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1.
Clin Transplant ; 35(7): e14378, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34060679

RESUMO

Monitoring of intestinal allograft function remains a challenge. While frequent endoscopies and biopsies are the gold standard, no single biomarker exists to screen for intestinal transplant rejection. The novel REG3α, an antimicrobial peptide secreted by intestinal enterocytes and Paneth cells, has been associated with inflammatory bowel disease as well as intestinal graft versus host disease. Our aim was to identify and describe a role of REG3α in monitoring or predicting acute allograft rejection after intestinal transplantation (ITx). Since 2019, we have incorporated REG3α into the standard monitoring of patients after ITx. We conducted a retrospective analysis of a prospectively maintained IRB-approved database and present, herein, the results of 2 adults with irreversible intestinal failure who underwent isolated ITx under this monitoring protocol. Increases in REG3α corresponded with acute allograft rejection in both cases and preceded acute allograft rejection by 1 week in one of the cases. We describe REG3α as a non-invasive marker of acute allograft rejection after adult isolated ITx which not only corresponded with acute allograft rejection but also preceded histopathological changes by 1 week.


Assuntos
Rejeição de Enxerto , Adulto , Aloenxertos , Biomarcadores , Rejeição de Enxerto/diagnóstico , Rejeição de Enxerto/etiologia , Humanos , Estudos Retrospectivos , Transplante Homólogo
2.
Pediatr Transplant ; 25(3): e13987, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33590644

RESUMO

The role of angiotensin II type-1 receptor (AT1R) antibodies in intestinal transplantation (ITx) is unclear. The aims were 1) to identify the prevalence of AT1R antibodies in pediatric ITx, compared to pediatric intestinal failure (IF), and 2) to determine whether AT1R antibodies were associated with graft dysfunction. 46 serum samples from 25 ITx patients (3 isolated ITx, 22 liver-inclusive ITx) were collected during routine visits >6 months apart and during episodes of graft dysfunction as a result of infectious enteritis or rejection. For comparison, samples were collected from 7 IF control patients. AT1R antibodies were considered positive for levels >17 U/mL. The median (range) AT1R antibody level for ITx patients was 40.0 U/mL (7.2-40.0), compared to 7.0 U/mL (5.7-40.0) for IF patients (p = .02). There was a trend toward higher prevalence of AT1R antibodies in ITx compared with IF patients (68% versus 29%, p = .09). Among ITx patients, the prevalence of AT1R antibodies was not different between periods of active graft dysfunction and normal health (83% versus 67%, p = .31). For 16 patients with >2 samples, AT1R antibodies remained positive in 67% cases, developed in 14% cases, disappeared in 10% cases, and remained negative in 10% cases. The changes in AT1R antibodies did not correlate with de/sensitizing events. This is the first study of AT1R antibodies in pediatric ITx. AT1R antibodies are highly prevalent after ITx and may be triggered by immune activation associated with the transplant. However, their pathogenicity and clinical utility remain in question.


Assuntos
Autoanticorpos/sangue , Insuficiência Intestinal/sangue , Intestinos/transplante , Receptor Tipo 1 de Angiotensina/imunologia , Adolescente , Criança , Pré-Escolar , Feminino , Antígenos HLA , Humanos , Masculino , Estudos Retrospectivos , Adulto Jovem
3.
Am J Physiol Gastrointest Liver Physiol ; 318(5): G931-G945, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32174134

RESUMO

Helicobacter pylori infection always induces gastritis, which may progress to ulcer disease or cancer. The mechanisms underlying mucosal injury by the bacteria are incompletely understood. Here, we identify a novel pathway for H. pylori-induced gastric injury, the impairment of maturation of the essential transport enzyme and cell adhesion molecule, Na-K-ATPase. Na-K-ATPase comprises α- and ß-subunits that assemble in the endoplasmic reticulum (ER) before trafficking to the plasma membrane. Attachment of H. pylori to gastric epithelial cells increased Na-K-ATPase ubiquitylation, decreased its surface and total levels, and impaired ion balance. H. pylori did not alter degradation of plasmalemma-resident Na-K-ATPase subunits or their mRNA levels. Infection decreased association of α- and ß-subunits with ER chaperone BiP and impaired assembly of α/ß-heterodimers, as was revealed by quantitative mass spectrometry and immunoblotting of immunoprecipitated complexes. The total level of BiP was not altered, and the decrease in interaction with BiP was not observed for other BiP client proteins. The H. pylori-induced decrease in Na-K-ATPase was prevented by BiP overexpression, stopping protein synthesis, or inhibiting proteasomal, but not lysosomal, protein degradation. The results indicate that H. pylori impairs chaperone-assisted maturation of newly made Na-K-ATPase subunits in the ER independently of a generalized ER stress and induces their ubiquitylation and proteasomal degradation. The decrease in Na-K-ATPase levels is also seen in vivo in the stomachs of gerbils and chronically infected children. Further understanding of H. pylori-induced Na-K-ATPase degradation will provide insights for protection against advanced disease.NEW & NOTEWORTHY This work provides evidence that Helicobacter pylori decreases levels of Na-K-ATPase, a vital transport enzyme, in gastric epithelia, both in acutely infected cultured cells and in chronically infected patients and animals. The bacteria interfere with BiP-assisted folding of newly-made Na-K-ATPase subunits in the endoplasmic reticulum, accelerating their ubiquitylation and proteasomal degradation and decreasing efficiency of the assembly of native enzyme. Decreased Na-K-ATPase expression contributes to H. pylori-induced gastric injury.


