scholarly journals Probiotics-Containing Yogurt Ingestion and H. pylori Eradication Can Restore Fecal Faecalibacterium prausnitzii Dysbiosis in H. pylori-Infected Children

Biomedicines ◽  
2020 ◽  
Vol 8 (6) ◽  
pp. 146
Author(s):  
Yao-Jong Yang ◽  
Peng-Chieh Chen ◽  
Fu-Ping Lai ◽  
Pei-Jane Tsai ◽  
Bor-Shyang Sheu
Keyword(s):  
Ex Vivo ◽  
Fecal Microbiota ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Bacterial Eradication ◽  
H Pylori ◽  
The 16S Rrna Gene ◽  
Ht 29 ◽  
Tgf Β1

This study investigated the compositional differences in fecal microbiota between children with and without H. pylori infection and tested whether probiotics-containing yogurt and bacterial eradication improve H. pylori-related dysbiosis. Ten H. pylori-infected children and 10 controls ingested probiotics-containing yogurt for 4 weeks. Ten-day triple therapy plus yogurt was given to the infected children on the 4th week. Fecal samples were collected at enrollment, after yogurt ingestion, and 4 weeks after successful H. pylori eradication for cytokines and microbiota analysis using ELISA and metagenomic sequencing of the V4 region of the 16S rRNA gene, respectively. The results showed H. pylori-infected children had significantly higher levels of fecal TGF-β1 than those who were not infected. Eight of 295 significantly altered OTUs in the H. pylori-infected children were identified. Among them, the abundance of F. prausnitzii was significantly lower in the H. pylori-infected children, and then increased after yogurt ingestion and successful bacterial eradication. We further confirmed probiotics promoted F. prausnitzii growth in vitro and in ex vivo using real-time PCR. Moreover, F. prausnitzii supernatant significantly ameliorated lipopolysaccharide-induced IL-8 in HT-29 cells. In conclusions, Probiotics-containing yogurt ingestion and H. pylori eradication can restore the decrease of fecal F. prausnitzii in H. pylori-infected children.

scholarly journals In vitro evaluation of lactic acid bacteria with probiotic activity isolated from local pickled leaf mustard from Wuwei in Anhui as substitutes for chemical synthetic additives

2021 ◽  
Vol 19 (1) ◽  
pp. 755-771
Author(s):  
Changjun Wu ◽  
Xiaopei Lin ◽  
Lin Tong ◽  
Chenwei Dai ◽  
Han Lv ◽  
...  
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Animal Experiments ◽  
Rrna Gene ◽  
Intestinal Microbes ◽  
Potential Resource ◽  
The 16S Rrna Gene ◽  
Ht 29

Abstract The extensive abuse of chemical synthetic additives has raised increased attention to food safety. As substitutes, probiotics play an important role in human health as they balance the intestinal microbes in host. This study was aimed to isolate and evaluate the potential probiotic activities of lactic acid bacteria (LAB) from a local pickled leaf mustard (PLM) from Wuwei city in Anhui province through in vitro experiments. A total of 17 LAB strains were obtained as probiotics. All the isolates were sensitive to chloramphenicol, tetracycline, erythromycin, and doxycycline but exhibited resistance to antibiotics (e.g., streptomycin, kanamycin, gentamicin, and vancomycin). Out of the 17 strains, 9 were sensitive to most of the antibiotics and had no cytotoxic activity on human colorectal adenocarcinoma cell line (HT-29) cells. The isolated AWP4 exhibited antibacterial activity against four indicator pathogen strains (ATCC8099: Escherichia coli, ATCC6538: Staphylococcus aureus, ATCC9120: Salmonella enteric, and BNCC192105: Shigella sonnei). Based on the phylogenetic analysis of the 16S rRNA gene, AWP4 belonged to Lactiplantibacillus plantarum. This study indicated that the Wuwei local PLM could be a potential resource to isolate beneficial LAB as probiotics. The data provide theoretical guidance for further animal experiments to estimate the probiotic effect and safety of Lpb. plantarum AWP4 in vivo.

