scholarly journals Combined Action of Shiga Toxin Type 2 and Subtilase Cytotoxin in the Pathogenesis of Hemolytic Uremic Syndrome

Toxins ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 536
Author(s):  
Romina S. Álvarez ◽  
Fernando D. Gómez ◽  
Elsa Zotta ◽  
Adrienne W. Paton ◽  
James C. Paton ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Cell Damage ◽  
Tubular Epithelial Cell ◽  
The Body ◽  
Epithelial Cell Line ◽  
Morphological Alterations ◽  
Proximal Tubular ◽  
Subtilase Cytotoxin

Shiga toxin-producing E. coli (STEC) produces Stx1 and/or Stx2, and Subtilase cytotoxin (SubAB). Since these toxins may be present simultaneously during STEC infections, the purpose of this work was to study the co-action of Stx2 and SubAB. Stx2 + SubAB was assayed in vitro on monocultures and cocultures of human glomerular endothelial cells (HGEC) with a human proximal tubular epithelial cell line (HK-2) and in vivo in mice after weaning. The effects in vitro of both toxins, co-incubated and individually, were similar, showing that Stx2 and SubAB contribute similarly to renal cell damage. However, in vivo, co-injection of toxins lethal doses reduced the survival time of mice by 24 h and mice also suffered a strong decrease in the body weight associated with a lowered food intake. Co-injected mice also exhibited more severe histological renal alterations and a worsening in renal function that was not as evident in mice treated with each toxin separately. Furthermore, co-treatment induced numerous erythrocyte morphological alterations and an increase of free hemoglobin. This work shows, for the first time, the in vivo effects of Stx2 and SubAB acting together and provides valuable information about their contribution to the damage caused in STEC infections.

scholarly journals Evaluation of Fab and F(ab′)2 Fragments and Isotype Variants of a Recombinant Human Monoclonal Antibody against Shiga Toxin 2

2010 ◽  
Vol 78 (3) ◽  
pp. 1376-1382 ◽  
Author(s):  
Donna E. Akiyoshi ◽  
Abhineet S. Sheoran ◽  
Curtis M. Rich ◽  
L. Richard ◽  
Susan Chapman-Bonofiglio ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Shiga Toxin ◽  
Chinese Hamster Ovary Cells ◽  
Human Monoclonal Antibody ◽  
Chinese Hamster ◽  
The Body ◽  
Neutralization Activity ◽  
Shiga Toxin 2

ABSTRACT 5C12 HuMAb is a human monoclonal antibody against the A subunit of Shiga toxin 2 (Stx2). We have previously shown that 5C12 HuMAb effectively neutralizes the cytotoxic effects of this toxin by redirecting its transport within the cell and also by neutralizing the toxin's ability to inhibit protein synthesis. The 5C12 HuMAb and its recombinant IgG1 version protect mice at a dose of 0.6 μg against a lethal challenge of Stx2. The contribution of the Fc region to this observed neutralization activity of the 5C12 antibody against Stx2 was investigated in this study. Using recombinant DNA technology, 5C12 isotype variants (IgG1, IgG2, IgG3, and IgG4) and antibody fragments [Fab, F(ab′)2] were expressed in Chinese hamster ovary cells and evaluated in vitro and in vivo. All four 5C12 isotype variants showed protection in vitro, with the IgG3 and IgG4 variants showing the highest protection in vivo. The Fab and F(ab′)2 fragments also showed protection in vitro but no protection in the mouse toxicity model. Similar results were obtained for a second HuMAb (5H8) against the B subunit of Stx2. The data suggest the importance of the Fc region for neutralization activity, but it is not clear if this is related to the stability of the full-length antibody or if the Fc region is required for effective elimination of the toxin from the body.

scholarly journals Human Recombinant Fab Fragment Neutralizes Shiga Toxin Type 2 Cytotoxic Effects in vitro and in vivo

Toxins ◽  
2018 ◽  
Vol 10 (12) ◽  
pp. 508 ◽  
Author(s):  
Daniela Luz ◽  
Maria Amaral ◽  
Flavia Sacerdoti ◽  
Alan Bernal ◽  
Wagner Quintilio ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Lethal Dose ◽  
Dependent Manner ◽  
Fab Fragment ◽  
Cell Viability Assay ◽  
Cytotoxic Effects ◽  
Morphological Alterations ◽  
Viability Assay