Assuntos
Retículo Endoplasmático/enzimologia , Células Epiteliais/enzimologia , Mucosa Gástrica/enzimologia , Gastrite/enzimologia , Proteínas de Choque Térmico/metabolismo , Infecções por Helicobacter/enzimologia , Helicobacter pylori/patogenicidade , ATPase Trocadora de Sódio-Potássio/metabolismo , Células Cultivadas , Retículo Endoplasmático/microbiologia , Chaperona BiP do Retículo Endoplasmático , Estabilidade Enzimática , Células Epiteliais/microbiologia , Mucosa Gástrica/microbiologia , Gastrite/genética , Gastrite/microbiologia , Infecções por Helicobacter/genética , Infecções por Helicobacter/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Proteólise , ATPase Trocadora de Sódio-Potássio/genética , Ubiquitinação
5.
J Pediatr Gastroenterol Nutr ; 68(5): 689-694, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30540707

RESUMO

OBJECTIVE: Recent studies show increased serum and esophageal IgG4 in patients with eosinophilic esophagitis (EoE), suggesting a possible IgG4-involved process. The role of IgG4 in pediatric EoE has not been extensively investigated. Our aim was to analyze IgG4 in esophageal tissue in children in parallel to that in adults with EoE. METHODS: In a retrospective institutional review board-approved study, we performed immunohistochemical staining of IgG4 in esophageal biopsy specimens from 39 subjects: children with EoE (n = 16), adults with EoE (n = 15), children with reflux esophagitis (n = 4), and pediatric controls (n = 4). We assessed the relationships between IgG4 staining and clinical, endoscopic, and histopathologic characteristics. RESULTS: Patients with EoE were significantly more likely to stain positively for IgG4 than children with reflux esophagitis or controls (P = 0.015). Fifteen of 31 (48%) EoE cases stained positively for IgG4. None of the reflux esophagitis or control cases stained positively. IgG4 staining had 48% sensitivity and 100% specificity for EoE. There was a trend toward IgG4 staining being associated with foreign body/food impaction (P = 0.153). There was a strong association between distal IgG4 staining and basal zone hyperplasia (P = 0.003). CONCLUSIONS: Our study suggests IgG4 is not a consistent finding of EoE at disease diagnosis. Although IgG4 staining was specific for EoE, it had a poor sensitivity with positive staining in only 48% of EoE patients. Further studies are warranted to fully elucidate the role of IgG4 in EoE.


Assuntos
Esofagite Eosinofílica/diagnóstico , Esofagite Péptica/diagnóstico , Esôfago/química , Imunoglobulina G/análise , Imuno-Histoquímica/estatística & dados numéricos , Adolescente , Adulto , Idoso , Estudos de Casos e Controles , Criança , Esofagoscopia/estatística & dados numéricos , Esôfago/patologia , Feminino , Humanos , Imuno-Histoquímica/métodos , Masculino , Pessoa de Meia-Idade , Reprodutibilidade dos Testes , Estudos Retrospectivos , Sensibilidade e Especificidade , Adulto Jovem
6.
J Bacteriol ; 200(14)2018 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-29735759

