scholarly journals Salmonella L-forms: formation in human bile in vitro and isolation culture from patients' gallbladder samples by a non-high osmotic isolation technique

2015 ◽  
Vol 21 (5) ◽  
pp. 470.e9-470.e16
Author(s):  
D.N. Wang ◽  
W.J. Wu ◽  
T. Wang ◽  
Y.Z. Pan ◽  
K.L. Tang ◽  
...  
Keyword(s):  
Isolation Technique ◽  
Human Bile

scholarly journals Liver Sinusoidal Endothelial Cell (Lsec) Isolation Following a Liver Perfusion Technique

2017 ◽  
Vol 16 (1) ◽  
Author(s):  
Shahida Saharudin ◽  
Norlelawati A. Talib ◽  
Nor Zamzila Abdullah ◽  
Jamalludin Ab. Rahman ◽  
Zunariah Buyong
Keyword(s):  
Liver Perfusion ◽  
Light And Electron Microscopy ◽  
Average Concentration ◽  
Liver Sinusoidal Endothelial Cell ◽  
Sprague Dawley ◽  
Perfusion Technique ◽  
Liver Sinusoidal Endothelial Cells ◽  
Isolation Technique ◽  
And Function

Introduction: Liver perfusion has been the standard method to digest and isolate liver cells including liver sinusoidal endothelial cells (LSEC). Poor cannulating skills through portal vein results in a waste of animal resource. Familiarization of both liver perfusion technique and adhering strictly to aseptic technique during cell handling ensure high cell yield, minimum morphology disruption and cell contamination. We aimed to present a method of liver perfusion procedure followed by the isolation of LSEC. Materials and method: The study was conducted with the approval of IACUC committee. Seven Sprague Dawley rats underwent these procedures under anaesthesia. Liver perfusion was done as previously described. Briefly, LSEC were isolated by liberase enzyme perfusion of the liver, isopycnic sedimentation in a two- step Percoll gradient and selective adherence. The purification and cultivation of LSEC was evaluated by light and electron microscopy. Results: Purity and viability of LSEC after selective adherence was 80.5 ± 3.5% and ≥ 95 %, respectively. The average concentration of the cells ranged from 32 - 75 x 106 per 400 gm rat. After 8 hours of culture, LSEC monolayers were contaminated with less than 5% of other cells. Conclusion: This method is reliable and reproducible for the isolation of LSEC to enable the study of structure and function of these cells in vitro. However, improvement on the perfusion skills and isolation technique are vital to ensure better cell purity.

Adenosine nucleotides in bile

1996 ◽  
Vol 270 (2) ◽  
pp. G246-G252 ◽  
Author(s):  
R. S. Chari ◽  
S. M. Schutz ◽  
J. E. Haebig ◽  
G. H. Shimokura ◽  
P. B. Cotton ◽  
...  
Keyword(s):  
Purinergic Receptors ◽  
Purinergic Receptor ◽  
Receptor Activation ◽  
Hepatoma Cells ◽  
Human Bile ◽  
Nucleotide Release ◽  
Cholangiocarcinoma Cell ◽  
Adenosine Nucleotides ◽  
Vitro Model

Activation of purinergic receptors by ATP stimulates Cl- efflux in biliary epithelial cells. To determine whether purinergic agonists are present under physiological conditions, we have assayed mammalian bile for nucleotides and assessed whether hepatoma and cholangiocarcinoma cell lines are capable of nucleotide release. Bile samples were collected from human, rat, and pig donors and assayed for nucleotide concentrations by luminometry. ATP, ADP, and AMP were present in bile from each species, and the average total nucleotide concentration in human bile was 5.21 +/- 0.91 microM (n = 16). In an in vitro model of HTC rat hepatoma cells or Mz-ChA-1 cholangiocarcinoma cells on a superfused column, nucleotides were present in the effluent from each cell type. Addition of alpha, beta-methyleneadenosine 5'-diphosphate (50 microM) to inhibit 5'-nucleotidase activity increased AMP concentrations two- to threefold. Exposure to forskolin (100 microM) or ionomycin (2 microM) stimulated nucleotide release from cholangiocarcinoma but not hepatoma cells. These studies indicate that adenosine nucleotides are present in bile in concentrations sufficient to activate purinergic receptors. Purinergic receptor activation by local nucleotide release might constitute an autocrine and/or paracrine mechanism for modulation of biliary secretion.