Shiga toxin (Stx) producing Escherichia coli (STEC) is responsible for causing hemolytic uremic syndrome (HUS), a life-threatening thrombotic microangiopathy characterized by thrombocytopenia, hemolytic anemia, and acute renal failure after bacterially induced hemorrhagic diarrhea. Until now, there has been neither an effective treatment nor method of prevention for the deleterious effects caused by Stx intoxication. Antibodies are well recognized as affinity components of therapeutic drugs; thus, a previously obtained recombinant human FabC11:Stx2 fragment was used to neutralize Stx2 in vitro in a Vero cell viability assay. Herein, we demonstrated that this fragment neutralized, in a dose-dependent manner, the cytotoxic effects of Stx2 on human glomerular endothelial cells, on human proximal tubular epithelial cells, and prevented the morphological alterations induced by Stx2. FabC11:Stx2 protected mice from a lethal dose of Stx2 by toxin-antibody pre-incubation. Altogether, our results show the ability of a new encouraging molecule to prevent Stx-intoxication symptoms during STEC infection.

scholarly journals Shiga Toxin 2 Targets the Murine Renal Collecting Duct Epithelium

2009 ◽  
Vol 77 (3) ◽  
pp. 959-969 ◽  
Author(s):  
Mitchell A. Psotka ◽  
Fumiko Obata ◽  
Glynis L. Kolling ◽  
Lisa K. Gross ◽  
Moin A. Saleem ◽  
...  
Keyword(s):  
Renal Failure ◽  
Shiga Toxin ◽  
Cell Damage ◽  
Collecting Duct ◽  
Signs And Symptoms ◽  
Filtration Barrier ◽  
Shiga Toxin 2 ◽  
Collecting Duct Epithelium

ABSTRACT Hemolytic-uremic syndrome (HUS) caused by Shiga toxin-producing Escherichia coli infection is a leading cause of pediatric acute renal failure. Bacterial toxins produced in the gut enter the circulation and cause a systemic toxemia and targeted cell damage. It had been previously shown that injection of Shiga toxin 2 (Stx2) and lipopolysaccharide (LPS) caused signs and symptoms of HUS in mice, but the mechanism leading to renal failure remained uncharacterized. The current study elucidated that murine cells of the glomerular filtration barrier were unresponsive to Stx2 because they lacked the receptor glycosphingolipid globotriaosylceramide (Gb3) in vitro and in vivo. In contrast to the analogous human cells, Stx2 did not alter inflammatory kinase activity, cytokine release, or cell viability of the murine glomerular cells. However, murine renal cortical and medullary tubular cells expressed Gb3 and responded to Stx2 by undergoing apoptosis. Stx2-induced loss of functioning collecting ducts in vivo caused production of increased dilute urine, resulted in dehydration, and contributed to renal failure. Stx2-mediated renal dysfunction was ameliorated by administration of the nonselective caspase inhibitor Q-VD-OPH in vivo. Stx2 therefore targets the murine collecting duct, and this Stx2-induced injury can be blocked by inhibitors of apoptosis in vivo.

scholarly journals Erythropoietin Inhibits Hypoxia–Induced Epithelial-To-Mesenchymal Transition via Upregulation of miR-200b in HK-2 Cells

2017 ◽  
Vol 42 (1) ◽  
pp. 269-280 ◽  
Author(s):  
Jiuxu Bai ◽  
Xiao Xiao ◽  
Xiaoling Zhang ◽  
Hanmin Cui ◽  
Junfeng Hao ◽  
...  
Keyword(s):  
Epithelial To Mesenchymal Transition ◽  
Epithelial Mesenchymal Transition ◽  
Interstitial Fibrosis ◽  
Tubular Epithelial Cell ◽  
Epithelial Cell Line ◽  
Protective Effects ◽  
Mesenchymal Transition ◽  
Proximal Tubular ◽  
Renal Tubular

Background/Aims: Renal tubular epithelial-mesenchymal transition (EMT) is regarded as an important factor leading to renal interstitial fibrosis. Erythropoietin (EPO) has been reported to attenuate renal fibrosis. The mechanism underlying this protective effect of EPO remains unclear. In this study, we aim to identify possible mechanisms of the EPO renoprotective effect. Methods: Hypoxia was induced in vitro by incubating human proximal tubular epithelial cell line HK-2 cells in 1% O2 and 5% CO2. Western blotting and reverse transcription polymerase chain reaction analyses were used to evaluate the expression of epithelial and mesenchymal markers in the cell samples. The expression of miR-200b in the HK-2 cells under hypoxia or treatment with EPO was examined. Results: EPO represses hypoxia-induced EMT by upregulating miR-200b in HK-2 cells. Overexpression of miR-200b represses the effect of ETS proto-oncogene 1 (Ets-1)-induced EMT in HK-2 cells. Conclusion: miR-200 mediates the protective effects of EPO on EMT in hypoxic HK-2 cells. EPO attenuated hypoxia-induced EMT by increasing miR-200 expression via the repression of Ets-1.