scholarly journals Novel strain-level resolution of Crohn’s disease mucosa-associated microbiota via an ex vivo combination of microbe culture and metagenomic sequencing

2021 ◽  
Author(s):  
J. J. Teh ◽  
E. M. Berendsen ◽  
E. C. Hoedt ◽  
S. Kang ◽  
J. Zhang ◽  
...  
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Ex Vivo ◽  
Taxonomic Composition ◽  
Strain Level ◽  
Patient Specific ◽  
Rrna Gene ◽  
Metagenomic Sequencing ◽  
Functional Characterisation ◽  
Bacterial Lineages

AbstractThe mucosa-associated microbiota is widely recognized as a potential trigger for Crohn’s disease pathophysiology but remains largely uncharacterised beyond its taxonomic composition. Unlike stool microbiota, the functional characterisation of these communities using current DNA/RNA sequencing approaches remains constrained by the relatively small microbial density on tissue, and the overwhelming amount of human DNA recovered during sample preparation. Here, we have used a novel ex vivo approach that combines microbe culture from anaerobically preserved tissue with metagenome sequencing (MC-MGS) to reveal patient-specific and strain-level differences among these communities in post-operative Crohn’s disease patients. The 16 S rRNA gene amplicon profiles showed these cultures provide a representative and holistic representation of the mucosa-associated microbiota, and MC-MGS produced both high quality metagenome-assembled genomes of recovered novel bacterial lineages. The MC-MGS approach also produced a strain-level resolution of key Enterobacteriacea and their associated virulence factors and revealed that urease activity underpins a key and diverse metabolic guild in these communities, which was confirmed by culture-based studies with axenic cultures. Collectively, these findings using MC-MGS show that the Crohn’s disease mucosa-associated microbiota possesses taxonomic and functional attributes that are highly individualistic, borne at least in part by novel bacterial lineages not readily isolated or characterised from stool samples using current sequencing approaches.

scholarly journals Spontaneous Mutations That Confer Antibiotic Resistance inHelicobacter pylori

2001 ◽  
Vol 45 (3) ◽  
pp. 727-733 ◽  
Author(s):  
Ge Wang ◽  
Trevor J. M. Wilson ◽  
Qin Jiang ◽  
Diane E. Taylor
Keyword(s):  
Mutant Allele ◽  
Rapid Development ◽  
Mutagenic Effect ◽  
Serial Passage ◽  
Sublethal Dose ◽  
23S Rrna ◽  
Rrna Gene ◽  
Spontaneous Mutations ◽  
H Pylori

ABSTRACT In this study, we systematically examined in vitro frequencies and spectra of the spontaneous mutations in Helicobacter pylori that confer resistance to clarithromycin (Clar), metronidazole (Mtzr), amoxicillin (Amxr), ciprofloxacin (Cipr), and rifampin (Rifr). The mutation rate of Rifror Cipr determined in a fluctuation assay is 1 × 10−8 to 2 × 10−8 per cell per division. In contrast, the mutation rates of Clar, Mtzr, and Amxr are much lower (<10−9). However, Mtzr mutants could be readily selected in vitro by using the serial passage method, suggesting that the mutagenic effect and selective effect of a sublethal dose of metronidazole contribute to the rapid development of Mtzr. Analysis of spontaneous Rifr, Clar, and Cipr mutants confirmed previous results indicating that mutations within therpoB gene, the 23S rRNA gene, and thegyrA gene, respectively, are responsible; also, several new mutant alleles were identified. Mtzrmutants resulted most frequently, but not always, from mutations in the rdxA gene. DNA fragments containing each mutant allele could readily transform susceptibleH. pylori strains to resistance, confirming that each mutant allele is responsible for the resistance phenotype.

scholarly journals Apoptotic cells induce CD103 expression and immunoregulatory function in myeloid dendritic cell precursors through integrin αv and TGF-β activation

2020 ◽  
Author(s):  
Ailiang Zhang ◽  
Helena Paidassi ◽  
Adam Lacy-Hulbert ◽  
John Savill
Keyword(s):  
Ex Vivo ◽  
Apoptotic Cell ◽  
Cell Interaction ◽  
Apoptotic Cells ◽  
Intestinal Epithelial ◽  
Mesenteric Lymph Nodes ◽  
Murine Bone Marrow ◽  
Enhanced Expression ◽  
Tgf Β1