RESUMO

Helicobacter pylori is an organism known to colonize the normal human stomach. Previous studies have shown that the bacterium does this by elevating its periplasmic pH via the hydrolysis of urea. However, the value of the periplasmic pH was calculated indirectly from the proton motive force equation. To measure the periplasmic pH directly in H. pylori, we fused enhanced green fluorescent protein (EGFP) to the predicted twin-arginine signal peptides of HydA and KapA from H. pylori and TorA from Escherichia coli The fusion proteins were expressed in the H. pylori genome under the control of the cagA promoter. Confocal microscopic and cell fractionation/immunoblotting analyses detected TorA-EGFP in the periplasm and KapA-EGFP in both the periplasm and cytoplasm, while the mature form of HydA-EGFP was seen at low levels in the periplasm, with major cytoplasmic retention of the precursor form. With H. pylori expressing TorA-EGFP, we established a system to directly measure periplasmic pH based on the pH-sensitive fluorimetry of EGFP. These measurements demonstrated that the addition of 5 mM urea has little effect on the periplasmic pH at a medium pH higher than pH 6.5 but rapidly increases the periplasmic pH to pH 6.1 at an acidic medium pH (pH 5.0), corresponding to the opening of the proton-gated channel, UreI, and confirming the basis of gastric colonization. Measurements of the periplasmic pH in an HP0244 (FlgS)-deficient mutant of H. pylori expressing TorA-EGFP revealed a significant loss of the urea-dependent increase in the periplasmic pH at an acidic medium pH, providing additional evidence that FlgS is responsible for recruitment of urease to the inner membrane in association with UreI.IMPORTANCEHelicobacter pylori has been identified as the major cause of chronic superficial gastritis and peptic ulcer disease. In addition, persistent infection with H. pylori, which, if untreated, lasts for the lifetime of an infected individual, predisposes one to gastric malignancies, such as adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. A unique feature of the neutralophilic bacterium H. pylori is its ability to survive in the extremely acidic environment of the stomach through its acid acclimation mechanism. The presented results on measurements of periplasmic pH in H. pylori based on fluorimetry of fully active green fluorescent protein fusion proteins exported with the twin-arginine translocase system provide a reliable and rapid tool for the investigation of acid acclimation in H. pylori.


Assuntos
Fluorometria/métodos , Proteínas de Fluorescência Verde/metabolismo , Helicobacter pylori/metabolismo , Antígenos de Bactérias , Proteínas de Bactérias , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/fisiologia , Helicobacter pylori/genética , Concentração de Íons de Hidrogênio , Mutação , Regiões Promotoras Genéticas , Ureia/metabolismo , Ureia/farmacologia
7.
J Cell Sci ; 129(12): 2394-406, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27142834

RESUMO

FXYD5 (also known as dysadherin), a regulatory subunit of the Na,K-ATPase, impairs intercellular adhesion by a poorly understood mechanism. Here, we determined whether FXYD5 disrupts the trans-dimerization of Na,K-ATPase molecules located in neighboring cells. Mutagenesis of the Na,K-ATPase ß1 subunit identified four conserved residues, including Y199, that are crucial for the intercellular Na,K-ATPase trans-dimerization and adhesion. Modulation of expression of FXYD5 or of the ß1 subunit with intact or mutated ß1-ß1 binding sites demonstrated that the anti-adhesive effect of FXYD5 depends on the presence of Y199 in the ß1 subunit. Immunodetection of the plasma membrane FXYD5 was prevented by the presence of O-glycans. Partial FXYD5 deglycosylation enabled antibody binding and showed that the protein level and the degree of O-glycosylation were greater in cancer than in normal cells. FXYD5-induced impairment of adhesion was abolished by both genetic and pharmacological inhibition of FXYD5 O-glycosylation. Therefore, the extracellular O-glycosylated domain of FXYD5 impairs adhesion by interfering with intercellular ß1-ß1 interactions, suggesting that the ratio between FXYD5 and α1-ß1 heterodimer determines whether the Na,K-ATPase acts as a positive or negative regulator of intercellular adhesion.


Assuntos
Glicoproteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Multimerização Proteica , Subunidades Proteicas/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Células A549 , Aminoácidos/metabolismo , Animais , Especificidade de Anticorpos , Adesão Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Cães , Células Epiteliais/metabolismo , Técnicas de Silenciamento de Genes , Glicosilação , Células HEK293 , Humanos , Canais Iônicos , Células Madin Darby de Rim Canino , Camundongos , Proteínas dos Microfilamentos , Ligação Proteica , Subunidades Proteicas/química , Ratos , ATPase Trocadora de Sódio-Potássio/química
8.
Clin Transplant ; 32(8): e13313, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29888807

RESUMO

Post-transplant lymphoproliferative disease (PTLD) has the highest incidence following intestinal transplantation (ITx). Our center has seen a recent increase in PTLD. Our aim was to review a single-center PTLD experience with a focus on clinical characteristics and outcomes. We completed a retrospective review of biopsy-proven PTLD cases using a prospectively maintained database of 115 ITx recipients transplanted between 1991 and 2014. Nineteen (17%) ITx recipients developed 25 PTLD cases during a median follow-up time of 6.4 (1.6-14.6) years. The incidence of early PTLD was 6% (n = 7). There was a trend toward increased risk of PTLD in children compared with adults (P = .11) and a significantly increased risk of PTLD in re-ITx compared with primary ITx recipients (P = .03). Most PTLD cases were diagnosed between 2010 and 2014 (n = 14). All early PTLD cases were EBV+ on in situ hybridization. Overall graft and patient survival are 68% and 74%, respectively. Second episodes of PTLD were diagnosed in 43% of surviving pediatric patients. Our program has a low incidence of early PTLD with overall excellent graft and patient survival following diagnosis. However, we have also seen a rising incidence of late PTLD. The cause of the increase is unknown as no major changes in immunosuppression protocols have occurred since 1999.


