Evaluation of flow-cytometry for the monitoring of infectious human rotavirus in water

1997 ◽  
Vol 35 (11-12) ◽  
pp. 451-453
Author(s):  
F. X. Abad ◽  
A. Bosch ◽  
J. Comas ◽  
D. Villalba ◽  
R. M. Pintó
Keyword(s):  
Flow Cytometry ◽  
Water Samples ◽  
Wild Type ◽  
Cell Monolayers ◽  
Naturally Occurring ◽  
Human Rotavirus ◽  
Faecal Samples

A method has been developed for the detection of infectious human rotavirus (HRV), based on infection of MA104 and CaCo-2 cell monolayers and ulterior flow cytometry. The sensitivity of the flow cytometry procedure for the cell-adapted HRV enabled the detection of 200 and 2 MPNCU in MA104 and CaCo-2 cells, respectively. Flow cytometry performed five days after infection of CaCo-2 enabled the detection of naturally occurring wild-type HRV in faecal samples and concentrated water samples.

scholarly journals Aged HCT-8 Cell Monolayers Support Cryptosporidium parvum Infection

2007 ◽  
Vol 73 (23) ◽  
pp. 7548-7551 ◽  
Author(s):  
Laura Y. Sifuentes ◽  
George D. Di Giovanni
Keyword(s):  
Water Quality ◽  
Cell Culture ◽  
Water Sample ◽  
Cryptosporidium Parvum ◽  
Water Samples ◽  
Sample Collection ◽  
Sample Processing ◽  
Cell Monolayers ◽  
Immunofluorescent Assay ◽  
Naturally Occurring

ABSTRACT Cell culture assays in various formats have been used to study the infectivity of Cryptosporidium spp. as well as to determine the infectivity of naturally occurring oocysts in water. Currently, cell culture assays for infectious Cryptosporidium spp. in water have largely been limited to practice in research laboratories. One obstacle to the routine use of Cryptosporidium cell culture assays for the analysis of water samples is the coordination of water sample collection and processing with readiness of cell culture monolayers. For most Cryptosporidium cell culture assays, monolayers are allowed to develop for 24 to 48 h to reach 80 to 100% confluence prior to inoculation. In this study, we used immunofluorescent assay microscopy to evaluate freshly confluent (2-day-old) and aged (8- to 67-day-old) HCT-8 cell monolayers for their ability to support Cryptosporidium parvum infection. HCT-8 monolayers as old as 67 days were clearly shown to support infection. In two of three experiments, aged monolayers (8- to 11-day-old and 11- to 22-day-old, respectively) developed the same number of C. parvum clusters of infection as freshly confluent monolayers. Results suggest that it may be possible to use cell monolayers from freshly confluent to 3 weeks old on hand for infectivity assays without having to schedule sample processing to coincide with development of freshly confluent monolayers. This would make Cryptosporidium cell culture assays much more feasible for water quality and utility laboratories.

Comparison of Cell Culture and a Poliovirus Gene Probe Assay for the Detection of Enteroviruses in Environmental Water Samples

1993 ◽  
Vol 27 (3-4) ◽  
pp. 311-314 ◽  
Author(s):  
Aaron B. Margolin ◽  
Charles P. Gerba ◽  
Kenneth J. Richardson ◽  
Jaime E. Naranjo
Keyword(s):  
Cell Culture ◽  
Activated Sludge ◽  
Water Samples ◽  
Cdna Probe ◽  
Culture Method ◽  
Tidal River ◽  
Nucleic Acid Hybridization ◽  
Treated Wastewater ◽  
Gene Probe ◽  
Naturally Occurring