scholarly journals Optimal Regimen of N-Acetylcysteine on Chromium-Induced Renal Cell Damage

Metabolites ◽  
2019 ◽  
Vol 9 (9) ◽  
pp. 172 ◽  
Author(s):  
I-Jeng Yeh ◽  
Tzu-Yi Wang ◽  
Jhong-Ching Lin ◽  
Tzeng-Jih Lin ◽  
Jung-San Chang ◽  
...  
Keyword(s):  
Renal Cell ◽  
Cell Damage ◽  
Tubular Epithelial Cell ◽  
Epithelial Cell Line ◽  
Expression Ratio ◽  
Delayed Treatment ◽  
Intracellular Ros ◽  
Proximal Tubular ◽  
Apoptosis Markers ◽  
Optimal Regimen

Chromium (Cr) is a well-known heavy metal that can cause renal damage. The production of reactive oxygen species (ROS) due to chromium-induced toxicity induces cell dysfunction, apoptosis, and death. N-acetylcysteine (NAC) is an antioxidant used as an antidote for chromium-induced toxicity. However, the optimal regimen and protective mechanisms of NAC are not fully understood in human renal cells. Our results showed that exposure to 10 μM K2Cr2O7, a toxic Cr(VI) compound, induced apoptosis and production of intracellular ROS in the human proximal tubular epithelial cell line HK-2. Supplements of 600 or 1000 µg/mL NAC inhibited intracellular ROS in HK-2 cells exposed to Cr(VI) and significantly increased cell viability within 2 h of Cr(VI)-induced cytotoxicity. Moreover, Cr(VI) induced the expression of apoptosis markers, including cleaved-caspase-3, cleaved-poly (ADP-ribose) polymerase, cleaved-caspase 8, and cleaved-caspase 9, and altered the expression ratio of Bax/Bcl-xL. Expression of apoptosis markers within 2 h of Cr(VI)-induced cytotoxicity in cells treated with 600 µg/mL NAC was significantly suppressed. However, delayed treatment with NAC at 4 h and 8 h after exposure to Cr did not suppress the activation of apoptotic pathways. In summary, our study reports the optimum timing and dose of NAC for the protection of human renal proximal tubular cells from Cr(VI)-induced cell death. The NAC treatment strategy described could be applied in clinical practice to suppress renal cell apoptosis, which in turn could rescue renal function.

scholarly journals Antioxidant Activity of Pigmented Rice and Impact on Health

JURNAL PANGAN ◽  
2019 ◽  
Vol 28 (1) ◽  
pp. 11-22
Author(s):  
Arfina Sukmawati Arifin
Keyword(s):  
Oxidative Stress ◽  
Diabetes Mellitus ◽  
Antioxidant Activity ◽  
Vascular Function ◽  
Healthy Lifestyle ◽  
Cell Damage ◽  
The Body ◽  
Food Sources

The high number of free radicals that are not balanced with the amount of antioxidants in the body triggers oxidative stress. Oxidative stress causes impaired vascular function, damage to proteins and lipids in membrane cell, and nucleic acid (DNA) mutations. Chronic cell damage has a negative effect on tissue that triggers various diseases such as neurodegenerative diseases (Alzheimer's, Parkinson's), cardiovascular diseases (hypertension, arteriosclerosis, and others), cataracts, retinal damage, maculopathy, rheumatoid arthritis, asthma, stroke, diabetes mellitus , immunodepression, cancer, aging, hyperoxia, dermatitis, and others. The application of a healthy lifestyle for example by consuming food sources of bioactive compounds can minimize health risks. Rice is the staple food of the Indonesian people. Some types of rice contain red and black pigments which are known to have high antioxidant activity compared to white rice. The pigment comes from anthocyanin and proanthocyanidin. Various studies in vitro and in vivo prove that anthocyanin and proantocyanidine act as antioxidants and potency as a preventative for various diseases such as cardiovascular, diabetes mellitus, and etc.

