Helicobacter pylori—Associated Dyspepsia in Paediatrics

Helicobacter pylori ubiquitously infects the human gastric mucosa since time immemorial,predictably before the man’s diaspora out of East Africa around 58,000 years ago [1].Colonization may have been somehow beneficial for human carriers, allowing the coevolutionof this gram-negative bacterium and its host over the centuries. Yet, at leastnowadays [2], this may not be a peaceful association, with infection almost invariablycausing an acute host immune response. However, in a fully adapted manner, H. pyloriavoids recognition and, thus, clearance, by the host immune system, with both infectionand the consequent gastritis persisting throughout the patients’ life. The clinical outcomeof this persistence is dependent on a sophisticated crosstalk between the host and thepathogen. If often asymptomatic, the H. pylori-associated non-ulcer dyspepsia is clearly thestrongest aetiological factor for severe gastric diseases that will develop late in adult life ina minority of infected patients, i.e., peptic ulcer disease, both gastric and duodenal ulcers,and gastric cancer, namely, adenocarcinoma and mucosa associated lymphoid tissue(MALT) lymphoma (reviewed in [3]). Peptic ulcer disease rarely occurs soon after H. pyloriinfection [4-8] that generally starts in childhood; this presumably reflects marked differencesin the virulence [9-16] and/or in the susceptibility of young patients [17-19].This chapter, focussing on the paediatric population, seeks to explore: the prevalence of H.pylori infection; the molecular mechanism used by H. pylori during colonization and infection;the role of this bacterium in the development of peptic ulcer-related organic dyspepsia; andthe genetic/proteome profile of the H. pylori-strains associated with peptic ulcer disease.BNP Paribas, Sociedade Portuguesa de Gastrenterologi

The Zebrafish Kupffer’s Vesicle: A Special Organ in a Model Organism to Study Human Diseases

The Kupffer’s vesicle (KV) is a small, ciliated organ transiently present during embryogenesis of the zebrafish and other teleosts. The KV is required to the establishment of visceral laterality, such as the heart on the left side, being also known by the name left-right organizer (LRO). The LRO is found in other vertebrates, including mice, rabbits, frogs and human embryos. Among these, the KV became an excellent model organ to investigate the early left-right events during development and in disease. Many ciliary molecular players associated to the human disease primary ciliary dyskinesia have been tested in the zebrafish looking at KV cilia and its downstream effects on flow and left-right markers. Additionally, given its morphology and molecular features, we proposed the KV as a model organ to study the molecular mechanisms of the renal cyst inflation that occurs in the autosomal dominant polycystic kidney disease. Although having no connection to the kidney, the KV mimics a renal cyst because it is a fluid-filled vesicle, lined by monociliated epithelial cells that express polycystin-2, which knockdown leads to the organ luminal enlargement through changes in ion/water epithelial transport. Here, we explore the usefulness of the zebrafish KV to model these diseases

Microtubule cytoskeleton perturbation induced by taxol and colchicine affects chaperonin containing TCP-1 (CCT) subunit gene expression in Tetrahymena cells

We report the existence of a CCTϵ subunit gene that encodes subunit ϵ of the chaperonin CCT (chaperonin containing TCP-1) in Tetrahymena pyriformis. This work focuses on the study of the effects of the microtubule polymerizing agent taxol and the depolymerizing agent colchicine on microtubule dynamics and their role in the regulation of tubulin and CCT subunit genes. Under taxol treatment some TpCCT and tubulin genes are distinctly expressed until 30 min of treatment. Cytoplasmic TpCCT mRNA levels slightly decrease while tubulin transcripts are increasing. In colchicine treated cells TpCCT and tubulin transcripts decrease in the initial 30 min of treatment and then start to increase. However, both antimitotic agents induce TpCCT and tubulin gene transcription. This induction does not correlate with increased steady-state levels of TpCCT proteins and seems to be necessary to replete cytoplasmic TpCCT mRNAs. Moreover, we found that TpCCTϵ and TpCCTα but not TpCCTη are present in the insoluble fraction after a postmitochondrial fractionation that contains components of the ciliate cortex structure, basal bodies and cilia. This suggests that some TpCCT subunits may be associated with these structures. The association of TpCCTϵ subunit is stimulated either by taxol or colchicine treatment. These observations support the idea that CCT subunits could have additional roles in vivo.info:eu-repo/semantics/publishedVersio