In the mammalian gut CD103+ve myeloid DCs are known to suppress inflammation threatened by luminal bacteria, but stimuli driving DC precursor differentiation towards this beneficial phenotype are incompletely understood. We isolated CD11+ve DCs from mesenteric lymph nodes (MLNs) of healthy mice; CD103+ve DCs were 8-24 folds more likely than CD103-ve DCs to exhibit extensive of prior phagocytosis of apoptotic intestinal epithelial cells. However, CD103+ve and CD103-ve MLN DCs exhibited similar ex vivo capacity to ingest apoptotic cells, indicating that apoptotic cells might drive immature DC differentiation towards the CD103+ve phenotype. When cultured with apoptotic cells, myeloid DC precursors isolated from murine bone marrow and characterised as lineage-ve CD103-ve, displayed enhanced expression of CD103 and β8 integrin and acquired increased capacity to induce Tregs after 7d in vitro. However, DC precursors isolated from α v -tie2 mice lacking α v integrins in the myeloid line exhibited reduced binding of apoptotic cells and complete deficiency in the capacity of apoptotic cells and/or latent TGF-β1 to enhance CD103 expression in culture, whereas active TGF-β1 increased DC precursor CD103 expression irrespective of α v expression. Fluorescence microscopy revealed clustering of α v integrin chains and latent TGF-β1 at points of contact between DC precursors and apoptotic cells. We conclude that myeloid DC precursors can deploy α v integrin to orchestrate binding of apoptotic cells, activation of latent TGF-β1 and acquisition of the immunoregulatory CD103+ve β8+ve DC phenotype. This implies that a hitherto unrecognised consequence of apoptotic cell interaction with myeloid phagocytes is programming that prevents inflammation.

scholarly journals TGF-β1 facilitates cell–cell communication in osteocytes via connexin43- and pannexin1-dependent gap junctions

2019 ◽  
Vol 5 (1) ◽  
Author(s):  
Wenjing Liu ◽  
Demao Zhang ◽  
Xin Li ◽  
Liwei Zheng ◽  
Chen Cui ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Lucifer Yellow ◽  
Cell Communication ◽  
Deep Understanding ◽  
Gap Junctional Intercellular Communication ◽  
Tgf Β1 ◽  
Cell Cell

Abstract Connexins and pannexins are two families of channel forming proteins that are able to pass small molecules to achieve communication between cells. While connexins have been recognized to mediate gap junctional intercellular communication (GJIC), pannexins are far less known. Our previous study reported the potential role of TGF-β1 in mediating of connexins in osteocytes in vitro. Herein, we aimed to elucidate the influence of TGF-β1 on cell–cell communication based on gap junctions assembled by connexins and pannexins in vitro and ex vivo. We first showed that TGF-β1 positively affected the elongation of dendritic processes of osteocytes. Our data indicated that TGF-β1 increased expressions of connexin43 (Cx43) and pannexin1 (panx1), which are indispensable for hemichannel formation in gap junctions, in osteocytes in vitro and ex vivo. TGF-β1 enhanced gap junction formation and impacted cell–cell communication in living osteocytes, as indicated by the scrape loading and Lucifer yellow transfer assays. TGF-β1 enhanced the expressions of Cx43 and panx1 via activation of ERK1/2 and Smad3/4 signalling. The TGF-β1-restored expressions of Cx43 and panx1 in osteocytes in the presence of an ERK inhibitor, U0126, further demonstrated the direct participation of Smad3/4 signalling. TGF-β1 increased the accumulation of Smad3 in the nuclear region (immunofluorescence assay) and promoted the enrichment of Smad3 at the binding sites of the promoters of Gja1 (Cx43) and Panx1 (ChIP assay), thereby initiating the enhanced gene expression. These results provide a deep understanding of the molecular mechanisms involved in the modulation of cell–cell communication in osteocytes induced by TGF-β1.

