (31.5% vs 11.2%; RR, 2.35; 95% CI, 1.38-4.01), with minimal heterogeneity (low certainty of evidence). There was no difference in the risk of serious adverse events with IMM or TNF antagonist withdrawal vs continued combination therapy. CONCLUSIONS: In patients with IBD in sustained corticosteroid-free clinical remission for >6 months on combination therapy, de-escalation with TNF antagonist withdrawal, but not IMM withdrawal, was associated with an increased risk of relapse.


Assuntos
Doença de Crohn , Doenças Inflamatórias Intestinais , Humanos , Imunossupressores/uso terapêutico , Inibidores do Fator de Necrose Tumoral/efeitos adversos , Fatores Imunológicos/efeitos adversos , Doença de Crohn/tratamento farmacológico , Recidiva , Indução de Remissão , Doenças Inflamatórias Intestinais/tratamento farmacológico
2.
Gastroenterology ; 162(7): 1975-1989, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35227778

RESUMO

BACKGROUND & AIMS: Epithelial wound healing is compromised and represents an unleveraged therapeutic target in inflammatory bowel disease (IBD). Intestinal epithelial cells exhibit plasticity that facilitates dedifferentiation and repair during the response to injury. However, it is not known whether epithelial cells of a neighboring organ can be activated to mediate re-epithelialization in acute colitis. Histological findings of a permanent squamous tissue structure in the distal colon in human IBD could suggest diverse cellular origins of repair-associated epithelium. Here, we tested whether skin-like cells from the anus mediate colonic re-epithelialization in murine colitis. METHODS: We studied dextran sulfate sodium-induced colitis and interleukin 10-deficient colitis in transgenic mice. We performed lineage tracing, 3-dimensional (3D) imaging, single-cell transcriptomics, and biophysical modeling to map squamous cell fates and to identify squamous cell types involved in colonic repair. RESULTS: In acute and chronic colitis, we found a large squamous epithelium, called squamous neo-epithelium of the colon (SNEC), near the anorectal junction. Neighboring squamous cells of the anus rapidly migrate into the ulcerated colon and establish this permanent epithelium of crypt-like morphology. These squamous cells derive from a small unique transition zone, distal to the border of colonic and anal epithelium, that resists colitic injury. The cells of this zone have a pre-loaded program of colonic differentiation and further upregulate key aspects of colonic epithelium during repair. CONCLUSION: Transitional anal cells represent unique reserve cells capable of rebuilding epithelial structures in the colon after colitis. Further study of these cells could reveal novel approaches to direct mucosal healing in inflammation and disease.


Assuntos
Carcinoma de Células Escamosas , Colite , Doenças Inflamatórias Intestinais , Canal Anal/patologia , Animais , Carcinoma de Células Escamosas/patologia , Colite/metabolismo , Colo/patologia , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Células Epiteliais/patologia , Humanos , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/patologia , Camundongos , Camundongos Endogâmicos C57BL , Reepitelização
3.
Am J Physiol Gastrointest Liver Physiol ; 321(3): G308-G324, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34260310

RESUMO

Intestinal mucosal healing is the primary therapeutic goal of medical treatments for inflammatory bowel disease (IBD). Epithelial stem cells are key players in the healing process. Lgr5+ stem cells maintain cellular turnover during homeostasis in the colonic crypt. However, they are lost and dispensable for repair in a wide variety of injury models, including dextran sulfate sodium (DSS) colitis, radiation, helminth infection, and T-cell activation. The direct loss of Lgr5+ cells activates a plasticity response in the epithelium in which other cell types can serve as stem cells. Whether this paradigm applies to mouse models of IBD remains unknown. In contrast to previously tested models, IBD models involve an inflammatory response rooted in the loss of immunologic tolerance to intestinal luminal contents including the microbiome. Here, we show the persistence of Lgr5+ cells in oxazolone, 2,4,6-trinitrobenzene sulfonic acid (TNBS), and Il10-/-, and Il10-/- Tnfr1-/- IBD models. This contrasts with results obtained from DSS-induced injury. Through high-throughput expression profiling, we find that these colitis models were associated with distinct patterns of cytokine expression. Direct exposure of colonic epithelial organoids to DSS, oxazolone, or TNBS resulted in increased apoptosis and loss of Lgr5+ cells. Targeted ablation of Lgr5+ cells resulted in severe exacerbation of chronic, antibody-induced IL-10-deficient colitis, but had only modest effects in TNBS-induced colitis. These results show that distinct mouse models of IBD-like colitis induce different patterns of Lgr5+ stem cell retention and function.NEW & NOTEWORTHY Acute intestinal injury and epithelial repair are associated with the loss of fast-cycling Lgr5+ stem cells and plasticity in the activation of formerly quiescent cell populations. In contrast, here we show in murine inflammatory bowel disease the persistence of the Lgr5+ stem cell population and its essential role in restricting the severity of chronic colitis. This demonstrates a diversity of stem cell responses to colitis.


