Measurement of Glutathione as a Tool for Oxidative Stress Studies by High Performance Liquid Chromatography.

BACKGROUND: Maintenance of the ratio of glutathione in the reduced (GSH) and oxidised (GSSG) state in cells is important in redox control, signal transduction and gene regulation, factors that are altered in many diseases. The accurate and reliable determination of GSH and GSSG simultaneously is a useful tool for oxidative stress determination. Measurement is limited primarily to the underestimation of GSH and overestimation GSSG as a result of auto-oxidation of GSH. The aim of this study was to overcome this limitation and develop, optimise and validate a reverse-phase high performance liquid chromatographic (HPLC) assay of GSH and GSSG for the determination of oxidant status in cardiac and chronic kidney diseases. METHODS: Fluorescence detection of the derivative, glutathione-O-pthaldialdehyde (OPA) adduct was used. The assay was validated by measuring the stability of glutathione and glutathione-OPA adduct under conditions that could affect the reproducibility including reaction time and temperature. Linearity, concentration range, limit of detection (LOD), limit of quantification (LOQ), recovery and extraction efficiency and selectivity of the method were assessed. RESULTS: There was excellent linearity for GSH (r2 = 0.998) and GSSG (r2 = 0.996) over concentration ranges of 0.1 µM-4 mM and 0.2 µM-0.4 mM respectively. The extraction of GSH from tissues was consistent and precise. The limit of detection for GSH and GSSG were 0.34 µM and 0.26 µM respectively whilst their limits of quantification were 1.14 µM and 0.88 µM respectively. CONCLUSION: These data validate a method for the simultaneous measurement of GSH and GSSG in samples extracted from biological tissues and offer a simple determination of redox status in clinical samples.

Expansion of cytotoxic tissue-resident CD8+ T cells and CCR6+CD161+ CD4+ T cells in the nasal mucosa following mRNA COVID-19 vaccination.

Vaccines against SARS-CoV-2 have shown high efficacy in clinical trials, yet a full immunologic characterization of these vaccines, particularly within the human upper respiratory tract, is less well known. Here, we enumerate and phenotype T cells in nasal mucosa and blood using flow cytometry before and after vaccination with the Pfizer-BioNTech COVID-19 vaccine (n = 21). Tissue-resident memory (Trm) CD8+ T cells expressing CD69+CD103+ increase in number ~12 days following the first and second doses, by 0.31 and 0.43 log10 cells per swab respectively (p = 0.058 and p = 0.009 in adjusted linear mixed models). CD69+CD103+CD8+ T cells in the blood decrease post-vaccination. Similar increases in nasal CD8+CD69+CD103- T cells are observed, particularly following the second dose. CD4+ cells co-expressing CCR6 and CD161 are also increased in abundance following both doses. Stimulation of nasal CD8+ T cells with SARS-CoV-2 spike peptides elevates expression of CD107a at 2- and 6-months (p = 0.0096) post second vaccine dose, with a subset of donors also expressing increased cytokines. These data suggest that nasal T cells may be induced and contribute to the protective immunity afforded by this vaccine.

Endocervical Regulatory T Cells Are Associated With Decreased Genital Inflammation and Lower HIV Target Cell Abundance.

Regulatory T cells (Tregs) play important roles in tissue homeostasis, but few studies have investigated tissue Tregs in the context of genital inflammation, HIV target cell density, and vaginal microbiota in humans. In women from Nairobi (n=64), the proportion of CD4+ CD25+ CD127low Tregs in the endocervix correlated with those in blood (r=0.31, p=0.01), with a higher Treg frequency observed in the endocervix (median 3.8 vs 2.0%, p<0.0001). Most Tregs expressed FOXP3 in both compartments, and CTLA-4 expression was higher on endocervical Tregs compared to blood (median 50.8 vs 6.0%, p<0.0001). More than half (34/62, 55%) of participants displayed a non-Lactobacillus dominant vaginal microbiota, which was not associated with endocervical Tregs or CD4+ T cell abundance. In a multivariable linear regression, endocervical Treg proportions were inversely associated with the number of elevated pro-inflammatory cytokines (p=0.03). Inverse Treg associations were also observed for specific cytokines including IL-1ß, G-CSF, Eotaxin, IL-1RA, IL-8, and MIP-1 ß. Higher endocervical Treg proportions were associated with lower abundance of endocervical CD4+ T cells (0.30 log10 CD4+ T cells per log10 Treg, p=0.00028), with a similar trend for Th17 cells (p=0.09). Selectively increasing endocervical Tregs may represent a pathway to reduce genital tract inflammation in women.

Impact of Intravenous Iron on Oxidative Stress and Mitochondrial Function in Experimental Chronic Kidney Disease.

BACKGROUND: Mitochondrial dysfunction is observed in chronic kidney disease (CKD). Iron deficiency anaemia (IDA), a common complication in CKD, is associated with poor clinical outcomes affecting mitochondrial function and exacerbating oxidative stress. Intravenous (iv) iron, that is used to treat anaemia, may lead to acute systemic oxidative stress. This study evaluated the impact of iv iron on mitochondrial function and oxidative stress. METHODS: Uraemia was induced surgically in male Sprague-Dawley rats and studies were carried out 12 weeks later in two groups sham operated and uraemic (5/6 nephrectomy) rats not exposed to i.v. iron versus sham operated and uraemic rats with iv iron. RESULTS: Induction of uraemia resulted in reduced iron availability (serum iron: 31.1 ± 1.8 versus 46.4 ± 1.4 µM), low total iron binding capacity (26.4 ± 0.7 versus 29.5 ± 0.8 µM), anaemia (haematocrit: 42.5 ± 3.0 versus 55.0 ± 3.0%), cardiac hypertrophy, reduced systemic glutathione peroxidase activity (1.12 ± 0.11 versus 1.48 ± 0.12 U/mL), tissue oxidative stress (oxidised glutathione: 0.50 ± 0.03 versus 0.36 ± 0.04 nmol/mg of tissue), renal mitochondrial dysfunction (proton/electron leak: 61.8 ± 8.0 versus 22.7 ± 5.77) and complex I respiration (134.6 ± 31.4 versus 267.6 ± 26.4 pmol/min/µg). Iron therapy had no effect on renal function and cardiac hypertrophy but improved anaemia and systemic glutathione peroxidase (GPx) activity. There was increased renal iron content and complex II and complex IV dysfunction. CONCLUSION: Iron therapy improved iron deficiency anaemia in CKD without significant impact on renal function or oxidant status.

Oxidative Stress and Cardiovascular Complications in Chronic Kidney Disease, the Impact of Anaemia.

Patients with chronic kidney disease (CKD) have significant cardiovascular morbidity and mortality as a result of risk factors such as left ventricular hypertrophy (LVH), oxidative stress, and inflammation. The presence of anaemia in CKD further increases the risk of LVH and oxidative stress, thereby magnifying the deleterious consequence in uraemic cardiomyopathy (UCM), and aggravating progression to failure and increasing the risk of sudden cardiac death. This short review highlights the specific cardio-renal oxidative stress in CKD and provides an understanding of the pathophysiology and impact of uraemic toxins, inflammation, and anaemia on oxidative stress.