scholarly journals 2599. Studying the Effects of Altering Histone Modification on Aspergillus fumigatus Virulence

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S903-S903
Author(s):  
Pam Lee ◽  
Hong Liu ◽  
Scott Filler
Keyword(s):  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Histone Modification ◽  
Histone Deacetylase ◽  
Cell Damage ◽  
Invasive Pulmonary Aspergillosis ◽  
Epithelial Cell Line ◽  
Saga Complex

Abstract Background As there are few drugs for treating invasive aspergillosis, there is an urgent need for new antifungal agents. Enzymes involved in histone modification are possible antifungal drug targets. We set out to investigate whether genes whose products are involved in histone modifications influence the virulence of Aspergillus fumigatus (Af). Methods Genes whose products were likely involved in histone modification were deleted in strain Af293 using CRISPR-Cas9. Virulence was assessed in a triamcinolone-treated mouse model of invasive pulmonary aspergillosis. The extent of Af-induced damage to the A549 pulmonary epithelial cell line was determined by Cr51 release assay. Results Af genes were selected for investigation based on their homology to genes encoding known histone modifying proteins and their high expression level in vivo. The genes were predicted to encode members of the COMPASS histone methyltransferase complex (cclA/bre2, set2/Afu5g06000), the SAGA histone acetyltransferase complex (spt3, spt8), and the RPDL histone deacetylase complex (hosA). The ΔcclA and Δset2 mutants had significant growth defects on rich media and were not tested further. The Δspt3 and Δspt8 mutants grew normally and had mild conidiation defects. The ΔhosA mutant had wild-type (WT) growth and conidiation in vitro. Mice infected with the WT strain had 100% mortality within 9 days whereas mice infected the Δspt3, Δspt8, and ΔhosA mutants had only 40% mortality by 21 days. The ΔhosA mutant also had impaired capacity to damage pulmonary epithelial cells in vitro. Conclusion Ccla and Set2, components of the COMPASS complex, are required for normal growth in vitro. Spt3 and Spt8, members of the SAGA complex, are required for normal conidiation and virulence. HosA, part of the RPD3L complex, is necessary for maximal virulence and induction of host cell damage. Our results suggest that the HosA histone deacetylase may be a promising drug target for treating invasive aspergillosis. Disclosures All authors: No reported disclosures.

Protective Effect of Schisandrin B Against Cyclosporine A-Induced Nephrotoxicity In Vitro and In Vivo

2012 ◽  
Vol 40 (03) ◽  
pp. 551-566 ◽  
Author(s):  
Shaohua Zhu ◽  
Yan Wang ◽  
Meiwan Chen ◽  
Jing Jin ◽  
Yuwen Qiu ◽  
...  
Keyword(s):  
Cell Viability ◽  
Cyclosporine A ◽  
Tubular Epithelial Cell ◽  
Epithelial Cell Line ◽  
Ros Generation ◽  
Therapeutic Effects ◽  
Schisandrin B ◽  
Protective Effects

Schisandrin B (Sch B) is an active ingredient of the fruit of Schisandra chinensis. It has many therapeutic effects arising from its tonic, sedative, antitussive and antiaging activities and is also used in the treatment of viral and chemical hepatitis. The aim of this study was to investigate the protective effects of Sch B on cyclosporine A (CsA)-induced nephrotoxicity in mice and HK-2 cells (a human proximal tubular epithelial cell line). After gavage with Sch B (20 mg/kg) or olive oil (vehicle), mice received CsA (30 mg/kg) by subcutaneous injection once daily for four weeks. Renal function, histopathology, and tissue glutathione (GSH) and malondialdehyde (MDA) levels were evaluated after the last treatment. The effects of Sch B on CsA–induced oxidative damage in HK-2 cells were investigated by measuring cell viability, the release of lactate dehydrogenase (LDH), the level of reactive oxygen species (ROS), and the cellular GSH and ATP concentrations. Cellular apoptosis was assessed by flow cytometry. Treatment with Sch B in CsA-treated mice significantly suppressed the elevation of blood urea nitrogen (BUN) and serum creatinine levels and attenuated the histopathological changes. Additionally, Sch B also decreased renal MDA levels and increased GSH levels in CsA-treated mice. Using an in vitro model, Sch B (2.5, 5 and 10 μM) significantly increased the cell viability and reduced LDH release and apoptosis induced by CsA (10 μM) in HK-2 cells. Furthermore, Sch B increased the intracellular GSH and ATP levels and attenuated CsA-induced ROS generation. In conclusion, Sch B appears to protect against CsA-induced nephrotoxicity by decreasing oxidative stress and cell death.