Cadmium and calcium uptake in isolated mitochondria-rich cell populations from the gills of the freshwater rainbow trout

2006 ◽  
Vol 291 (1) ◽  
pp. R170-R176 ◽  
Author(s):  
Fernando Galvez ◽  
Denise Wong ◽  
Chris M. Wood
Keyword(s):  
Rainbow Trout ◽  
Cellular Localization ◽  
Calcium Uptake ◽  
High Capacity ◽  
Cell Types ◽  
Isolation Technique ◽  
Pavement Cells ◽  
Gill Cells

A novel cell isolation technique was used to characterize cadmium and calcium uptake in distinct populations of gill cells from the adult rainbow trout ( Oncorhynchus mykiss). A specific population of mitochondria-rich (MR) cell, termed the PNA+ MR cell (PNA is peanut lectin agglutinin), was found to accumulate over threefold more 109Cd than did PNA− MR cells, pavement cells (PV cells), and mucous cells during a 1-h in vivo exposure at 2.4 μg/l 109Cd. In vitro 109Cd exposures, performed in standard PBS and Cl−-free PBS, at concentrations from 1 to 16 μg/l 109Cd, were also carried out to further characterize Cd2+ uptake kinetics. As observed during in vivo experiments, PNA+ MR cells accumulated significantly more 109Cd than did other cell types when exposures were performed by an in vitro procedure in PBS. Under such conditions, Cd2+ accumulation kinetics in all cell types could be described with Michaelis-Menten relationships, with Km values of ∼3.0 μg/l Cd (27 nM) for both MR cell subtypes and 8.6 μg/l Cd (77 nM) for PV cells. In similar experiments performed in Cl−-free conditions, a significant reduction in 109Cd accumulation in PNA+ MR cells was seen but not in PNA− MR or in PV cells. In vitro 45Ca fluxes were also performed to determine the cellular localization of Ca2+ transport in these functionally distinct populations of gill cells. 45Ca uptake was most pronounced in PNA+ MR cells, with levels over threefold higher than those found in either PNA− MR or in PV cells. Results from the present study suggest that the PNA+ MR cell type is a high-affinity and high-capacity site for apical entry of Cd2+ and Ca2+ in the gill epithelium of rainbow trout.

scholarly journals Cholangiocyte organoids from human bile retain a local phenotype and can repopulate bile ducts in vitro

2021 ◽  
Vol 11 (12) ◽  
Author(s):  
Floris J. M. Roos ◽  
Haoyu Wu ◽  
Jorke Willemse ◽  
Ruby Lieshout ◽  
Laura A. Muñoz Albarinos ◽  
...  
Keyword(s):  
Bile Ducts ◽  
Human Bile

scholarly journals Cultivation and Genomic Characterization of the Bile Bacterial Species From Cholecystitis Patients

2021 ◽  
Vol 12 ◽  
Author(s):  
Qiulong Yan ◽  
Siyi Zhang ◽  
Shenghui Li ◽  
Guangyang Wang ◽  
Aiqin Zhang ◽  
...  
Keyword(s):  
Bile Acids ◽  
Bacterial Species ◽  
Fluoroquinolone Resistance ◽  
Metagenomic Sequencing ◽  
Multidrug Resistance Proteins ◽  
Aminoglycoside Resistance ◽  
Human Bile ◽  
Resistance Proteins ◽  
Bile Bacteria