Screening of Prophage Sequences Among Helicobacter Pylori

Until recently, Helicobacter pylori was considered a bacterium without prophages. The presence of an incomplete prophage sequence in strain B38 and a complete prophage sequence in strain B45 showed otherwise. Using a PCR strategy, based on degenerated primers designed after aligning bacteriophage integrase genes from H. pylori strains B38 and B45, and H. acinonychis prophage II, we found that integrase sequence was present in 21.4% (73/341) of the H. pylori clinical strains tested. The phylogenetic analysis of the sequenced region revealed that strains cluster according to their geographic origin, but not to their pathology. We have applied the same methodology to additional 147 European strains and 77 African strains, determining the presence of integrase sequence in 25.2% (37/147) of the former and in 19.5% (15/77) of the latter. Currently, we have a total of 565 strains screened for the presence of integrase gene, with 125 positive for this sequence (22.1%). To understand if these integrase sequences belong to reminiscent or complete prophages we are also screening for the presence of other prophage coding sequences. Among integrase positive strains, we found 19.2% (5/26) positive strains for the primase sequence and 53.3% (8/15) for the presence of the end of the phage. Presently, we are running the sequencing of the PCR amplified products in order to conduct the phylogenetic analysis. The results reinforce the abundance of prophages sequences in H. pylori and suggest that the majority of them belong to reminiscent prophages integrated within the bacterium genome

Pkd2 Affects Cilia Length and Impacts LR Flow Dynamics and Dand5

Funding: This work was supported by the Fundação para a Ciência e a Tecnologia (FCT-ANR/BEX-BID/0153/2012 research grant). SL is funded by an FCT CEEC contract for Principal Investigator. The work was also supported by Sociedade Portuguesa de Nefrologia (research grant 2015), iNOVA4Health – UID/Multi/04462/2013 (a program financially supported by Fundação para a Ciência e Tecnologia/ Ministério da Educação e Ciência, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement) and Fundação para a Ciência e Tecnologia (research grant – PTDC/BEXBID/1411/2014). MR-R was supported by national funds through Fundação para a Ciência e Tecnologia, in the context of the celebration of the program contract foreseen in the numbers 4, 5, and 6 of article 23 of D.L. no. 57/2016 of 29 August, as amended by Law no. 57/2017 of 19 July. Zebrafish used as animal model were reared and maintained in the CEDOC Fish Facility, with the support from the research consortia Congento, co-financed by Lisboa Regional Operational Program (Lisboa2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and Fundação para a Ciência e TecnologiaThe left-right (LR) field recognizes the importance of the mechanism involving the calcium permeable channel Polycystin-2. However, whether the early LR symmetry breaking mechanism is exclusively via Polycystin-2 has not been tested. For that purpose, we need to be able to isolate the effects of decreasing the levels of Pkd2 protein from any eventual effects on flow dynamics. Here we demonstrate that curly-up (cup) homozygous mutants have abnormal flow dynamics. In addition, we performed one cell stage Pkd2 knockdowns and LR organizer specific Pkd2 knockdowns and observed that both techniques resulted in shorter cilia length and abnormal flow dynamics. We conclude that Pkd2 reduction leads to LR defects that cannot be assigned exclusively to its putative role in mediating mechanosensation because indirectly, by modifying cell shape or decreasing cilia length, Pkd2 deficit affects LR flow dynamics.publishersversionpublishe

New NCI-N87-derived human gastric epithelial line after human telomerase catalytic subunit over-expression