scholarly journals Assessing the Effects of Ginger Extract on Polyphenol Profiles and the Subsequent Impact on the Fecal Microbiota by Simulating Digestion and Fermentation In Vitro

Nutrients ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 3194
Author(s):  
Jing Wang ◽  
Yong Chen ◽  
Xiaosong Hu ◽  
Fengqin Feng ◽  
Luyun Cai ◽  
...  
Keyword(s):  
Gut Microbiota ◽  
Ph Value ◽  
Illumina Miseq ◽  
Short Chain Fatty Acids ◽  
Fecal Microbiota ◽  
Rrna Gene ◽  
Gut Health ◽  
Ginger Extract ◽  
Mixed Culture Fermentation

The beneficial effects of ginger polyphenols have been extensively reported. However, their metabolic characteristics and health effects on gut microbiota are poor understood. The purpose of this study was to investigate the digestion stability of ginger polyphenols and their prebiotic effects on gut microbiota by simulating digestion and fermentation in vitro. Following simulated digestion in vitro, 85% of the polyphenols were still detectable, and the main polyphenol constituents identified in ginger extract are 6-, 8-, and 10-gingerols and 6-shogaol in the digestive fluids. After batch fermentation, the changes in microbial populations were measured by 16S rRNA gene Illumina MiSeq sequencing. In mixed-culture fermentation with fecal inoculate, digested ginger extract (GE) significantly modulated the fecal microbiota structure and promoted the growth of some beneficial bacterial populations, such as Bifidobacterium and Enterococcus. Furthermore, incubation with GE could elevate the levels of short-chain fatty acids (SCFAs) accompanied by a decrease in the pH value. Additionally, the quantitative PCR results showed that 6-gingerol (6G), as the main polyphenol in GE, increased the abundance of Bifidobacterium significantly. Therefore, 6G is expected to be a potential prebiotic that improves human health by promoting gut health.

Ex vivo treatment of proliferating human cord blood stem cells with stroma-derived factor–1 enhances their ability to engraft NOD/SCID mice

Blood ◽  
2002 ◽  
Vol 99 (9) ◽  
pp. 3454-3457 ◽  
Author(s):  
Hanno Glimm ◽  
Patrick Tang ◽  
Ian Clark-Lewis ◽  
Christof von Kalle ◽  
Connie Eaves
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Transforming Growth Factor ◽  
Ex Vivo ◽  
Severe Combined Immunodeficiency ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Steel Factor ◽  
Tgf Β1

Abstract Ex vivo proliferation of hematopoietic stem cells (HSCs) is important for cellular and gene therapy but is limited by the observation that HSCs do not engraft as they transit S/G2/M. Recently identified candidate inhibitors of human HSC cycling are transforming growth factor-β1(TGF-β1) and stroma-derived factor–1 (SDF-1). To determine the ability of these factors to alter the transplantability of human HSCs proliferating in vitro, lin− cord blood cells were first cultured for 96 hours in serum-free medium containing Flt3 ligand, Steel factor, interleukin-3, interleukin-6, and granulocyte colony-stimulating factor. These cells were then transferred to medium containing Steel factor and thrombopoietin with or without SDF-1 and/or TGF-β1 for 48 hours. Exposure to SDF-1 but not TGF-β1 significantly increased (&gt; 2-fold) the recovery of HSCs able to repopulate nonobese diabetic/severe combined immunodeficiency mice. These results suggest new strategies for improving the engraftment activity of HSCs stimulated to proliferate ex vivo.

scholarly journals Influence of Conditioned Media on the Re-Differentiation Capacity of Human Chondrocytes in 3D Spheroid Cultures

2020 ◽  
Vol 9 (9) ◽  
pp. 2798
Author(s):  
Annett Klinder ◽  
Sophie Kussauer ◽  
Bettina Hiemer ◽  
Andreas Wree ◽  
Rainer Bader ◽  
...  
Keyword(s):  
Conditioned Medium ◽  
Culture Media ◽  
Ex Vivo ◽  
Cartilage Matrix ◽  
Human Chondrocytes ◽  
Growth Media ◽  
Cell Cultivation ◽  
Cell Based Therapy ◽  
Tgf Β1