Assuntos
Colite/metabolismo , Doenças Inflamatórias Intestinais/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/citologia , Animais , Proliferação de Células/fisiologia , Modelos Animais de Doenças , Epitélio/metabolismo , Homeostase/fisiologia , Mucosa Intestinal/metabolismo , Camundongos , Regeneração/fisiologia , Células-Tronco/metabolismo
4.
Am J Physiol Gastrointest Liver Physiol ; 320(6): G990-G1001, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33826403

RESUMO

Proinflammatory macrophages are essential drivers of colitis and express the growth factor receptor ErbB4. This study tested the role of ErbB4 and its specific ligand, NRG4, in regulating macrophage function. We show that endogenous NRG4-ErbB4 signaling limits macrophage production of proinflammatory cytokines in vitro and limits colitis severity in vivo and thus is a potential target for therapeutic intervention.


Assuntos
Inflamação/metabolismo , Macrófagos/metabolismo , Neurregulinas/metabolismo , Receptor ErbB-4/metabolismo , Transdução de Sinais/fisiologia , Animais , Colite/metabolismo , Colo/metabolismo , Citocinas/metabolismo , Inflamação/genética , Interleucina-10/genética , Interleucina-10/metabolismo , Ativação de Macrófagos/fisiologia , Camundongos , Camundongos Knockout
5.
Am J Physiol Gastrointest Liver Physiol ; 319(1): G1-G10, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32421359

RESUMO

The development of modern methods to induce optical transparency ("clearing") in biological tissues has enabled the three-dimensional (3D) reconstruction of intact organs at cellular resolution. New capabilities in visualization of rare cellular events, long-range interactions, and irregular structures will facilitate novel studies in the alimentary tract and gastrointestinal systems. The tubular geometry of the alimentary tract facilitates large-scale cellular reconstruction of cleared tissue without specialized microscopy setups. However, with the rapid pace of development of clearing agents and current relative paucity of research groups in the gastrointestinal field using these techniques, it can be daunting to incorporate tissue clearing into experimental workflows. Here, we give some advice and describe our own experience bringing tissue clearing and whole mount reconstruction into our laboratory's investigations. We present a brief overview of the chemical concepts that underpin tissue clearing, what sorts of questions whole mount imaging can answer, how to choose a clearing agent, an example of how to clear and image alimentary tissue, and what to do after obtaining the image. This short review will encourage other gastrointestinal researchers to consider how utilizing tissue clearing and creating 3D "maps" of tissue might deepen the impact of their studies.


Assuntos
Trato Gastrointestinal/patologia , Técnicas de Cultura de Tecidos , Animais , Microambiente Celular/fisiologia , Humanos , Imageamento Tridimensional/métodos , Pesquisa
6.
Infect Immun ; 87(7)2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31010817

RESUMO

The symbiotic relationship between the gut microbiome and the host provides a nutrient-rich environment for gut microbes and has beneficial effects on host health. Although the composition of the gut microbiome is known to be influenced by both host genetics and environmental factors, host effects on the activities and functions of the gut microbial communities remain poorly understood. Intestinal epithelial cells exert front-line responses to gut microbes and contribute to maintaining a healthy intestinal homeostasis. Here, seeking to elucidate whether intestinal epithelial cells modulate Lactobacillus rhamnosus GG (LGG) functions, we examined the production of p40, an LGG-derived secretory protein that protects intestinal epithelial cells against inflammation. We found that growth medium conditioned with colonic epithelial cell-derived components promotes p40 protein synthesis and secretion by LGG and enhances LGG-stimulated protective responses in intestinal epithelial cells. Furthermore, when LGG was cultured with the colonic luminal contents from healthy mice, p40 production was upregulated but was attenuated with luminal contents from mice with intestinal inflammation. Importantly, the colonic epithelial cell-derived components potentiated LGG-produced p40 levels in a mouse model of colitis and enhanced LGG-mediated amelioration of intestinal inflammation in this model. Notably, we found that colonic epithelial cell-secreted extracellular vesicles participate in communicating with LGG and that heat shock protein 90 (HSP90) in these vesicles might mediate the promotion of p40 production. These results reveal a previously unrecognized mechanism by which the anti-inflammatory effect of LGG is reinforced by intestinal epithelial cells and thereby maintains intestinal health.


Assuntos
Proteínas de Bactérias/metabolismo , Células Epiteliais/microbiologia , Mucosa Intestinal/microbiologia , Lacticaseibacillus rhamnosus/metabolismo , Vesículas Secretórias/microbiologia , Animais , Proteínas de Bactérias/genética , Células Epiteliais/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Mucosa Intestinal/metabolismo , Lacticaseibacillus rhamnosus/genética , Camundongos , Camundongos Endogâmicos C57BL , Vesículas Secretórias/metabolismo
7.
J Cell Sci ; 130(1): 90-96, 2017 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-27026527

RESUMO

Cell shedding from the intestinal villus is a key element of tissue turnover that is essential to maintain health and homeostasis. However, the signals regulating this process are not well understood. We asked whether shedding is controlled by epidermal growth factor receptor (EGFR), an important driver of intestinal growth and differentiation. In 3D ileal enteroid culture and cell culture models (MDCK, IEC-6 and IPEC-J2 cells), extrusion events were suppressed by EGF, as determined by direct counting of released cells or rhodamine-phalloidin labeling of condensed actin rings. Blockade of the MEK-ERK pathway, but not other downstream pathways such as phosphoinositide 3-kinase (PI3K) or protein kinase C (PKC), reversed EGF inhibition of shedding. These effects were not due to a change in cell viability. Furthermore, EGF-driven MAPK signaling inhibited both caspase-independent and -dependent shedding pathways. Similar results were found in vivo, in a novel zebrafish model for intestinal epithelial shedding. Taken together, the data show that EGF suppresses cell shedding in the intestinal epithelium through a selective MAPK-dependent pathway affecting multiple extrusion mechanisms. EGFR signaling might be a therapeutic target for disorders featuring excessive cell turnover, such as inflammatory bowel diseases.


