Genetic diversity and seroprevalence of Toxoplasma gondii in COVID­19 patients; a first case-control study in Iran.

BACKGROUND: Toxoplasmosis is a serious or life-threatening disease in immunosuppressed patients and pregnant women. This study examined the likely association between Toxoplasma gondii infection and COVID-19 patients with moderate illness. METHODS: Seventy blood samples were collected from patients at the Health Reference Laboratory of Tabriz, Northwest Iran from April 2021 to September 2021. In addition, 70 healthy subjects of the same age (37 ± 15 years) and sex distribution were ethnically matched. Sera samples were examined for the detection of anti-Toxoplasma antibodies using ELISA. Nested-PCR targets were amplified based on the B1 and GRA6 genes. GRA6 amplicons were subjected to sequencing and phylogenetic analysis. RESULTS: The seroprevalence of toxoplasmosis based on IgG titer was 35.7% in the COVID­19 patients and 27.1% in the control group, representing not to be associated with the Toxoplasma seropositivity in COVID­19 patients (P = 0.18) compared to healthy subjects. Anti-T. gondii IgM was not found in any of the patients and healthy individuals. According to PCR amplification of the B1 and GRA6 genes, the frequency of T. gondii in COVID-19 patients was 14.2% (10/70). However, no T. gondii infection was detected in the healthy group. The CD4+T cell count was relatively lower in toxoplasmosis-infected patients (430-450 cells/mm3) than in control group (500-1500 cells/mm3). High genetic diversity (Hd: 0.710) of the type I strain of T. gondii was characterized in the patients. Present results showed that consumption of raw vegetables and close contact with stray cats can increase the transmission of T. gondii to COVID-19 patients (P < 0.01). CONCLUSIONS: The current study revealed that T. gondii type I infection is unequivocally circulating among the COVID-19 patients in Tabriz; However, no significant association was observed between the occurrence of Toxoplasma and the severity of COVID-19. To make more accurate health decisions, multicenter investigations with a larger sample size of different ethnic groups of the Iranian population are needed.

Evaluation of Activity Kinetic Parameters of SK319cys, As a New Cysteine Variant of Streptokinase: A Comparative Study.

BACKGROUND: Despite the extensive use of streptokinase in thrombolytic therapy, its administration may have some shortcomings like allergic reactions and relatively low half life. Specific PEGylation on cysteine at desired sites of streptokinase may alleviate these deficiencies and improve the quality of treatment. OBJECTIVE: This study was carried out to create a new cystein variant of streptokinase and compare its activity with formerly mutated SK263cys, SK45cys and intact streptokinase (Ski) to introduce superior candidates for specific PEGylation. METHOD: In silico study was carried out to select appropriate amino acid for cysteine substitution and accordingly mutagenesis was carried out by SOEing PCR. The mutated gene was cloned in E. coli, expressed, and purified by affinity chromatography. Activity of the purified proteins was assayed and kinetic parameters of enzymatic reaction were analyzed. RESULTS: According to in silico data, Arginine319 was selected for substitution with cysteine. SK319cys was achieved with 98% purity after cloning, expression and purification. It was shown that the enzymatic efficiency of SK319Cys and SK263cys was increased 18 and 21%, respectively, when compared to SKi (79.4 and 81.3 vs. 67.1µM-1min-1), while SK45cys showed 7% activity decrease (62.47µM-1min-1) compared to SKi. According to time-based activity assay, SK319Cys and SK263cys exhibited higher activity at lower substrate concentrations (100 and 200 µM), but at higher concentrations of substrate (400 and 800 µM), the proteins showed a very close trend of activity. CONCLUSION: SK319cys, as the new cysteine variant of streptokinase, together with SK263cys and SK45cys can be considered as appropriate molecules for specific PEGylation.

Relationship between serum high sensitivity C-reactive protein with angiographic severity of coronary artery disease and traditional cardiovascular risk factors.

Serum high-sensitivity C-reactive protein (hs-CRP) is predictive of coronary artery disease (CAD). The aim of this study was to examine the possible association of hs-CRP with presence and severity of CAD and traditional CAD risk factors. This case-control study was carried out on 2,346 individuals from September 2011 to May 2013. Of these 1,187 had evidence of coronary disease, and were subject to coronary angiography, and the remainder were healthy controls (n = 1,159). Characteristics were determined using standard laboratory techniques and serum Hs-CRP levels were estimated using enzyme-linked immunosorbent assay (ELISA) kits, and severity of CAD was assessed according to the score of obstruction in coronary artery. Serum hs-CRP levels were higher in those with severe coronary disease, who had stenosis ≥ 50% stenosis of at least one coronary artery (all p < 0.001 vs. individuals in healthy control), and correlated significantly with the score for coronary artery disease (all p < 0.01). After adjustment for conventional risk factors, regression analysis revealed that smoking habits, fasting blood glucose, total cholesterol, high-density lipoprotein, hs-CRP, blood pressure, anxiety, dietary intake of vitamin E, and cholesterol remained as independent determinants for angiographic severity of CAD. The area under the receiving operating characteristic (ROC) curve for serum hs-CRP was 0.869 (CI 95% 0.721-0.872, p < 0.001). The optimal values for the cut-off point was a serum hs-CRP of 2.78 mg/l (sensitivity 80.20%, specificity 85%) to predict severity of CAD. Increased serum hs-CRP levels are significantly associated with angiographic severity of CAD, suggesting its value as a biomarkers for predicting CAD.

Serum concentrations of MCP-1 and IL-6 in combination predict the presence of coronary artery disease and mortality in subjects undergoing coronary angiography.