Nucleic acid hybridization provides a rapid non-cell culture method for the detection of enteric viruses in water. The purpose of this work was to compare the detection of naturally occurring enteroviruses by cell culture with their detection by a poliovirus gene probe in various types of water samples. Samples of activated sludge effluent, tertiary treated wastewater (activated sludge, filtration and passage through reverse osmosis), ground water, surface water and tidal river water were processed through 1 MDS Virozorb filters to concentrate any naturally occurring virus. Viruses were eluted from the filters with pH 9.5 beef extract and reduced in volume by flocculation to 20-30 ml. These concentrates were then assayed in the BGM cell line by the cytopathogenic effects (CPE) method and by a poliovirus cDNA probe (base pairs 115-7440) labeled with 32P. A total of 233 samples were assayed in this manner. In slightly more than 93% of the samples gene probe and cell culture yielded the same results. Of these samples 36 were positive by gene probe and 28 by cell culture assay. Positive samples for gene probe were confirmed by treatment with NaOH or RNAse and then reprobed. Samples demonstrating CPE upon primary passage were confirmed positive by subsequent passage of cell lysate on a new monolayer of BGM cells. Ten samples were positive by gene probe and negative by cell culture, and 4 samples were negative by gene probe and positive by cell culture.

scholarly journals Semi-Synthesis of Small Molecules of Aminocarbazoles: Tumor Growth Inhibition and Potential Impact on p53

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1637
Author(s):  
Solida Long ◽  
Joana B. Loureiro ◽  
Carla Carvalho ◽  
Luís Gales ◽  
Lucília Saraiva ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Small Molecules ◽  
Tumor Cells ◽  
Mutant P53 ◽  
Wild Type ◽  
Naturally Occurring ◽  
Growth Inhibitory ◽  
Growth Inhibitory Activity ◽  
Amino Derivatives ◽  
Carbazole Alkaloids

The tumor suppressor p53 is inactivated by mutation in approximately 50% of human cancers. Small molecules that bind and stabilize those mutants may represent effective anticancer drugs. Herein, we report the tumor cell growth inhibitory activity of carbazole alkaloids and amino derivatives, as well as their potential activation of p53. Twelve aminocarbazole alkaloids were semi-synthesized from heptaphylline (1), 7-methoxy heptaphylline (2), and 7-methoxymukonal (3), isolated from Clausena harmandiana, using a reductive amination protocol. Naturally-occurring carbazoles 1–3 and their amino derivatives were evaluated for their potential effect on wild-type and mutant p53 activity using a yeast screening assay and on human tumor cell lines. Naturally-occurring carbazoles 1–3 showed the most potent growth inhibitory effects on wild-type p53-expressing cells, being heptaphylline (1) the most promising in all the investigated cell lines. However, compound 1 also showed growth inhibition against non-tumor cells. Conversely, semi-synthetic aminocarbazole 1d showed an interesting growth inhibitory activity in tumor cells expressing both wild-type and mutant p53, exhibiting low growth inhibition on non-tumor cells. The yeast assay showed a potential reactivation of mutant p53 by heptaphylline derivatives, including compound 1d. The results obtained indicate that carbazole alkaloids may represent a promising starting point to search for new mutp53-reactivating agents with promising applications in cancer therapy.

scholarly journals Responsiveness to exogenous cAMP of a Saccharomyces cerevisiae strain conferred by naturally occurring alleles of PDE1 and PDE2.

Genetics ◽  
1993 ◽  
Vol 135 (2) ◽  
pp. 321-326 ◽  
Author(s):  
H Mitsuzawa
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Slow Growth ◽  
Loss Of Function ◽  
Wild Type ◽  
Triple Mutant ◽  
Apparent Absence ◽  
Camp Pathway ◽  
Naturally Occurring ◽  
Elevated Activity ◽  
Growth Phenotype

Abstract The Saccharomyces cerevisiae strain P-28-24C, from which cAMP requiring mutants derived, responded to exogenously added cAMP. Upon the addition of cAMP, this strain showed phenotypes shared by mutants with elevated activity of the cAMP pathway. Genetic analysis involving serial crosses of this strain to a strain with another genetic background revealed that the responsiveness to cAMP results from naturally occurring loss-of-function alleles of PDE1 and PDE2, which encode low and high affinity cAMP phosphodiesterases, respectively. In addition, P-28-24C was found to carry a mutation conferring slow growth that lies in CYR1, which encodes adenylate cyclase, and the slow growth phenotype caused by the cyr1 mutation was suppressed by the pde2 mutation. Therefore P-28-24C is fortuitously a pde1 pde2 cyr1 triple mutant. Responsiveness to cAMP conferred by pde mutations suggests that S. cerevisiae cells are permeable to cAMP to some extent and that the apparent absence of effect of exogenously added cAMP on wild-type cells is due to immediate degradation by cAMP phosphodiesterases.