Assuntos
Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais/metabolismo , Intestinos/citologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Animais , Inibidores de Caspase/farmacologia , Caspases/metabolismo , Cães , Células Epiteliais/efeitos dos fármacos , Células Madin Darby de Rim Canino , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Peixe-Zebra , Proteínas rho de Ligação ao GTP/metabolismo
8.
J Biol Chem ; 291(39): 20462-72, 2016 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-27507810

RESUMO

EGF receptor (EGFR) in tumor cells serves as a tumor promoter. However, information about EGFR activation in macrophages in regulating M2 polarization and tumor development is limited. This study aimed to investigate the effects of EGFR activation in macrophages on M2 polarization and development of gastrointestinal tumors. IL-4, a cytokine to elicit M2 polarization, stimulated release of an EGFR ligand, HB-EGF, and transactivation and down-regulation of EGFR in Raw 264.7 cells and peritoneal macrophages from WT mice. Knockdown of HB-EGF in macrophages inhibited EGFR transactivation by IL-4. IL-4-stimulated STAT6 activation, Arg1 and YM1 gene expression, and HB-EGF production were further enhanced by inhibition of EGFR activity in Raw 264.7 cells using an EGFR kinase inhibitor and in peritoneal macrophages from Egfr(wa5) mice with kinase inactive EGFR and by knockdown of EGFR in peritoneal macrophages from Egfr(fl/fl) LysM-Cre mice with myeloid cell-specific EGFR deletion. Chitin induced a higher level of M2 polarization in peritoneal macrophages in Egfr(fl/fl) LysM-Cre mice than that in Egfr(fl/fl) mice. Accordingly, IL-4-conditioned medium stimulated growth and epithelial-to-mesenchymal transition in gastric epithelial and colonic tumor cells, which were suppressed by that from Raw 264.7 cells with HB-EGF knockdown but promoted by that from Egfr(wa5) and Egfr(fl/fl) LysM-Cre peritoneal macrophages. Clinical assessment revealed that the number of macrophages with EGFR expression became less, indicating decreased inhibitory effects on M2 polarization, in late stage of human gastric cancers. Thus, IL-4-stimulated HB-EGF-dependent transactivation of EGFR in macrophages may mediate inhibitory feedback for M2 polarization and HB-EGF production, thereby inhibiting gastrointestinal tumor growth.


Assuntos
Receptores ErbB/biossíntese , Neoplasias Gastrointestinais/metabolismo , Regulação Neoplásica da Expressão Gênica , Macrófagos Peritoneais/metabolismo , Ativação Transcricional , Animais , Linhagem Celular Tumoral , Receptores ErbB/genética , Neoplasias Gastrointestinais/genética , Neoplasias Gastrointestinais/patologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Humanos , Interleucina-4/genética , Interleucina-4/metabolismo , Macrófagos Peritoneais/patologia , Camundongos , Camundongos Knockout , Células RAW 264.7
9.
Gastroenterology ; 149(4): 993-1005.e2, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26072395

RESUMO

BACKGROUND & AIMS: Tumor necrosis factor receptor 2 (TNFR2, Tnfrsf1b) regulates multiple aspects of immune function, but little is known about its role in the immunopathogenesis of inflammatory bowel disease (IBD). We investigated whether TNFR2 restricts the activity of specific immune cell subtypes to protect against the development of colitis in mice. METHODS: Tnfr2(-/-) mice were crossed with interleukin (Il) 10(-/-) mice, which spontaneously develop colitis, to generate Il10(-/-)Tnfr2(-/-) mice. Colonic tissues were collected from Il10(-/-)Tnfr2(-/-) mice along with Il10(-/-) mice (controls) and analyzed by flow cytometry and histology. Bone marrow was transplanted into Il10(-/-) and Il10(-/-)Tnfr2(-/-) mice from Il10(-/-) or Il10(-/-)Tnfr2(-/-) donors by intravenous injection. CD8(+) T cells were neutralized in Il10(-/-)Tnfr2(-/-) mice by intraperitoneal injection of anti-CD8 or isotype control antibodies. Colitis was induced in Rag2(-/-) mice by intravenous injections of naïve CD8(+) T cells isolated from C57BL/6 or Tnfr2(-/-) mice. RESULTS: Il10(-/-)Tnfr2(-/-) mice spontaneously developed more severe colitis compared with Il10(-/-) controls, characterized by selective expansion of colonic CD8(+) T cells. Transplantation of TNFR2-deficient bone marrow resulted in significantly increased incidence and severity of colitis. Transcriptome analyses showed that the expression of genes regulated by TNFR2 were specific to CD8(+) T cells and included genes associated with risk for IBD. Depletion of CD8(+) T cells from Il10(-/-)Tnfr2(-/-) mice prevented colonic inflammation. Adoptive transfer of TNFR2-null naïve CD8(+) T cells compared with CD8(+) T cells from control mice increased the severity of colitis that developed in Rag2(-/-) mice. CONCLUSIONS: TNFR2 protects mice from colitis by inhibiting the expansion of colonic CD8(+) T cells. TNFR2 regulates expression of genes that regulate CD8(+) T cells and have been associated with susceptibility to IBD. Disruption in TNFR2 signaling might therefore be associated with pathogenesis. Strategies to increase levels or activity of TNFR2 and thereby reduce the activity of CD8(+) T cells might be developed to treat IBD patients with CD8(+) T cell dysfunction.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Colite/prevenção & controle , Colo/metabolismo , Imunidade Celular , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Transferência Adotiva , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/transplante , Proliferação de Células , Colite/genética , Colite/imunologia , Colite/metabolismo , Colite/patologia , Colo/imunologia , Colo/patologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Regulação da Expressão Gênica , Interleucina-10/genética , Interleucina-10/metabolismo , Ativação Linfocitária , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Tipo II do Fator de Necrose Tumoral/deficiência , Receptores Tipo II do Fator de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/imunologia , Transdução de Sinais , Fatores de Tempo
10.
J Immunol ; 192(3): 1013-23, 2014 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-24391216