Assuntos
Intestinos/transplante , Transtornos Linfoproliferativos/mortalidade , Transtornos Linfoproliferativos/patologia , Complicações Pós-Operatórias/mortalidade , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Lactente , Transtornos Linfoproliferativos/cirurgia , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Prospectivos , Estudos Retrospectivos , Taxa de Sobrevida
9.
Helicobacter ; 23(3): e12490, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29696729

RESUMO

BACKGROUND: The pathogen Helicobacter pylori encounters many stressors as it transits to and infects the gastric epithelium. Gastric acidity is the predominate stressor encountered by the bacterium during initial infection and establishment of persistent infection. H. pylori initiates a rapid response to acid to maintain intracellular pH and proton motive force appropriate for a neutralophile. However, acid sensing by H. pylori may also serve as a transcriptional trigger to increase the levels of other pathogenic factors needed to subvert host defenses such as acid acclimation, antioxidants, flagellar synthesis and assembly, and CagA secretion. MATERIALS AND METHODS: Helicobacter pylori were acid challenged at pH 3.0, 4.5, 6.0 vs nonacidic pH for 4 hours in the presence of urea, followed by RNA-seq analysis and qPCR. Cytoplasmic pH was monitored under the same conditions. RESULTS: About 250 genes were induced, and an equal number were repressed at acidic pHs. Genes encoding for antioxidant proteins, flagellar structural proteins, particularly class 2 genes, T4SS/Cag-PAI, Fo F1 -ATPase, and proteins involved in acid acclimation were highly expressed at acidic pH. Cytoplasmic pH decreased from 7.8 at pHout of 8.0 to 6.0 at pHout of 3.0. CONCLUSIONS: These results suggest that increasing extracellular or intracellular acidity or both are detected by the bacterium and serve as a signal to initiate increased production of protective and pathogenic factors needed to counter host defenses for persistent infection. These changes are dependent on degree of acidity and time of acid exposure, triggering a coordinated response to the environment required for colonization.


Assuntos
Ácidos/metabolismo , Regulação Bacteriana da Expressão Gênica , Helicobacter pylori/genética , Estômago/microbiologia , Transcriptoma/genética , Adaptação Fisiológica , Proteínas de Bactérias/genética , Meios de Cultura , Helicobacter pylori/fisiologia , Concentração de Íons de Hidrogênio , Família Multigênica , Proteoma/genética , RNA Bacteriano/genética , Urease/genética , Fatores de Virulência/genética
10.
Mod Pathol ; 30(11): 1567-1576, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28752846

RESUMO

One of the major challenges in biomarker development is the collection of tumor tissue of adequate quality for analysis. A prospective clinical trial was initiated to collect tissues from triple negative breast cancers prior to and after neoadjuvant chemotherapy in order to study the mechanisms of chemoresistance. Sixty patients had pre-chemotherapy biopsies performed by either a surgeon or a radiologist, while those with residual tumor after chemotherapy had research-only biopsies and/or surgical samples collected in liquid nitrogen, RNA-later and formalin. We examined each core for tumor cellularity, stromal content, and necrosis after which, RNA and DNA extraction was performed. We found that biopsies collected with ultrasound guidance were more likely to contain tumor than those collected by the surgeon. Patient reluctance to undergo research-only biopsies after chemotherapy was not a problem. Pre-chemotherapy tumor biopsies frequently did not contain any tumor cells (15%) or did not have ≥50% tumor content (63%). Indeed, 50% of patients had at least 2 pre-chemotherapy core biopsies with <50% tumor content. After chemotherapy, 30% of biopsy or surgical samples in patients with incomplete response did not contain any tumor. Finally, RNA-later not only made histopathological assessment of tumor content difficult, but yielded less DNA than fresh snap frozen samples. We recommend that high-quality tissue procurement can be best accomplished if at least three image-guided core biopsies be obtained per sample, each of these cores be examined for tumor cellularity and that at least some of them be freshly snap frozen in liquid nitrogen.