scholarly journals Characterization of TNF receptor subtype expression and signaling on pulmonary endothelial cells in mice

2011 ◽  
Vol 300 (5) ◽  
pp. L781-L789 ◽  
Author(s):  
Szabolcs Bertok ◽  
Michael R. Wilson ◽  
Anthony D. Dorr ◽  
Justina O. Dokpesi ◽  
Kieran P. O'Dea ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Adhesion Molecules ◽  
Surface Expression ◽  
Tnf Receptor ◽  
Wild Type ◽  
Tnf Receptors ◽  
Pulmonary Endothelial Cells ◽  
Microvascular Inflammation

TNF plays a crucial role in the pathogenesis of acute lung injury. However, the expression profile of its two receptors, p55 and p75, on pulmonary endothelium and their influence on TNF signaling during lung microvascular inflammation remain uncertain. Using flow cytometry, we characterized the expression profile of TNF receptors on the surface of freshly harvested pulmonary endothelial cells (PECs) from mice and found expression of both receptors with dominance of p55. To investigate the impact of stimulating individual TNF receptors, we treated wild-type and TNF receptor knockout mice with intravenous TNF and determined surface expression of adhesion molecules (E-selectin, VCAM-1, ICAM-1) on PECs by flow cytometry. TNF-induced upregulation of all adhesion molecules was substantially attenuated by absence of p55, whereas lack of p75 had a similar but smaller effect that varied between adhesion molecules. Selective blockade of individual TNF receptors by specific antibodies in wild-type primary PEC culture confirmed that the in vivo findings were due to direct effects of TNF receptor inhibition on endothelium and not other cells (e.g., circulating leukocytes). Finally, we found that PEC surface expression of p55 dramatically decreased in the early stages of endotoxemia following intravenous LPS, while no change in p75 expression was detected. These data demonstrate a crucial in vivo role of p55 and an auxiliary role of p75 in TNF-mediated adhesion molecule upregulation on PECs. It is possible that the importance of the individual receptors varies at different stages of pulmonary microvascular inflammation following changes in their relative expression.

Deep Learning-enabled Holographic Imaging Flow-Cytometry for Label-Free Detection of Giardia Lamblia in Water Samples

2021 ◽  
Author(s):  
Zoltán Göröcs ◽  
David Baum ◽  
Fang Song ◽  
Kevin de Haan ◽  
Hatice Ceylan Koydemir ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Deep Learning ◽  
Water Samples ◽  
Giardia Lamblia ◽  
Label Free ◽  
Holographic Imaging ◽  
Imaging Flow Cytometry ◽  
Label Free Detection

Enteropathogenic Escherichia coli adherence to intestinal epithelial monolayers diminishes barrier function

1995 ◽  
Vol 268 (2) ◽  
pp. G374-G379 ◽  
Author(s):  
J. Spitz ◽  
R. Yuhan ◽  
A. Koutsouris ◽  
C. Blatt ◽  
J. Alverdy ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Host Cell ◽  
Barrier Function ◽  
Cell Function ◽  
Cytoskeletal Proteins ◽  
Calcium Stores ◽  
Intestinal Epithelial ◽  
Enteropathogenic Escherichia Coli ◽  
Wild Type ◽  
Cell Monolayers