RESUMO

Macrophages regulate innate immunity to maintain intestinal homeostasis and play pathological roles in intestinal inflammation. Activation of the epidermal growth factor receptor (EGFR) promotes cellular proliferation, differentiation, survival, and wound closure in several cell types. However, the impact of EGFR in macrophages remains unclear. This study was to investigate whether EGFR activation in macrophages regulates cytokine production and intestinal inflammation. We found that EGFR was activated in colonic macrophages in mice with dextran sulfate sodium (DSS)-induced colitis and in patients with ulcerative colitis. DSS-induced acute colitis was ameliorated, and recovery from colitis was promoted in Egfr(fl/fl)LysM-Cre mice with myeloid cell-specific deletion of EGFR, compared with LysM-Cre mice. DSS treatment increased IL-10 and TNF levels during the acute phase of colitis, and increased IL-10 but reduced TNF levels during the recovery phase in Egfr(fl/fl)LysM-Cre mice. An anti-IL-10 neutralizing Ab abolished these effects of macrophage-specific EGFR deletion on DSS-induced colitis in Egfr(fl/fl)LysM-Cre mice. LPS stimulated EGFR activation and inhibition of EGFR kinase activity enhanced LPS-stimulated NF-κB activation in RAW 264.7 macrophages. Furthermore, induction of IL-10 production by EGFR kinase-blocked RAW 264.7 cells, in response to LPS plus IFN-γ, correlated with decreased TNF production. Thus, although selective deletion of EGFR in macrophages leads to increases in both pro- and anti-inflammatory cytokines in response to inflammatory stimuli, the increase in the IL-10 level plays a role in suppressing proinflammatory cytokine production, resulting in protection of mice from intestinal inflammation. These results reveal an integrated response of macrophages regulated by EGFR in intestinal inflammatory disorders.


Assuntos
Colite/imunologia , Citocinas/biossíntese , Receptores ErbB/fisiologia , Macrófagos/imunologia , Adolescente , Adulto , Idoso , Animais , Linhagem Celular , Colite/induzido quimicamente , Colite/metabolismo , Colite Ulcerativa/metabolismo , Colo/imunologia , Colo/patologia , Colo/fisiologia , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/genética , Citocinas/genética , Sulfato de Dextrana/toxicidade , Receptores ErbB/deficiência , Receptores ErbB/genética , Feminino , Regulação da Expressão Gênica/imunologia , Humanos , Imunidade Inata , Inflamação , Interferon gama/farmacologia , Interleucina-10/antagonistas & inibidores , Interleucina-10/biossíntese , Interleucina-10/imunologia , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Células Mieloides/metabolismo , NF-kappa B/metabolismo , Neutrófilos/metabolismo , Quinazolinas/farmacologia , Regeneração , Transdução de Sinais/imunologia , Baço/imunologia , Baço/patologia , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/genética , Tirfostinas/farmacologia , Adulto Jovem
11.
Am J Physiol Gastrointest Liver Physiol ; 308(3): G161-70, 2015 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-25477373

RESUMO

Tumor necrosis factor (TNF) and its receptors TNFR1 and TNFR2 are major therapeutic targets for inflammatory bowel disease. Research advances have demonstrated that TNF produces pleiotropic responses in the gastrointestinal (GI) tract. Although in excess TNF can contribute to GI pathology, TNF is also a critical protective factor to promote GI homeostasis following injury and inflammation. Genetic studies using candidate and genome-wide association study approaches have identified variants in TNF or its receptors that are associated with Crohn's disease or ulcerative colitis in multiple populations, although the basis for these associations remains unclear. This review considers the efficacy and mechanism of anti-TNF therapies for inflammatory bowel disease to reconcile the many disparate aspects of TNF research and to consider the potential protective effects of TNF signaling in GI health.