Assuntos
Biomarcadores Tumorais/análise , Biópsia Guiada por Imagem/métodos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Ultrassonografia de Intervenção/métodos , Adulto , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biópsia/métodos , Quimioterapia Adjuvante , Feminino , Humanos , Pessoa de Meia-Idade , Terapia Neoadjuvante
11.
Biochem J ; 473(12): 1703-18, 2016 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27048593

RESUMO

Septins are a family of cytoskeletal GTP-binding proteins that assemble into membrane-associated hetero-oligomers and organize scaffolds for recruitment of cytosolic proteins or stabilization of membrane proteins. Septins have been implicated in a diverse range of cancers, including gastric cancer, but the underlying mechanisms remain unclear. The hypothesis tested here is that septins contribute to cancer by stabilizing the receptor tyrosine kinase ErbB2, an important target for cancer treatment. Septins and ErbB2 were highly over-expressed in gastric cancer cells. Immunoprecipitation followed by MS analysis identified ErbB2 as a septin-interacting protein. Knockdown of septin-2 or cell exposure to forchlorfenuron (FCF), a well-established inhibitor of septin oligomerization, decreased surface and total levels of ErbB2. These treatments had no effect on epidermal growth factor receptor (EGFR), emphasizing the specificity and functionality of the septin-ErbB2 interaction. The level of ubiquitylated ErbB2 at the plasma membrane was elevated in cells treated with FCF, which was accompanied by a decrease in co-localization of ErbB2 with septins at the membrane. Cathepsin B inhibitor, but not bafilomycin or lactacystin, prevented FCF-induced decrease in total ErbB2 by increasing accumulation of ubiquitylated ErbB2 in lysosomes. Therefore, septins protect ErbB2 from ubiquitylation, endocytosis and lysosomal degradation. The FCF-induced degradation pathway is distinct from and additive with the degradation induced by inhibiting ErbB2 chaperone Hsp90. These results identify septins as novel regulators of ErbB2 expression that contribute to the remarkable stabilization of the receptor at the plasma membrane of cancer cells and may provide a basis for the development of new ErbB2-targeting anti-cancer therapies.


Assuntos
Receptor ErbB-2/metabolismo , Septinas/metabolismo , Neoplasias Gástricas/metabolismo , Western Blotting , Linhagem Celular Tumoral , Cromatografia Líquida , Citoesqueleto/metabolismo , Humanos , Imunoprecipitação , Compostos de Fenilureia/farmacologia , Ligação Proteica/efeitos dos fármacos , Piridinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/fisiologia , Receptor ErbB-2/genética , Septinas/antagonistas & inibidores , Septinas/genética , Transdução de Sinais/fisiologia , Espectrometria de Massas em Tandem , Ubiquitinação/efeitos dos fármacos
12.
J Biol Chem ; 290(9): 5280-97, 2015 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-25575596

RESUMO

Septins are a family of 14 cytoskeletal proteins that dynamically form hetero-oligomers and organize membrane microdomains for protein complexes. The previously reported interactions with SNARE proteins suggested the involvement of septins in exocytosis. However, the contradictory results of up- or down-regulation of septin-5 in various cells and mouse models or septin-4 in mice suggested either an inhibitory or a stimulatory role for these septins in exocytosis. The involvement of the ubiquitously expressed septin-2 or general septin polymerization in exocytosis has not been explored to date. Here, by nano-LC with tandem MS and immunoblot analyses of the septin-2 interactome in mouse brain, we identified not only SNARE proteins but also Munc-18-1 (stabilizes assembled SNARE complexes), N-ethylmaleimide-sensitive factor (NSF) (disassembles SNARE complexes after each membrane fusion event), and the chaperones Hsc70 and synucleins (maintain functional conformation of SNARE proteins after complex disassembly). Importantly, α-soluble NSF attachment protein (SNAP), the adaptor protein that mediates NSF binding to the SNARE complex, did not interact with septin-2, indicating that septins undergo reorganization during each exocytosis cycle. Partial depletion of septin-2 by siRNA or impairment of septin dynamics by forchlorfenuron inhibited constitutive and stimulated exocytosis of secreted and transmembrane proteins in various cell types. Forchlorfenuron impaired the interaction between SNAP-25 and its chaperone Hsc70, decreasing SNAP-25 levels in cultured neuroendocrine cells, and inhibited both spontaneous and stimulated acetylcholine secretion in mouse motor neurons. The results demonstrate a stimulatory role of septin-2 and the dynamic reorganization of septin oligomers in exocytosis.


Assuntos
Encéfalo/metabolismo , Exocitose , Proteoma/metabolismo , Septinas/metabolismo , Animais , Western Blotting , Encéfalo/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Cães , Feminino , Células HEK293 , Humanos , Células Madin Darby de Rim Canino , Masculino , Camundongos Endogâmicos BALB C , Microscopia Confocal , Células PC12 , Compostos de Fenilureia/farmacologia , Ligação Proteica/efeitos dos fármacos , Multimerização Proteica , Proteômica , Piridinas/farmacologia , Interferência de RNA , Ratos , Septinas/química , Septinas/genética , Proteína 25 Associada a Sinaptossoma/metabolismo
13.
Helicobacter ; 21(1): 69-81, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25997502