The mechanism by which enteropathogenic Escherichia coli (EPEC) causes diarrhea remains elusive. Several alterations within the host cell have been demonstrated to occur following EPEC attachment including increases in intracellular Ca2+ concentration and rearrangement and phosphorylation of several cytoskeletal proteins. The consequences of these intracellular perturbations on host cell function, however, have not been determined. The aim of this study was to examine the effect of EPEC adherence on intestinal epithelial barrier function. T84 cell monolayers were infected with either wild-type EPEC or a nonadherent isogenic derivative. Transepithelial electrical resistance, a measure of barrier function, decreased 33.5 +/- 6.4% after a 6-h incubation with the wild-type strain. Electron microscopy revealed ultrastructurally normal cells, and lactate dehydrogenase release assays failed to demonstrate cytotoxicity. Dual 22Na+ and [3H]mannitol flux studies localized the permeability defect to tight junctions. In addition, cumulative flux of the paracellular marker mannitol was four- to fivefold greater across monolayers infected with wild-type EPEC. Sequestration of intracellular calcium stores by dantrolene completely abrogated the resistance drop associated with EPEC attachment. These data demonstrate that adherence of EPEC to intestinal epithelial cell monolayers disrupts tight junction barrier function via a calcium-requiring event.

scholarly journals 947 Recombinant myxoma virus MC509-N1 demonstrates antitumor efficacy as monotherapy and in combination with immune checkpoint inhibitors

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A996-A996
Author(s):  
Enkhtaivan Gansukh ◽  
Tommy Alain ◽  
Tae-Geuk Kim ◽  
Ye-Na Namgung ◽  
Ka-Yeon Son ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Tumor Microenvironment ◽  
Tumor Growth ◽  
Immune Checkpoint ◽  
Myxoma Virus ◽  
Wild Type ◽  
Anticancer Efficacy ◽  
Immune Profile ◽  
Immune State ◽  
Inhibition Of Tumor Growth

BackgroundThere are several obstacles to effective cancer immunotherapy including the heterogenic immune profile and the state of the tumor microenvironment. Oncolytic virotherapy provides an opportunity to overcome some of these limitations through high viral replication and the expression of therapeutic transgenes (TGs) within the tumor tissue. Myxoma virus (MYXV) belongs to the family of Poxviridae and represents a potent oncolytic virus and a safe platform as this virus is non-pathogenic in any hosts apart from lagomorphs. Importantly, MYXV has a high capacity of encoding for multiple TG payloads. Here we engineered MC509-N1, a novel double-encoding transgenes (TG1 and TG2) oncolytic MYXV designed for intravenous (IV) injection. The therapeutic TG1 acts to modify and remodel the immune state of the tumor microenvironment, and TG2 allows for prolonged self-evasion from the host immune defense.MethodsTransgenes expression upon infection was detected by ELISA and by flow cytometry. To determine anticancer efficacy, syngeneic B16F10 melanoma or MC38 colorectal cancer-bearing C57BL/6 mice were injected with MC509-N1 intratumorally or IV with or without immune checkpoint inhibitor (ICI). Tumor growth and survival was monitored after treatment and the immune profile within the tumor microenvironment was analyzed by flow cytometry. Mice cured of their tumors from the original treatment were rechallenged with primary tumor cells to examine anticancer immunity.ResultsCells upon infection with MC509-N1 were found to express both transgenes at high levels and stimulate downstream mechanisms. Importantly, the engineering of both transgenes did not affect MC509-N1 infectivity and productivity as compared to wild-type MYXV. Intratumoral injections of MC509-N1 effectively suppressed tumor growth and improved overall survival of both syngeneic cancer models. Furthermore, MC509-N1 therapy effectively modulated the immune profile within the tumor microenvironment, especially the ratio between tumor infiltrated CD8+ cytotoxic T cells and CD4+FoxP3+ T regulatory cells. In addition, IV injections of MC509-N1 showed improved inhibition of tumor growth compared to wild type MYXV. The combination therapy of MC509-N1 with the ICI anti-PD-L1 further promoted inhibition of tumor growth as demonstrated by higher rate of complete regression and improved survival rate. Furthermore, rechallenge experiments revealed that this combination regimen established specific anticancer immune memory and protected from cancer recurrence.ConclusionsOur results demonstrate that the novel engineered MC509-N1 exhibits potent anticancer efficacy, adequately modulates the immune state of the tumor microenvironment, and acts synergistically to eliminate cancer in combination with ICI.

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