C3a receptor activation promotes uric acid or LPS-induced CCL2 production in proximal tubular epithelial cells

2019 ◽  
Author(s):  
Danyu You ◽  
Congwei Luo ◽  
Liyan Yang ◽  
Jiong Cui ◽  
Yi Chen ◽  
...  
Keyword(s):  
Uric Acid ◽  
Receptor Activation ◽  
Tubular Epithelial Cell ◽  
Enzyme Linked Immunosorbent Assay ◽  
Epithelial Cell Line ◽  
Ccl2 Expression ◽  
Proximal Tubular ◽  
Ccl2 Production

Abstract Background To identify the role of the interaction of complement fragment 3a (C3a) and its receptor C3aR in uric acid (UA) or lipopolysaccharide (LPS)-induced CCL2 expression in human renal proximal tubular epithelial cell line HK-2.Methods HK-2 cells were cultured in vitro and treated with UA or LPS to induce the production of CCL2. To determine the role of C3a-C3aR interaction in CCL2 production, HK-2 cells exposed to UA or LPS were pretreated with the small molecule sb290157 (1μmol/l) or recombinant C3a protein (100nmol/l) to block or amplify C3a-C3aR interaction. The expression of CCL2, C3 and C3aR were detected by real-time quantitative PCR (Q-PCR), enzyme-linked immunosorbent assay (ELISA) and Western Blotting.Results 12 hours of UA (150μmol/L) stimulation or 8 hours of LPS (5μg/ml) treatment significantly induced CCL2 and C3 mRNA transcription in HK-2 cells. The expression of CCL2 induced by UA or LPS could be abrogated by C3aR blockade. C3a stimulation alone has little effect on inducing CCL2 expression in HK-2 cells. However, when it stimulated the HK-2 cells together with UA or LPS, it could remarkably potentiate UA or LPS-induced CCL2 expression.Conclusion The activation of C3aR provides an important co-stimulating signal for CCL2 production in HK-2 cells and blocking the interaction of C3a-C3aR will significantly inhibit UA or LPS induced CCL2 production.

scholarly journals Exosomes function as nanoparticles to transfer miR-199a-3p to reverse chemoresistance to cisplatin in hepatocellular carcinoma

2020 ◽  
Vol 40 (7) ◽  
Author(s):  
Kun Zhang ◽  
Chu-xiao Shao ◽  
Jin-de Zhu ◽  
Xin-liang Lv ◽  
Chao-yong Tu ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Normal Liver ◽  
The Body ◽  
Cell Counting ◽  
Epithelial Cell Line ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Micro Rnas

Abstract Hepatocellular carcinoma (HCC) is a frequently seen malignant tumor globally. The occurrence of cisplatin (DDP) resistance is one of the main reasons for the high mortality of HCC patients. Therefore, it is of great theoretical significance and application value to explore the mechanism of chemotherapy resistance. Drug resistance can be modulated by exosomes containing mRNAs, micro RNAs (miRNAs) and other non-coding RNA (ncRNAs). Exosomal miR-199a-3p (Exo-miR-199a-3p) was subjected to extraction and verification. Whether exo-miR-199a-3p could make HCC cells sensitive to DDP in vitro was verified via flow cytometry, Cell Counting Kit-8 (CCK-8) assay, immunofluorescence assay and Transwell assay. Intravenous injection of exo-miR-199a-3p and intraperitoneal injection of DDP were carried out in vivo. Moreover, the possible targets of miR-199a-3p were screened through bioinformatics analysis, which were ascertained by Western blotting (WB). Then, miR-199a-3p levels in human normal liver epithelial cell line HL-7702 and HCC cell lines HuH7 and HuH7/DDP were elevated in a concentration-dependent manner. Exo-miR-199a-3p has abilities to adjust underlying targets and conjugate cells, to repress cells to invade, stimulate their apoptosis and abate their ability. Additionally, the caudal injection of exo-miR-199a-3p reversed the chemoresistance of tumors and slowed down their growth in the body owing to the up-regulation of miR-199a-3p and down-regulation of underlying target proteins in tumors. Finally, exo-miR-199a-3p was found to overturn the HCC’s resistance to DDP, and it may function in DDP-refractory HCC therapy as an underlying option in the future.

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