The microbes in human bile are closely related to gallbladder health and other potential disorders. Although the bile microbial community has been investigated by recent studies using amplicon or metagenomic sequencing technologies, the genomic information of the microbial species resident in bile is rarely reported. Herein, we isolated 138 bacterial colonies from the fresh bile specimens of four cholecystitis patients using a culturome approach and genomically characterized 35 non-redundant strains using whole-genome shotgun sequencing. The bile bacterial isolates spanned 3 classes, 6 orders, 10 families, and 14 genera, of which the members of Enterococcus, Escherichia–Shigella, Lysinibacillus, and Enterobacter frequently appeared. Genomic analysis identified three species, including Providencia sp. D135, Psychrobacter sp. D093, and Vibrio sp. D074, which are not represented in existing reference genome databases. Based on the genome data, the functional capacity between bile and gut isolates was compared. The bile strains encoded 5,488 KEGG orthologs, of which 4.9% were specific to the gut strains, including the enzymes involved in biofilm formation, two-component systems, and quorum-sensing pathways. A total of 472 antibiotic resistance genes (ARGs) were identified from the bile genomes including multidrug resistance proteins (42.6%), fluoroquinolone resistance proteins (12.3%), aminoglycoside resistance proteins (9.1%), and β-lactamase (7.2%). Moreover, in vitro experiments showed that some bile bacteria have the capabilities for bile salt deconjugation or biotransformation (of primary bile acids into secondary bile acids). Although the physiological or pathological significance of these bacteria needs further exploration, our works expanded knowledge about the genome, diversity, and function of human bile bacteria.

The Abundance and Organization of Salmonella Extracellular Polymeric Substances in Gallbladder-Mimicking Environments and In Vivo

2021 ◽  
Author(s):  
Mark M. Hahn ◽  
Juan F. González ◽  
Regan Hitt ◽  
Lauren Tucker ◽  
John S. Gunn
Keyword(s):  
Spatial Organization ◽  
Extracellular Polymeric Substances ◽  
Wild Type ◽  
Chronic Infections ◽  
Human Bile ◽  
Cholesterol Gallstones ◽  
Coated Surfaces ◽  
Biofilm Development

Salmonella enterica serovar Typhi ( S. Typhi ) causes chronic infections by establishing biofilms on cholesterol gallstones. Production of extracellular polymeric substances (EPSs) is key to biofilm development and biofilm architecture depends on which EPSs are made. The presence and spatial distribution of Salmonella EPSs produced in vitro and in vivo were investigated in S. Typhi murium and S. Typhi biofilms by confocal microscopy. Comparisons between serovars and EPS-mutant bacteria were examined by growth on cholesterol-coated surfaces, with human gallstones in ox or human bile, and in mice with gallstones. On cholesterol-coated surfaces, major differences in EPS biomass were not found between serovars. Co-culture biofilms containing wild-type (WT) and EPS-mutant bacteria demonstrated WT compensation for EPS mutations. Biofilm EPS analysis from gallbladder-mimicking conditions found that culture in human bile more consistently replicated the relative abundance and spatial organization of each EPS on gallstones from the chronic mouse model than culture in ox bile. S. Typhi murium biofilms cultured in vitro on gallstones in ox bile exhibited co-localized pairings of curli fimbriae/lipopolysaccharide and O antigen capsule/cellulose while these associations were not present in S. Typhi biofilms or in mouse gallstone biofilms. In general, inclusion of human bile with gallstones in vitro replicated biofilm development on gallstones in vivo , demonstrating its strength as a model for studying biofilm parameters or EPS-directed therapeutic treatments.

Effects of adherence factors and human bile on bacterial attachment and biliary stent blockage: An in vitro study

2002 ◽  
Vol 56 (1) ◽  
pp. 72-77 ◽  
Author(s):  
Joseph W. Leung ◽  
Yan-lei Liu ◽  
Rapheal C.Y. Chan ◽  
Thomas K.W. Ling ◽  
Augustine F. Cheng
Keyword(s):  
In Vitro Study ◽  
Bacterial Attachment ◽  
Biliary Stent ◽  
Vitro Study ◽  
Human Bile
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