AIM: To establish a cellular model correctly mimicking the gastric epithelium to overcome the limitation in the study of Helicobacter pylori (H. pylori ) infection. METHODS: Aiming to overcome this limitation, clones of the heterogenic cancer-derived NCI-N87 cell line were isolated, by stably-transducing it with the human telomerase reverse-transcriptase (hTERT) catalytic subunit gene. The clones were first characterized regarding their cell growth pattern and phenotype. For that we measured the clones' adherence properties, expression of cell-cell junctions' markers (ZO-1 and E-cadherin) and ability to generate a sustained transepithelial electrical resistance. The gastric properties of the clones, concerning expression of mucins, zymogens and glycan contents, were then evaluated by haematoxylin and eosin staining, Periodic acid Schiff (PAS) and PAS/Alcian Blue-staining, immunocytochemistry and Western blot. In addition, we assessed the usefulness of the hTERT-expressing gastric cell line for H. pylori research, by performing co-culture assays and measuring the IL-8 secretion, by ELISA, upon infection with two H. pylori strains differing in virulence. RESULTS: Compared with the parental cell line, the most promising NCI-hTERT-derived clones (CL5 and CL6) were composed of cells with homogenous phenotype, presented higher relative telomerase activities, better adhesion properties, ability to be maintained in culture for longer periods after confluency, and were more efficient in PAS-reactive mucins secretion. Both clones were shown to produce high amounts of MUC1, MUC2 and MUC13. NCI-hTERT-CL5 mucins were shown to be decorated with blood group H type 2 (BG-H), Lewis-x (Lex), Ley and Lea and, in a less extent, with BG-A antigens, but the former two antigens were not detected in the NCI-hTERT-CL6. None of the clones exhibited detectable levels of MUC6 nor sialylated Lex and Lea glycans. Entailing good gastric properties, both NCIhTERT- clones were found to produce pepsinogen-5 and human gastric lipase. The progenitor-like phenotype of NCI-hTERT-CL6 cells was highlighted by large nuclei and by the apical vesicular-like distribution of mucin 5AC and Pg5, supporting the accumulation of mucus-secreting and zymogens-chief mature cells functions. CONCLUSION: These traits, in addition to resistance to microaerobic conditions and good responsiveness to H. pylori co-culture, in a strain virulence-dependent manner, make the NCI-hTERT-CL6 a promising model for future in vitro studies.info:eu-repo/semantics/publishedVersio

New NCI-N87-derived human gastric epithelial line after human telomerase catalytic subunit over-expression

Supported by Grants from the Fundacao para a Ciencia e a Tecnologia (FCT, Portugal), No. PPCDT/SAL-IMI/57297/2004 and No. PTDC/BIM-MEC/1051/2012; The Swedish Cancer foundation; The Swedish Research Council, No. K2010-79X-21372-01-3; Forska utan djurforsok, Animal Free Research; and by Research fellowship 2011 from the Sociedade Portuguesa de Gastrenterologia (Portugal).AIM: To establish a cellular model correctly mimicking the gastric epithelium to overcome the limitation in the study of Helicobacter pylori (H. pylori) infection. METHODS: Aiming to overcome this limitation, clones of the heterogenic cancer-derived NCI-N87 cell line were isolated, by stably-transducing it with the human telomerase reverse-transcriptase (hTERT) catalytic subunit gene. The clones were first characterized regarding their cell growth pattern and phenotype. For that we measured the clones' adherence properties, expression of cell-cell junctions' markers (ZO-1 and E-cadherin) and ability to generate a sustained transepithelial electrical resistance. The gastric properties of the clones, concerning expression of mucins, zymogens and glycan contents, were then evaluated by haematoxylin and eosin staining, Periodic acid Schiff (PAS) and PAS/Alcian Blue-staining, immunocytochemistry and Western blot. In addition, we assessed the usefulness of the hTERT-expressing gastric cell line for H. pylori research, by performing co-culture assays and measuring the IL-8 secretion, by ELISA, upon infection with two H. pylori strains differing in virulence. RESULTS: Compared with the parental cell line, the most promising NCI-hTERT-derived clones (CL5 and CL6) were composed of cells with homogenous phenotype, presented higher relative telomerase activities, better adhesion properties, ability to be maintained in culture for longer periods after confluency, and were more efficient in PAS-reactive mucins secretion. Both clones were shown to produce high amounts of MUC1, MUC2 and MUC13. NCI-hTERT-CL5 mucins were shown to be decorated with blood group H type 2 (BG-H), Lewis-x (Le(x)), Le(y) and Le(a) and, in a less extent, with BG-A antigens, but the former two antigens were not detected in the NCI-hTERT-CL6. None of the clones exhibited detectable levels of MUC6 nor sialylated Le(x) and Le(a) glycans. Entailing good gastric properties, both NCI-hTERT-clones were found to produce pepsinogen-5 and human gastric lipase. The progenitor-like phenotype of NCI-hTERT-CL6 cells was highlighted by large nuclei and by the apical vesicular-like distribution of mucin 5AC and Pg5, supporting the accumulation of mucus-secreting and zymogens-chief mature cells functions. CONCLUSION: These traits, in addition to resistance to microaerobic conditions and good responsiveness to H. pylori co-culture, in a strain virulence-dependent manner, make the NCI-hTERT-CL6 a promising model for future in vitro studies.publishersversionpublishe