Assuntos
Anticorpos Monoclonais/uso terapêutico , Doenças Inflamatórias Intestinais/tratamento farmacológico , Fatores de Necrose Tumoral/imunologia , Animais , Anticorpos Monoclonais/imunologia , Fármacos Gastrointestinais/uso terapêutico , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/metabolismo , Humanos , Doenças Inflamatórias Intestinais/imunologia , Receptores do Fator de Necrose Tumoral/imunologia , Fatores de Necrose Tumoral/metabolismo
12.
Am J Physiol Gastrointest Liver Physiol ; 308(9): G721-35, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25721303

RESUMO

The mucosal layer of the colon is a unique and dynamic site where host cells interface with one another and the microbiome, with major implications for physiology and disease. However, the cellular mechanisms mediating colonic regeneration, inflammation, dysplasia, and dysbiosis remain undercharacterized, partly because the use of thin tissue sections in many studies removes important volumetric context. To address these challenges in visualization, we have developed the deep mucosal imaging (DMI) method to reconstruct continuous extended volumes of mouse colorectal mucosa at cellular resolution. Use of ScaleA2 and SeeDB clearing agents enabled full visualization of the colonic crypt, the fundamental unit of adult colon. Confocal imaging of large colorectal expanses revealed epithelial structures involved in repair, inflammation, tumorigenesis, and stem cell function, in fluorescent protein-labeled, immunostained, paraffin-embedded, or human biopsy samples. We provide freely available software to reconstruct and explore on computers with standard memory allocations the large DMI datasets containing in toto representations of distal colonic mucosal volume. Extended-volume imaging of colonic mucosa through the novel, extensible, and readily adopted DMI approach will expedite mechanistic investigations of intestinal physiology and pathophysiology at intracrypt to multicrypt length scales.


Assuntos
Colite/patologia , Colo/patologia , Mucosa Intestinal/patologia , Microscopia Confocal/métodos , Reto/patologia , Animais , Colite/induzido quimicamente , Colite/genética , Colite/metabolismo , Colo/metabolismo , Sulfato de Dextrana , Modelos Animais de Doenças , Frutose , Genes Reporter , Glicerol/análogos & derivados , Humanos , Processamento de Imagem Assistida por Computador , Mucosa Intestinal/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Reto/metabolismo , Software , Soluções
13.
Gastroenterology ; 146(7): 1739-51.e14, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24530706

RESUMO

BACKGROUND & AIMS: The gastric cancer-causing pathogen Helicobacter pylori up-regulates spermine oxidase (SMOX) in gastric epithelial cells, causing oxidative stress-induced apoptosis and DNA damage. A subpopulation of SMOX(high) cells are resistant to apoptosis, despite their high levels of DNA damage. Because epidermal growth factor receptor (EGFR) activation can regulate apoptosis, we determined its role in SMOX-mediated effects. METHODS: SMOX, apoptosis, and DNA damage were measured in gastric epithelial cells from H. pylori-infected Egfr(wa5) mice (which have attenuated EGFR activity), Egfr wild-type mice, or in infected cells incubated with EGFR inhibitors or deficient in EGFR. A phosphoproteomic analysis was performed. Two independent tissue microarrays containing each stage of disease, from gastritis to carcinoma, and gastric biopsy specimens from Colombian and Honduran cohorts were analyzed by immunohistochemistry. RESULTS: SMOX expression and DNA damage were decreased, and apoptosis increased in H. pylori-infected Egfr(wa5) mice. H. pylori-infected cells with deletion or inhibition of EGFR had reduced levels of SMOX, DNA damage, and DNA damage(high) apoptosis(low) cells. Phosphoproteomic analysis showed increased EGFR and erythroblastic leukemia-associated viral oncogene B (ERBB)2 signaling. Immunoblot analysis showed the presence of a phosphorylated (p)EGFR-ERBB2 heterodimer and pERBB2; knockdown of ErbB2 facilitated apoptosis of DNA damage(high) apoptosis(low) cells. SMOX was increased in all stages of gastric disease, peaking in tissues with intestinal metaplasia, whereas pEGFR, pEGFR-ERBB2, and pERBB2 were increased predominantly in tissues showing gastritis or atrophic gastritis. Principal component analysis separated gastritis tissues from patients with cancer vs those without cancer. pEGFR, pEGFR-ERBB2, pERBB2, and SMOX were increased in gastric samples from patients whose disease progressed to intestinal metaplasia or dysplasia, compared with patients whose disease did not progress. CONCLUSIONS: In an analysis of gastric tissues from mice and patients, we identified a molecular signature (based on levels of pEGFR, pERBB2, and SMOX) for the initiation of gastric carcinogenesis.