Cytokines play a key role in the pathogenesis of coronary artery disease (CAD). The aim of current study was to investigate the relationship between the serum concentrations of 12 cytokines with mortality and extent of CAD in individuals undergoing angiography and healthy controls. 342 CAD patients were recruited and divided into 2 groups: those with ≥50% occlusion in at least one coronary artery [Angiography (+)] or <50% obstruction in coronary arteries [Angiography (-)]. Also 120 healthy subjects were enrolled as control group. Lipid profile, fasting blood glucose, body mass index, and blood pressure were evaluated in all the subjects. An Evidence Investigator® was used for measuring 12 cytokines (IL-1α, IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, MCP-1, IFN-γ, EGF, VEGF) using sandwich chemiluminescent assays. Univariate analysis, multivariate regression models, ROC, and Kaplan-Meier survival curves were used for exploring the candidate markers in CAD patients. Serum level of IFN-γ, IL-4, MCP-1, EGF, IL-6, and IL-8 were markedly higher in angiogram-positive patients, while VEGF concentrations were significantly (P < 0.05) lower, compared to control group. ROC analysis for MCP-1 showed that a cut-off of 61.95 pg/mL had 91% sensitivity and 91% specificity for predicting CAD patients. Moreover, >2.16 pg/mL IL-6 had a > 94% sensitivity and 70% specificity in predicting 2 years mortality in the subjects with a serum MCP-1 > 61.95 pg/ mL, and patients having IL-6/MCP-1 combination had a shorter survival.Our findings demonstrate that CAD patients with serum MCP-1 and IL-6 levels of >61.95 and >2.16 pg/mL had a higher mortality with 94.1% sensitivity and 70.5% specificity for predicting mortality in CAD patients.

Mechanism of vasorelaxation induced by Achillea wilhelmsii in rat isolated thoracic aorta.

BACKGROUND: Achillea wilhelmsii (A. wilhelmsii) is used in Iraninan folk medicine for the treatment of hypertension; also, in previous reports, the hypotensive and antihypertensive effects of this plant have been indicated. The aim of the present study is to investigate the vasorelaxant effect of the hydroalcholic extract of A. wilhelmsii and its underlying mechanisms in isolated rat aorta. MATERIALS AND METHODS: The effect of the hydroalcholic A. wilhelmsii extract was tested on the contractile response of Wistar rat aorta induced by potassium chloride (KCl) and phenylephrine (PE) using a pressure transducer that is connected to the PowerLab. RESULTS: The cumulative concentrations of A. wilhelmsii (0.5-8 mg/ml) induced a vasorelaxation both in endothelium-intact and endothelium-denuded aortas precontracted by high K(+) (6 × 10(-2) M) or 10(-6) M PE. A. wilhelmsii, at a concentration of 4 mg/ml, reduced Ca(2+)-induced contraction (P < 0.001 vs. control) after PE or KCl had generated a stable contraction in the Ca(2+)-free solution. Furthermore, after incubation with diltiazem, the vasorelaxant effect of A. wilhelmsii reduced in the endothelium-denuded aortas precontracted by PE or KCl (P < 0.001 vs. control). In contrast, A. wilhelmsii-induced relaxation was not affected by glibenclamide, BaCl2, ruthenium red, methylene blue, or heparin. CONCLUSIONS: The results showed that A. wilhelmsii had a vasorelaxation effect, which was not endothelium-dependent. The relaxation was mediated by inhibition of extracellular Ca(2+) influx through voltage- and receptor-operated Ca(2+) channels (VDDCs and ROCCs) in vascular smooth muscle cells.

Relationship between coronary and renal artery disease and associated risk factors in hypertensive and diabetic patients undergoing coronary angiography.

AIMS: Performing simultaneous renal angiography in patients undergoing coronary angiography for suspected coronary artery disease (CAD) is suitable for those who have a high probability for renal artery stenosis (RAS), thus better recognition of all potential candidates could have paramount importance. METHODS AND RESULTS: In a cross sectional study, 260 consecutive hypertensive and/or diabetic patients (135 males, 125 females with average age of 57.1 and 57.2 years respectively) underwent simultaneous coronary and renal angiography. RAS was identified in 55 patients (21.2%). Significant RAS (> 50%) was present in 37 patients (14.2%). Female sex (P=0.01), older age (62.1+/-10 vs 56.3+/-8.9 years, p=0.001), higher serum creatinine level (1.3+/-0.69 vs 0.98+/-0.35 mg/dl p=0.017), reduced estimated glomerular filtration rate (eGFR) (58.6+/-25.4 vs 81.8+/-28.1 ml/min/1.73 m2, p< 0.001), increased levels of intra-arterial systolic blood pressure (169.8+/-31.1 vs 155.1+/-28.4 mmHg, p=0.004) and pulse pressure (90.9+/-26.2 vs 77.5+/-21.9, p=<0.001) during catheterisation, history of hypertension alone (p=0.007) or accompanied with diabetes mellitus (DM) (p=0.014) and multi vessel CAD (> 2 vessels, p=0.002) were associated with significant RAS in univariate analysis and normal coronary arteries was a strong negative predictive factor (negative predictive value=95%). There was no significant relationship between involved location of coronary arteries, history of DM alone, history of dyslipidaemia and smoking with RAS. In multivariate model, female sex [odds ratio (OR) 0.3; 95% confidence interval (CI) 0.12-0.80, P=0.016], multivessel CAD (OR 1.88; 95% CI 1.25-2.83, P=0.002) and reduced eGFR (OR 0.96; 95% CI 0.95-0.99, P=0.002) were independent predictors of RAS. CONCLUSIONS: Considering the limitations of non-invasive techniques, it seems worthwhile from both diagnostic and prognostic standpoints to perform simultaneous renal angiography following coronary angiography in patients with multivessel CAD, especially if other mentioned risk predictors are also present.