Zebrafish model as a screen to prevent cyst inflation in autosomal dominant polycystic kidney disease

Funding Information: Funding: This work was supported by Sociedade Portuguesa de Nefrologia, iNOVA4Health-UID/Multi/04462/2013 (a program financially supported by Fundação para a Ciência e Tecnolo-gia (FCT)/Ministério da Educação e Ciência, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement) and FCT-ANR/BEX-BID/0153/2012 and PTDC/BEX-BID/1411/2014 research grants. M.R.R. was supported by national funds through FCT, first with an iNOVA post-doctoral fellowship and, more recently, with a contract in the context of the celebration of the program contract foreseen in the numbers 4, 5, and 6 of article 23.◦ of D.L. no. 57/2016 of 29 August, as amended by Law no. 57/2017 of 19 July. S.S.L. had a FCT-Investigator contract, followed by NOVA NMS contracts and, at the moment, a FCT CEEC (Concurso Estímulo ao Emprego Científico) contract, as principal investigator. This article is supported by the LYSOCIL project. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 811087. Zebrafish used as an animal model were reproduced and maintained in the CEDOC Fish Facility, with the support from the research infrastructure Congento, co-financed by Lisboa Regional Operational Programme (Lisboa2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and FCT under the project LISBOA-01-0145-FEDER-022170. Funding Information: This work was supported by Sociedade Portuguesa de Nefrologia, iNOVA4Health-UID/Multi/04462/2013 (a program financially supported by Funda??o para a Ci?ncia e Tecnolo-gia (FCT)/Minist?rio da Educa??o e Ci?ncia, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement) and FCT-ANR/BEX-BID/0153/2012 and PTDC/BEX-BID/1411/2014 research grants. M.R.R. was supported by national funds through FCT, first with an iNOVA post-doctoral fellowship and, more recently, with a contract in the context of the celebration of the program contract foreseen in the numbers 4, 5, and 6 of article 23.? of D.L. no. 57/2016 of 29 August, as amended by Law no. 57/2017 of 19 July. S.S.L. had a FCT-Investigator contract, followed by NOVA NMS contracts and, at the moment, a FCT CEEC (Concurso Est?mulo ao Emprego Cient?fico) contract, as principal investigator. This article is supported by the LYSOCIL project. This project has received funding from the European Union?s Horizon 2020 research and innovation programme under grant agreement No 811087. Zebrafish used as an animal model were reproduced and maintained in the CEDOC Fish Facility, with the support from the research infrastructure Congento, co-financed by Lisboa Regional Operational Programme (Lisboa2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) and FCT under the project LISBOA-01-0145-FEDER-022170. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland.In autosomal dominant polycystic kidney disease (ADPKD), kidney cyst growth requires the recruitment of CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel that is defective in cystic fibrosis. We have been studying cyst inflation using the zebrafish Kupffer’s vesicle (KV) as model system because we previously demonstrated that knocking down polycystin 2 (PC2) induced a CFTR-mediated enlargement of the organ. We have now quantified the PC2 knock-down by showing that it causes a 73% reduction in the number of KV cilia expressing PC2. According to the literature, this is an essential event in kidney cystogenesis in ADPKD mice. Additionally, we demonstrated that the PC2 knockdown leads to a significant accumulation of CFTR-GFP at the apical region of the KV cells. Furthermore, we determined that KV enlargement is rescued by the injection of Xenopus pkd2 mRNA and by 100 µM tolvaptan treatment, the unique and approved pharmacologic approach for ADPKD management. We expected vasopressin V2 receptor antagonist to lower the cAMP levels of KV-lining cells and, thus, to inactivate CFTR. These findings further support the use of the KV as an in vivo model for screening compounds that may prevent cyst enlargement in this ciliopathy, through CFTR inhibition.publishersversionpublishe