RESUMO

BACKGROUND: The pH-sensitive Helicobacter pylori ArsRS two-component system (TCS) aids survival of this neutralophile in the gastric environment by directly sensing and responding to environmental acidity. ArsS is required for acid-induced trafficking of urease and its accessory proteins to the inner membrane, allowing rapid, urea-dependent cytoplasmic and periplasmic buffering. Expression of ArsR, but not its phosphorylation, is essential for bacterial viability. The aim of this study was to characterize the roles of ArsS and ArsR in the response of H. pylori to acid. MATERIALS AND METHODS: Wild-type H. pylori and an arsR(D52N) phosphorylation-deficient strain were incubated at acidic or neutral pH. Gene and protein expression, survival, membrane trafficking of urease proteins, urease activity, and internal pH were studied. RESULTS: Phosphorylation of ArsR is not required for acid survival. ArsS-driven trafficking of urease proteins to the membrane in acid, required for recovery of internal pH, is independent of ArsR phosphorylation. ArsR phosphorylation increases expression of the urease gene cluster, and the loss of negative feedback in a phosphorylation-deficient mutant leads to an increase in total urease activity. CONCLUSIONS: ArsRS has a dual function in acid acclimation: regulation of urease trafficking to UreI at the cytoplasmic membrane, driven by ArsS, and regulation of urease gene cluster expression, driven by phosphorylation of ArsR. ArsS and ArsR work through phosphorylation-dependent and phosphorylation-independent regulatory mechanisms to impact acid acclimation and allow gastric colonization. Furthering understanding of the intricacies of acid acclimation will impact the future development of targeted, nonantibiotic treatment regimens.


Assuntos
Ácidos/metabolismo , Proteínas de Bactérias/metabolismo , Infecções por Helicobacter/microbiologia , Helicobacter pylori/metabolismo , Proteínas de Bactérias/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Regulação Bacteriana da Expressão Gênica , Helicobacter pylori/genética , Humanos , Fosforilação , Transporte Proteico , Urease/genética , Urease/metabolismo
14.
Curr Gastroenterol Rep ; 18(7): 33, 2016 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-27177639

RESUMO

Helicobacter pylori infects about 50 % of the world's population, causing at a minimum chronic gastritis. A subset of infected patients will ultimately develop gastric or duodenal ulcer disease, gastric adenocarcinoma, or MALT (mucosa-associated lymphoid tissue) lymphoma. Eradication of H. pylori requires complex regimens that include acid suppression and multiple antibiotics. The efficacy of treatment using what were once considered standard regimens have declined in recent years, mainly due to widespread development of antibiotic resistance. Addition of bismuth to standard triple therapy regimens, use of alternate antibiotics, or development of alternative regimens using known therapies in novel combinations have improved treatment efficacy in specific populations, but overall success of eradication remains less than ideal. Novel regimens under investigation either in vivo or in vitro, involving increased acid suppression ideally with fewer antibiotics or development of non-antibiotic treatment targets, show promise for future therapy.


Assuntos
Gastrite/tratamento farmacológico , Gastrite/microbiologia , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori , Antibacterianos/uso terapêutico , Bismuto/uso terapêutico , Doença Crônica , Farmacorresistência Bacteriana , Quimioterapia Combinada , Humanos , Inibidores da Bomba de Prótons/uso terapêutico
15.
J Pediatr Gastroenterol Nutr ; 61(6): 636-40, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26020374

RESUMO

OBJECTIVES: Biopsies remain the criterion standard in the diagnosis of intestinal transplant (ITx) rejection, and gastrointestinal endoscopy plays a pivotal role in patient management. Herein, we describe a single-center 23-year endoscopic experience in pediatric ITx recipients. METHODS: A retrospective review of endoscopy and pathology reports of all ITx recipients <18 years old transplanted between 1991 and 2013 was performed with the aim of describing the procedural indications, findings, and complications. RESULTS: A total of 1770 endoscopic procedures within 1014 sessions were performed. A combination of esophagogastroduodenoscopy and ileoscopy was the most common procedure (36%). Increased stool output (35%) and surveillance endoscopy (32%) were the most common indications. A total of 162 episodes of biopsy-proven rejection were diagnosed. The first episode of rejection occurred at a median of 1 month after ITx. Of histology-proven rejections, 45% had normal-appearing endoscopies. The rate of procedural complications, including but not limited to bleeding and perforation, was 1.8%. CONCLUSIONS: Endoscopy with biopsy plays a significant role in the care of ITx recipients. Multiple procedures are required for graft surveillance, diagnosis of rejection, subsequent treatment, and follow-up of therapy. The gross endoscopic appearance, particularly in mild to moderate acute cellular rejection, does not correlate well with histology. Complex anatomy, complication rates that are higher than patients with non-ITx pediatric endoscopy, and timely histologic interpretation by experienced pathologists are reasons that these procedures should be performed at centers accustomed to caring for ITx recipients. The field would benefit from the development of a noninvasive biomarker to reliably and efficiently detect rejection.