A major challenge of cell-based therapy for cartilage lesions is the preservation of the chondrogenic phenotype during ex vivo cell cultivation. In this in vitro study, the chondro-inductive capacity of two different hyaline cartilage-conditioned cell culture media on human chondrocytes in 3D spheroids was determined. Media were conditioned by incubation of 200 mg/mL vital or devitalized cartilage matrix in growth media over 35 days. The media were analyzed for the content of soluble procollagen type (Col) II and glycosaminoglycans (GAGs) as well as released TGF-β1, IGF-1 and IGFBP3. Unconditioned medium served as a negative control while the positive medium control was supplemented with TGF-β1 and IGF-1. Spheroid cultures prepared from human chondrocytes were cultivated at 37 °C, 5% CO2 and 21% O2 in the respective media and controls. After 14 and 35 days, the deposition of ECM components was evaluated by histological analysis. Vital cartilage-conditioned medium contained significantly higher levels of Col II and active TGF-β1 compared to medium conditioned with the devitalized cartilage matrix. Despite these differences, the incubation with vital as well as devitalized cartilage conditioned medium led to similar results in terms of deposition of proteoglycans and collagen type II, which was used as an indicator of re-differentiation of human chondrocytes in spheroid cultures. However, high density 3D cell cultivation showed a positive influence on re-differentiation.

Study of In Vitro Proliferation Potential of CD133 Positive Cells Derived from Umbilical Cord Blood.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4039-4039
Author(s):  
Ri Zhang ◽  
Wenjin Gao ◽  
Yuanyuan Sun ◽  
Jingcheng Miao ◽  
Xueguang Zhang
Keyword(s):  
Cord Blood ◽  
Progenitor Cells ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Hematopoietic Stem ◽  
Low Concentration ◽  
Tgf Β1

Abstract Transforming growth factor-beta 1 (TGF-β1) is known to maintain primitive human hematopoietic stem/progenitor cells with polyfunctional role in a quiescent state and CD133 is a new stem cell antigen that may provide an alternative to CD34 for the selection and expansion of hematopoietic cells for transplantation. To investigate the specific effect of TGF-β1 on proliferation and differentiation of CD133 positive cells derived from umbilical cord blood (UCB) during short-term culture in vitro, CD133 positive cells from 20 fresh UCB samples were selected using Miltenyi Biotec’s CliniMACS separation device and were cultured in IMDM medium with 20% FCS in the presence of a cytokine combination of SCF, IL-6, thrombopoietin, IL-3 and Flt3-ligand for up to 2 weeks and TGF-β1 with low concentration was also added to the mediumon day 4. The proliferative response was assessed at day 7, day 10 and day 14 by evaluating the following parameters: nucleated cells (NC), clonogenic progenitors (CFU-GEMM,CFU-GM and BFU-E), and immunophenotypes (CD133 and CD34). The results showed that efficacious expansion of various hematopoietic stem/progenitor cells was constantly observed during the culture. The fold expansion of NC on day7, day10 and day14 expansion were 33.59,224.26 and 613.48, respectively. The fold expansion of CFU-GEMM, CFU-GM and BFU-E on day 10 were 24.89, 41.62 and 49.28, respectively, obviously higher than that without ex vivo expansion (P<0.05). The expansions of CD133+, CD133+CD34+ and CD34+ subpopulation on day 14 were up to 25.83-fold, 16.16-fold and 60.54-fold, respectively. Furthermore the expansion systems with TGF-β1 showed more CD133+ cells than control at every time points. Our datas suggested that the CD133+ cells from human UCB have great expansion potential for ex-vivo expansion. The low concentration of TGF-β1 may delay over-differentiation of hematopoietic stem/progenitor cells.

Th17 Immune Response in H. Pylori Infection ex vivo and in vitro

2017 ◽  
Vol 152 (5) ◽  
pp. S666
Author(s):  
Arne Kandulski ◽  
Wiebke Schirrmeister ◽  
Cosima Langner ◽  
Alexander Link ◽  
Peter Malfertheiner
Keyword(s):  
Immune Response ◽  
Ex Vivo ◽  
H Pylori
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