BabA-mediated adherence of pediatric ulcerogenic H. pylori strains to gastric mucins at neutral and acidic pH

Funding This work was supported by the Familjen Erling-Perssons Stiftelse;Fundação para a Ciência e a Tecnologia [FCTPTDC/BIM-MEC/1051/2012];Swedish research council [521-2011-2370];Swedish research council [621-2014-4361]; Swedish cancer society;Åke Wibergs ;Ragnar Söderbergs stiftelse;Stiftelserna Wilhelm och Martina Lundgrens;Svenska Forskningsrådet Formas [221-2013-590];Svenska Forskningsrådet Formas [221-2011-1036].Helicobacter pylori infection can result in non-ulcer dyspepsia (NUD), peptic ulcer disease (PUD), adenocarcinoma, and gastric lymphoma. H. pylori reside within the gastric mucus layer, mainly composed of mucins carrying an array of glycan structures that can serve as bacterial adhesion epitopes. The aim of the present study was to characterize the binding ability, adhesion modes, and growth of H. pylori strains from pediatric patients with NUD and PUD to gastric mucins. Our results showed an increased adhesion capacity of pediatric PUD H. pylori strains to human and rhesus monkey gastric mucins compared to the NUD strains both at neutral and acidic pH, regardless if the mucins were positive for Lewis b (Leb), Sialyl-Lewis x (SLex) or LacdiNAc. In addition to babA positive strains being more common among PUD associated strains, H. pylori babA positive strains bound more avidly to gastric mucins than NUD babA positive strains at acidic pH. Binding to Leb was higher among babA positive PUD H. pylori strains compared to NUD strains at neutral, but not acidic, pH. PUD derived babA-knockout mutants had attenuated binding to mucins and Leb at acidic and neutral pH, and to SLex and DNA at acidic pH. The results highlight the role of BabA-mediated adherence of pediatric ulcerogenic H. pylori strains, and points to a role for BabA in adhesion to charged structures at acidic pH, separate from its specific blood group binding activity.publishersversionpublishe

Ulcerogenic Helicobacter pylori Strains Isolated from Children: A Contribution to Get Insight into the Virulence of the Bacteria

Infection with Helicobacter pylori is the major cause for the development of peptic ulcer disease (PUD). In children, with no other etiology for the disease, this rare event occurs shortly after infection. In these young patients, habits of smoking, diet, consumption of alcohol and non-steroid anti-inflammatory drugs and stress, in addition to the genetic susceptibility of the patient, represent a minor influence. Accordingly, the virulence of the implicated H. pylori strain should play a crucial role in the development of PUD. Corroborating this, our in vitro infection assays comparing a pool of five H. pylori strains isolated from children with PUD to a pool of five other pediatric clinical isolates associated with non-ulcer dyspepsia (NUD) showed the greater ability of PUD strains to induce a marked decrease in the viability of gastric cells and to cause severe damage in the cells cytoskeleton as well as an impairment in the production/secretion of mucins. To uncover virulence features, we compared the proteome of these two groups of H. pylori strains. Two-dimensional gel electrophoresis followed by mass-spectrometry allowed us to detect 27 differentially expressed proteins between them. In addition to the presence of genes encoding well established virulence factors, namely cagA, vacAs1, oipA “on” status, homB and jhp562 genes, the pediatric ulcerogenic strains shared a proteome profile characterized by changes in the abundance of: motility-associated proteins, accounting for higher motility; antioxidant proteins, which may confer increased resistance to inflammation; and enzymes involved in key steps in the metabolism of glucose, amino acids and urea, which may be advantageous to face fluctuations of nutrients. In conclusion, the enhanced virulence of the pediatric ulcerogenic H. pylori strains may result from a synergy between their natural ability to better adapt to the hostile human stomach and the expression of the established virulence factors