Assuntos
Dano ao DNA , Células Epiteliais/enzimologia , Receptores ErbB/metabolismo , Mucosa Gástrica/enzimologia , Infecções por Helicobacter/enzimologia , Helicobacter pylori/metabolismo , Receptor ErbB-2/metabolismo , Animais , Apoptose , Linhagem Celular , Sobrevivência Celular , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Técnicas de Cocultura , Colômbia , Progressão da Doença , Ativação Enzimática , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Receptores ErbB/deficiência , Receptores ErbB/genética , Mucosa Gástrica/microbiologia , Mucosa Gástrica/patologia , Gastrite/enzimologia , Gastrite/microbiologia , Gastrite/patologia , Infecções por Helicobacter/genética , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Helicobacter pylori/patogenicidade , Honduras , Humanos , Metaplasia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Fosforilação , Lesões Pré-Cancerosas/enzimologia , Lesões Pré-Cancerosas/microbiologia , Lesões Pré-Cancerosas/patologia , Análise de Componente Principal , Multimerização Proteica , Receptor ErbB-2/genética , Transdução de Sinais , Neoplasias Gástricas/enzimologia , Neoplasias Gástricas/microbiologia , Neoplasias Gástricas/patologia , Tennessee , Poliamina Oxidase
14.
J Biol Chem ; 288(42): 30742-30751, 2013 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-24043629

RESUMO

p40, a Lactobacillus rhamnosus GG (LGG)-derived soluble protein, ameliorates intestinal injury and colitis, reduces apoptosis, and preserves barrier function by transactivation of the EGF receptor (EGFR) in intestinal epithelial cells. The aim of this study is to determine the mechanisms by which p40 transactivates the EGFR in intestinal epithelial cells. Here we show that p40-conditioned medium activates EGFR in young adult mouse colon epithelial cells and human colonic epithelial cell line, T84 cells. p40 up-regulates a disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) catalytic activity, and broad spectrum metalloproteinase inhibitors block EGFR transactivation by p40 in these two cell lines. In ADAM17-deficient mouse colonic epithelial (ADAM17(-/-) MCE) cells, p40 transactivation of EGFR is blocked, but can be rescued by re-expression with WT ADAM17. Furthermore, p40 stimulates release of heparin binding (HB)-EGF, but not transforming growth factor (TGF)α or amphiregulin, in young adult mouse colon cells and ADAM17(-/-) MCE cells overexpressing WT ADAM17. Knockdown of HB-EGF expression by siRNA suppresses p40 effects on transactivating EGFR and Akt, preventing apoptosis, and preserving tight junction function. The effects of p40 on HB-EGF release and ADAM17 activation in vivo are examined after administration of p40-containing pectin/zein hydrogel beads to mice. p40 stimulates ADAM17 activity and EGFR activation in colonic epithelial cells and increases HB-EGF levels in blood from WT mice, but not from mice with intestinal epithelial cell-specific ADAM17 deletion. Thus, these data define a mechanism of a probiotic-derived soluble protein in modulating intestinal epithelial cell homeostasis through ADAM17-mediated HB-EGF release, leading to transactivation of EGFR.


Assuntos
Proteínas de Bactérias/metabolismo , Células Epiteliais/metabolismo , Receptores ErbB/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Mucosa Intestinal/metabolismo , Lacticaseibacillus rhamnosus/metabolismo , Probióticos/metabolismo , Ativação Transcricional , Proteínas ADAM/biossíntese , Proteínas ADAM/genética , Proteína ADAM17 , Animais , Linhagem Celular Tumoral , Ativação Enzimática/genética , Células Epiteliais/citologia , Células Epiteliais/microbiologia , Receptores ErbB/genética , Regulação Enzimológica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Mucosa Intestinal/citologia , Mucosa Intestinal/microbiologia , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador alfa/metabolismo , Regulação para Cima/genética
15.
Clin Gastroenterol Hepatol ; 12(12): 2026-32, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24732284

RESUMO

BACKGROUND & AIMS: Pediatric functional abdominal pain has been linked to functional gastrointestinal disorders (FGIDs) in adulthood, but little is known about patient characteristics in childhood that increase the risk for FGID in young adulthood. We investigated the contribution of gastrointestinal symptoms, extraintestinal somatic symptoms, and depressive symptoms in pediatric patients with functional abdominal pain and whether these predicted FGIDs later in life. METHODS: In a longitudinal study, consecutive new pediatric patients, diagnosed with functional abdominal pain in a subspecialty clinic, completed a comprehensive baseline evaluation of the severity of their physical and emotional symptoms. They were contacted 5 to 15 years later and evaluated, based on Rome III symptom criteria, for abdominal pain-related FGIDs, including irritable bowel syndrome, functional dyspepsia, functional abdominal pain syndrome, and abdominal migraine. Controlling for age, sex, baseline severity of abdominal pain, and time to follow-up evaluation, multivariable logistic regression was used to evaluate the association of baseline gastrointestinal, extraintestinal somatic, and depressive symptoms in childhood with FGID in adolescence and young adulthood. RESULTS: Of 392 patients interviewed an average of 9.2 years after their initial evaluation, 41% (n = 162) met symptom criteria for FGID; most met the criteria for irritable bowel syndrome. Extraintestinal somatic and depressive symptoms at the initial pediatric evaluation were significant predictors of FGID later in life, after controlling for initial levels of GI symptoms. Age, sex, and abdominal pain severity at initial presentation were not significant predictors of FGID later in life. CONCLUSIONS: In pediatric patients with functional abdominal pain, assessment of extraintestinal and depressive symptoms may be useful in identifying those at risk for FGID in adolescence and young adulthood.