Assuntos
Endoscopia Gastrointestinal/métodos , Rejeição de Enxerto , Intestinos/cirurgia , Transplante de Órgãos , Adolescente , Biópsia , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Intestinos/patologia , Masculino , Estudos Retrospectivos
16.
Breast J ; 20(3): 258-66, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24673796

RESUMO

Idiopathic granulomatous mastitis (IGM) is an infrequently reported benign breast disease of unknown etiology. Surgical treatment has been widely advocated but can be disfiguring. We describe demographic and clinico-pathologic features of women with IGM in a safety net hospital, and evaluate steroid therapy as a breast-conserving modality of treatment. We also examine a possible ethnic predominance in Hispanic women. We conducted an observational prospective cohort study of all women with biopsy-proven granulomatous mastitis in the breast clinics of an urban safety net public hospital from 2006 to 2010. Demographic, ethnic and clinical data, treatment history, and response to treatment were collected. Patients were followed up prospectively to determine the type of treatment prescribed, complete resolution of disease, and median time to resolution. A nested case-control study was conducted to examine Hispanic prevalence using chi-square statistic. The mean age was 35 years. 80% were Hispanic. 80% presented with a painful breast mass. 59% initially received antibiotics with incomplete resolution. 90% women were prescribed oral steroids, 3% underwent surgical treatment, and 6% remained under observation with spontaneous resolution. Of those who received steroid, 80% had complete resolution of disease with a median time to complete resolution of 159 days (IQR 120-241 days). Ethnicity data in a nested case-control study revealed that women in the IGM group were more likely to be Hispanic than in the control group with an odds ratio of 3 (95% CI 1.42-6.24, p-value 0.0032). IGM is a benign but locally aggressive breast disease. Treatment with steroids is an effective breast-conserving option. Predominance in Hispanic women of childbearing age suggests a common genetic, environmental, immunologic, or infectious etiology and warrants further study with a multi-disciplinary approach.


Assuntos
Mastite Granulomatosa/tratamento farmacológico , Prednisona/uso terapêutico , Adulto , Idoso , Anti-Inflamatórios/uso terapêutico , Estudos de Casos e Controles , Feminino , Hispânico ou Latino , Humanos , México/etnologia , Pessoa de Meia-Idade , Estudos Prospectivos , Resultado do Tratamento , Adulto Jovem
17.
Biomolecules ; 14(7)2024 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-39062486

RESUMO

Helicobacter pylori is a highly prevalent human gastric pathogen that causes gastritis, ulcer disease, and gastric cancer. It is not yet fully understood how H. pylori injures the gastric epithelium. The Na,K-ATPase, an essential transporter found in virtually all mammalian cells, has been shown to be important for maintaining the barrier function of lung and kidney epithelia. H. pylori decreases levels of Na,K-ATPase in the plasma membrane of gastric epithelial cells, and the aim of this study was to demonstrate that this reduction led to gastric injury by impairing the epithelial barrier. Similar to H. pylori infection, the inhibition of Na,K-ATPase with ouabain decreased transepithelial electrical resistance and increased paracellular permeability in cell monolayers of human gastric cultured cells, 2D human gastric organoids, and gastric epithelium isolated from gerbils. Similar effects were caused by a partial shRNA silencing of Na,K-ATPase in human gastric organoids. Both H. pylori infection and ouabain exposure disrupted organization of adherens junctions in human gastric epithelia as demonstrated by E-cadherin immunofluorescence. Functional and structural impairment of epithelial integrity with a decrease in Na,K-ATPase amount or activity provides evidence that the H. pylori-induced downregulation of Na,K-ATPase plays a role in the complex mechanism of gastric disease induced by the bacteria.


Assuntos
Mucosa Gástrica , Infecções por Helicobacter , Helicobacter pylori , Ouabaína , ATPase Trocadora de Sódio-Potássio , ATPase Trocadora de Sódio-Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/genética , Humanos , Animais , Ouabaína/farmacologia , Infecções por Helicobacter/metabolismo , Infecções por Helicobacter/microbiologia , Infecções por Helicobacter/patologia , Mucosa Gástrica/metabolismo , Mucosa Gástrica/microbiologia , Mucosa Gástrica/patologia , Mucosa Gástrica/efeitos dos fármacos , Gerbillinae , Membrana Celular/metabolismo , Membrana Celular/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/efeitos dos fármacos , Organoides/metabolismo , Organoides/microbiologia
18.
Gastroenterol Clin North Am ; 53(3): 453-459, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-39068006

RESUMO

The history of intestinal transplantation can be traced back to the turn of the twentieth century. Although advancements have been made, the intestine still presents a greater challenge to transplantation than does that of other solid organs, experiencing higher rates of graft rejection and lower long-term survival. Increasingly, intestinal re-transplantation (re-ITx) is seen as a viable option and is now the fourth most common indication for ITx. Changes to immunosuppression protocols, technical modifications, and infectious disease monitoring have contributed to improved outcomes. The authors review the literature on re-ITx in regard to the history, management considerations, and future directions.


