Use of flow cytometry and monochlorobimane to quantitate intracellular glutathione concentrations in feline leukocytes

Webb, Craig B.; Bedwell, Cathy L.; Guth, Amanda; Avery, P.; Dow, Steven

doi:10.1016/j.vetimm.2006.02.009

Cited by 22 publications

(24 citation statements)

References 27 publications

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“…After treatment, the cells were washed with PBS and detached with 0.25% trypsin with EDTA. Cells were incubated with either 5 M carboxy-H2DCFDA for 30 min at 37°C or with 40 M mBCl for 20 min at room temperature in the dark, as previously done (27). Data were collected on more than 50,000 cells per sample using a Gallios flow cytometer (Beckman Coulter, Brea, CA) and analyzed using FlowJo software version 7.6.5 (Tree Star, Ashland, OR).…”

Section: Biochemicalsmentioning

confidence: 99%

Interaction of Interferon Gamma-Induced Reactive Oxygen Species with Ceftazidime Leads to Synergistic Killing of Intracellular Burkholderia pseudomallei

Mosovsky

Silva

Troyer

et al. 2014

Antimicrob Agents Chemother

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Burkholderia pseudomallei, a facultative intracellular pathogen, causes severe infections and is inherently refractory to many antibiotics. Previous studies from our group have shown that interferon gamma (IFN-␥) interacts synergistically with the antibiotic ceftazidime to kill bacteria in infected macrophages. The present study aimed to identify the underlying mechanism of that interaction. We first showed that blocking reactive oxygen species (ROS) pathways reversed IFN-␥-and ceftazidime-mediated killing, which led to our hypothesis that IFN-␥-induced ROS interacted with ceftazidime to synergistically kill Burkholderia bacteria. Consistent with this hypothesis, we also observed that buthionine sulfoximine (BSO), another inducer of ROS, could substitute for IFN-␥ to similarly potentiate the effect of ceftazidime on intracellular killing. Next, we observed that IFN-␥ induced ROS-mediated killing of intracellular but not extracellular bacteria. On the other hand, ceftazidime effectively reduced extracellular bacteria but was not capable of intracellular killing when applied at 10 g/ml. We investigated the exact role of IFN-␥-induced ROS responses on intracellular bacteria and notably observed a lack of actin polymerization associated with Burkholderia bacteria in IFN-␥-treated macrophages, which led to our finding that IFN-␥-induced ROS blocks vacuolar escape. Based on these results, we propose a model in which synergistically reduced bacterial burden is achieved primarily through separate and compartmentalized killing: intracellular killing by IFN-␥-induced ROS responses and extracellular killing by ceftazidime. Our findings suggest a means of enhancing antibiotic activity against Burkholderia bacteria through combination with drugs that induce ROS pathways or otherwise target intracellular spread and/or replication of bacteria.

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Section: Biochemicalsmentioning

confidence: 99%

Interaction of Interferon Gamma-Induced Reactive Oxygen Species with Ceftazidime Leads to Synergistic Killing of Intracellular Burkholderia pseudomallei

Mosovsky

Silva

Troyer

et al. 2014

Antimicrob Agents Chemother

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“…Endogenous glutathione (GSH) levels were measured by monochlorobimane staining as described (Webb et al, 2006).…”

Section: Detection Of Intracellular Glutathione Levelsmentioning

confidence: 99%

Thiazolide-induced apoptosis in colorectal cancer cells is mediated via the Jun kinase–Bim axis and reveals glutathione-S-transferase P1 as Achilles’ heel

et al. 2011

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“…Previous studies in cats with anemia and hematological abnormalities revealed the high production of free radicals (Snow 1962;Christopher 1989;Christopher et al 1990;Webb et al 2006). A study in 20 CKD cats demonstrated a higher reduced glutathione to oxidized glutathione (GSH/GSSG) ratio than the controls, while superoxide dismutase (SOD) values were the same (Keegan and Webb 2010).…”

Section: Introductionmentioning

confidence: 99%

Changes in reduced glutathione, oxidized glutathione, and glutathione peroxidase in cats with naturally occurring chronic kidney disease

Piyarungsri

Pusoonthornthum

2016

Comp Clin Pathol

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Chronic kidney disease (CKD) is one of the most common diseases in geriatric cats. The objective of this study was to determine oxidative stress in CKD cats by measuring changes in reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione peroxidase (GPx), and reduced glutathione to oxidized glutathione (GSH/GSSG) ratio in cats with naturally occurring chronic kidney disease (CKD). Thirteen clinically normal client-owned aged-matched cats and 23 naturally occurring CKD cats were included. Completed blood count, blood urea nitrogen, creatinine, GSH, GPx, GSSG, and GSH/GSSG ratio were measured on the first day of diagnosis. The results showed that CKD cats had significantly lower GSH, GPx, and GSH/GSSG ratio levels (2.77 ± 0.27 mM, 2.32 ± 0.40 nmol/min and 148.26 ± 34.19) than the clinically normal client-owned age-matched cats (4.23 ± 0.67 mM, 6.68 ± 0.79 nmol/min and 312.64 ± 76.80). The GSSG in the CKD cats (35.20 ± 4.37 μM) was significantly higher than in the clinically normal client-owned age-matched cats (19.66 ± 2.75 μM). The results indicated that cats with naturally occurring CKD experience oxidative stress.

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Use of flow cytometry and monochlorobimane to quantitate intracellular glutathione concentrations in feline leukocytes

Cited by 22 publications

References 27 publications

Interaction of Interferon Gamma-Induced Reactive Oxygen Species with Ceftazidime Leads to Synergistic Killing of Intracellular Burkholderia pseudomallei

Interaction of Interferon Gamma-Induced Reactive Oxygen Species with Ceftazidime Leads to Synergistic Killing of Intracellular Burkholderia pseudomallei

Thiazolide-induced apoptosis in colorectal cancer cells is mediated via the Jun kinase–Bim axis and reveals glutathione-S-transferase P1 as Achilles’ heel

Changes in reduced glutathione, oxidized glutathione, and glutathione peroxidase in cats with naturally occurring chronic kidney disease

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