Assuntos
Dor Abdominal/diagnóstico , Dor Abdominal/epidemiologia , Dor Abdominal/fisiopatologia , Adolescente , Criança , Comorbidade , Depressão/diagnóstico , Depressão/epidemiologia , Feminino , Gastroenteropatias/diagnóstico , Gastroenteropatias/epidemiologia , Humanos , Estudos Longitudinais , Masculino , Prognóstico , Adulto Jovem
16.
Gut ; 62(1): 73-82, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22267598

RESUMO

BACKGROUND: Necrotising enterocolitis (NEC) is the most common gastrointestinal emergency in premature infants. Immaturity of gastrointestinal immune regulation may predispose preterm infants to NEC as FOXP3 T regulatory cells (Treg) are critical for intestinal immune homoeostasis. OBJECTIVE: To investigate the hypothesis that abnormal developmental regulation of lamina propria Treg would define premature infants with NEC. DESIGN: Lamina propria mononuclear cell populations from surgically resected ileum from 18 patients with NEC and 30 gestational age-matched non-NEC surgical controls were prospectively isolated. Polychromatic flow cytometry was performed to phenotype and analyse lamina propria T cell populations. The cytokine gene expression profile in NEC tissue was compared with that of non-NEC controls. RESULTS: The total number of Treg, CD4, or CD8 T cells in each ileum section was independent of gestational age, age or postmenstrual age and similar between patients with NEC and controls. In contrast, the ratio of Treg to CD4 T cells or Treg to CD8 T cells was significantly lower in NEC ileum than in infants without NEC (medians 2.9% vs 6.6%, p=0.001 and medians 6.6% vs 25.9%, p<0.001, respectively). For any given number of CD4 or CD8 T cells, Treg were, on average, 60% lower in NEC ileum than in controls. NEC tissue cytokine gene expression profiles were characteristic of inhibited Treg development or function. Treg/CD4 and Treg/CD8 ratios recovered between initial resection for NEC and reanastomosis. CONCLUSION: The proportion of lamina propria Treg is significantly reduced in the ileum of premature infants with NEC and may contribute to the excessive inflammatory state of this disease.


Assuntos
Linfócitos T CD8-Positivos/metabolismo , Enterocolite Necrosante/imunologia , Fatores de Transcrição Forkhead/metabolismo , Doenças do Prematuro/imunologia , Mucosa Intestinal/imunologia , Linfócitos T Reguladores/metabolismo , Biomarcadores/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Estudos de Casos e Controles , Feminino , Citometria de Fluxo , Perfilação da Expressão Gênica , Humanos , Recém-Nascido , Recém-Nascido Prematuro , Contagem de Linfócitos , Masculino , Estudos Prospectivos
17.
Inflamm Bowel Dis ; 30(3): 402-409, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-37229656

RESUMO

BACKGROUND: Intestinal ultrasound (IUS) is a noninvasive tool to assess bowel inflammation. There is a paucity of data on its accuracy in pediatric patients. AIM: The aim of this study is to evaluate the diagnostic performance of bowel wall thickness (BWT) measured using IUS compared with endoscopic disease activity in children suspected of having inflammatory bowel disease (IBD). METHODS: We conducted a single-center cross-sectional pilot study of pediatric patients suspected to have previously undiagnosed IBD. Endoscopic inflammation was graded using segmental scores of the Simple Endoscopic Score for Crohn's Disease (SES-CD) and the Ulcerative Colitis Endoscopic Index of Severity (UCEIS) and classified as having healthy, mild, or moderate/severe disease activity. Association between BWT and endoscopic severity was assessed using the Kruskal-Wallis test. The diagnostic performance of BWT to detect active disease at endoscopy was evaluated using the area under the receiver operating characteristic curve; sensitivity and specificity were calculated. RESULTS: In all, 174 bowel segments in 33 children were assessed by IUS and ileocolonoscopy. An elevated median BWT was associated with increased bowel segment disease severity, classified by the SES-CD (P < .001) and the UCEIS (P < .01). Using a cutoff value of 1.9 mm, we found that the BWT had an area under the receiver operating characteristic curve of 0.743 (95% CI, 0.67-0.82), a sensitivity of 64% (95% CI, 53%-73%), and a specificity of 76% (95% CI, 65%-85%) to detect inflamed bowel. CONCLUSION: Increasing BWT is associated with increasing endoscopic activity in pediatric IBD. Our study suggests that the optimal BWT cutoff value for detecting active disease may be less than that seen in adults. Additional pediatric studies are needed.


Increasing bowel wall thickness (BWT) is associated with increasing IBD endoscopic scores on colonoscopy. There is moderate to fair agreement between the prediction of IBD diagnosis and Paris classification using intestinal ultrasound (IUS). Bowel wall thickness cutoff values to detect inflamed bowel segments are likely lower for children with IBD than for adults, although further studies with wider age ranges are needed to confirm this finding.