Assuntos
Rejeição de Enxerto , Intestinos , Reoperação , Humanos , Intestinos/transplante , Rejeição de Enxerto/prevenção & controle , Imunossupressores/uso terapêutico , Transplante de Órgãos/métodos , Sobrevivência de Enxerto
19.
Am J Physiol Gastrointest Liver Physiol ; 305(10): G731-9, 2013 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-23989011

RESUMO

Gastric infection by Helicobacter pylori is the most common cause of ulcer disease and gastric cancer. The mechanism of progression from gastritis and inflammation to ulcers and cancer in a fraction of those infected is not definitively known. Significant acidity is unique to the gastric environment and is required for ulcer development. The interplay between gastric acidity and H. pylori pathogenesis is important in progression to advanced disease. The aim of this study was to characterize the impact of acid on gastric epithelial integrity and cytokine release and how H. pylori infection alters these responses. Human gastric epithelial (HGE-20) cells were grown on porous inserts, and survival, barrier function, and cytokine release were studied at various apical pH levels in the presence and absence of H. pylori. With apical acidity, gastric epithelial cells demonstrate increased barrier function, as evidenced by increased transepithelial electrical resistance (TEER) and decreased paracellular permeability. This effect is reduced in the presence of wild-type, but not urease knockout, H. pylori. The epithelial inflammatory response is also modulated by acidity and H. pylori infection. Without H. pylori, epithelial IL-8 release decreases in acid, while IL-6 release increases. In the presence of H. pylori, acidic pH diminishes the magnitude of the previously reported increase in IL-8 and IL-6 release. H. pylori interferes with the gastric epithelial response to acid, contributing to altered barrier function and inflammatory response. H. pylori diminishes acid-induced tightening of cell junctions in a urease-dependent manner, suggesting that local pH elevation promotes barrier compromise and progression to mucosal damage.


Assuntos
Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Helicobacter pylori/metabolismo , Linhagem Celular Tumoral , Meios de Cultura/química , Impedância Elétrica , Fenômenos Eletrofisiológicos , Regulação Bacteriana da Expressão Gênica , Helicobacter pylori/genética , Humanos , Concentração de Íons de Hidrogênio
20.
Helicobacter ; 18(5): 363-72, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23600974

RESUMO

BACKGROUND: Helicobacter pylori, a neutralophile, colonizes the acidic environment of the human stomach by employing acid acclimation mechanisms that regulate periplasmic and cytoplasmic pH. The regulation of urease activity is central to acid acclimation. Inactive urease apoenzyme, UreA/B, requires nickel for activation. Accessory proteins UreE, F, G, and H are required for nickel insertion into apoenzyme. The ExbB/ExbD/TonB complex transfers energy from the inner to outer membrane, providing the driving force for nickel uptake. Therefore, the aim of this study was to determine the contribution of ExbD to pH homeostasis. MATERIALS AND METHODS: A nonpolar exbD knockout was constructed and survival, growth, urease activity, and membrane potential were determined in comparison with wildtype. RESULTS: Survival of the ΔexbD strain was significantly reduced at pH 3.0. Urease activity as a function of pH and UreI activation was similar to the wildtype strain, showing normal function of the proton-gated urea channel, UreI. The increase in total urease activity over time in acid seen in the wildtype strain was abolished in the ΔexbD strain, but recovered in the presence of supraphysiologic nickel concentrations, demonstrating that the effect of the ΔexbD mutant is due to loss of a necessary constant supply of nickel. In acid, ΔexbD also decreased its ability to maintain membrane potential and periplasmic buffering in the presence of urea. CONCLUSIONS: ExbD is essential for maintenance of periplasmic buffering and membrane potential by transferring energy required for nickel uptake, making it a potential nonantibiotic target for H. pylori eradication.


Assuntos
Adaptação Fisiológica , Proteínas de Bactérias/metabolismo , Helicobacter pylori/fisiologia , Homeostase , Proteínas de Bactérias/genética , Membrana Celular/fisiologia , Técnicas de Inativação de Genes , Helicobacter pylori/efeitos dos fármacos , Helicobacter pylori/genética , Helicobacter pylori/crescimento & desenvolvimento , Humanos , Concentração de Íons de Hidrogênio , Potenciais da Membrana , Viabilidade Microbiana/efeitos dos fármacos , Urease/metabolismo
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