Assuntos
Colite Ulcerativa , Doenças Inflamatórias Intestinais , Adulto , Humanos , Criança , Estudos Transversais , Projetos Piloto , Colite Ulcerativa/diagnóstico por imagem , Inflamação , Gravidade do Paciente
18.
Adv Ther ; 41(3): 901-914, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38286962

RESUMO

Dysbiosis corresponds to the disruption of a formerly stable, functionally complete microbiota. In the gut, this imbalance can lead to adverse health outcomes in both the short and long terms, with a potential increase in the lifetime risks of various noncommunicable diseases and disorders such as atopy (like asthma), inflammatory bowel disease, neurological disorders, and even behavioural and psychological disorders. Although antibiotics are highly effective in reducing morbidity and mortality in infectious diseases, antibiotic-associated diarrhoea is a common, non-negligible clinical sign of gut dysbiosis (and the only visible one). Re-establishment of a normal (functional) gut microbiota is promoted by completion of the clinically indicated course of antibiotics, the removal of any other perturbing external factors, the passage of time (i.e. recovery through the microbiota's natural resilience), appropriate nutritional support, and-in selected cases-the addition of probiotics. Systematic reviews and meta-analyses of clinical trials have confirmed the strain-specific efficacy of some probiotics (notably the yeast Saccharomyces boulardii CNCM I-745 and the bacterium Lactobacillus rhamnosus GG) in the treatment and/or prevention of antibiotic-associated diarrhoea in children and in adults. Unusually for a probiotic, S. boulardii is a eukaryote and is not therefore directly affected by antibiotics-making it suitable for administration in cases of antibiotic-associated diarrhoea. A robust body of evidence from clinical trials and meta-analyses shows that the timely administration of an adequately dosed probiotic (upon initiation of antibiotic treatment or within 48 h) can help to prevent or resolve the consequences of antibiotic-associated dysbiosis (such as diarrhoea) and promote the resilience of the gut microbiota and a return to the pre-antibiotic state. A focus on the prescription of evidence-based, adequately dosed probiotics should help to limit unjustified and potentially ineffective self-medication.


Assuntos
Lacticaseibacillus rhamnosus , Probióticos , Saccharomyces boulardii , Adulto , Criança , Humanos , Antibacterianos/efeitos adversos , Diarreia/induzido quimicamente , Diarreia/prevenção & controle , Disbiose/induzido quimicamente , Disbiose/terapia , Probióticos/uso terapêutico , Saccharomyces cerevisiae , Metanálise como Assunto , Revisões Sistemáticas como Assunto
19.
Am J Physiol Gastrointest Liver Physiol ; 305(2): G196-203, 2013 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-23681474

RESUMO

Chronic infection of the gastric mucosa by Helicobacter pylori is associated with an increased risk of developing gastric cancer; however, the vast majority of infected individuals never develop this disease. One H. pylori virulence factor that increases gastric cancer risk is the cag pathogenicity island, which encodes a bacterial type IV secretion system. Cyclooxygenase-2 (COX-2) expression is induced by proinflammatory stimuli, leading to increased prostaglandin E2 (PGE2) secretion by gastric epithelial cells. COX-2 expression is increased in gastric tissue from H. pylori-infected persons. H. pylori also activates the epidermal growth factor receptor (EGFR) in gastric epithelial cells. We now demonstrate that H. pylori-induced activation of COX-2 in gastric cells is dependent upon EGFR activation, and that a functional cag type IV secretion system and direct bacterial contact are necessary for full induction of COX-2 by gastric epithelial cells. PGE2 secretion is increased in cells infected with H. pylori, and this induction is dependent on a functional EGFR. Increased apoptosis in response to H. pylori occurs in cells treated with a COX-2 inhibitor, as well as COX-2-/- cells, indicating that COX-2 expression promotes cell survival. In vivo, COX-2 induction by H. pylori is significantly reduced in mice deficient for EGFR activation compared with wild-type mice with a fully functional receptor. Collectively, these findings indicate that aberrant activation of the EGFR-COX-2 axis may lower the threshold for carcinogenesis associated with chronic H. pylori infection.


Assuntos
Ciclo-Oxigenase 2/metabolismo , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , Infecções por Helicobacter/metabolismo , Helicobacter pylori , Estômago/citologia , Animais , Linhagem Celular , Ciclo-Oxigenase 2/genética , Dinoprostona , Receptores ErbB/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Infecções por Helicobacter/microbiologia , Camundongos , Camundongos Knockout
20.
iScience ; 26(10): 107829, 2023 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-37736049

RESUMO

Colonic epithelial repair is a key determinant of health. Repair involves changes in epithelial differentiation, an extensive proliferative response, and upregulation of regeneration-associated "fetal-like" transcripts, including Ly6a (Sca-1), that represent Yap1 and interferon targets. However, little is known about how this regenerative program terminates and how homeostasis is restored during injury and inflammation. Here we show that, after the initial entry into the regenerative state, the subsequent upregulation of tumor necrosis factor (TNF) receptor 2 (R2, TNFR2, Tnfrsf1b) clears the regenerative signaling and restores homeostatic patterns of epithelial differentiation. Targeted deletion of epithelial TNFR2 in vivo and in colonoid cultures revealed persistent expression of Ly6a, hyperproliferation, and reduced secretory differentiation. Moreover, mice lacking epithelial TNFR2 also failed to complete colon ulcer healing, suggesting that partial resolution of regenerative signaling is essential for the completion of the repair process. These results demonstrate how epithelial cells dynamically leverage a colitis-associated cytokine to choreograph repair.

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