Martin Beránek

High incidence of thrombosis and venous thromboembolism was reported in patients with COVID-19. In this study, we focused on analysis of thrombophilic mutations performed without a standard DNA extraction step. In one hundred of COVID-19 positive outpatients, real-time PCR for Leiden mutation in the FV gene and G20210A mutation in the FII gene was carried out from DNA extracts and modified whole blood samples, and their cycle threshold (Ct) values were evaluated. In the extracts, healthy homozygotes (wt/wt), heterozygotes (M/wt), and homozygous carriers of Leiden mutation (M/M) provided median Ct values of 18.5, 19.4/22.0, and 20.9. In the whole blood, Ct values were 25.3 (wt/wt), 24.8/27.2 (M/wt), and 26.9 (M/M). Median Ct values for G20210A in the extracts were 19.6 for homozygotes (wt/wt), and 19.7/20.4 for heterozygous carriers. The whole blood samples provided Ct values of 23.9 in healthy homozygotes and 26.3/27.2 in heterozygotes for G20210A mutation. No homozygous subjects for G20210A and no double heterozygotes (for Leiden and G20210A mutations) were found. Despite significant differences in the Ct values, genotyping showed complete result concordance of the DNA extracts and the whole blood samples. The integrity and amplificability of DNA molecules in the whole blood samples during 28 days of deep freezing, interrupted by four cycles of thawing, did not significantly change. In conclusion, we demonstrated a new protocol for the detection of the thrombophilic mutations via real time PCR on the modified whole blood of COVID-19 positive patients. The blood modification was reliable, easy, cheap, and saving costs and turnaround time of the whole laboratory process.

We compared the efficiency of real-time PCR analysis of FII (c.*97G>A, G20210A) and FV Leiden (c.1601G>A) thrombophilic mutations in the samples obtained from venous blood treated with various anti­coagulant agents (EDTA, heparin, and sodium fluoride with potassium oxalate), or from clotted venous blood; one hundred samples of wild-type subjects were tested. Genomic DNA extracts and whole blood specimens modified by 90 °C heating were analysed by real-time PCR analysis; cycle threshold values were subsequently evaluated. Real-time PCR analysis for the FII gene assay performed in DNA extracts from EDTA blood samples revealed a median Ct value of 19.3. Similar Ct values were apparent in the DNA extracts obtained from the heparinized blood and sodium fluoride with potassium oxalate-treated samples: 18.5 and 18.9, respectively. Signi­ficantly higher Ct values were found in extracts from clotted blood with medians of 20.6 (tubes with inert separation gel) and 20.5 (tubes without the gel, both P < 0.001). The data on the FV real-time PCR analysis were very comparable to the FII assay. In the modified whole blood, the samples treated with heparin salts showed significantly lower Ct values (P < 0.001) in both assays when compared with the samples with EDTA, sodium fluoride with potassium oxalate, and with the samples with clotted blood. Our results indicate that real-time PCR analyses of thrombophilic mutations were not negatively influenced by the presence of heparin salts in collection tubes. Blood samples with various anticoagulants might be exchangeable for each other when DNA analysis of thrombophilic mutations is required.

Background: Psoriasis is a pathological condition characterized by immune system dysfunction and inflammation. Patients with psoriasis are more likely to develop a wide range of disorders associated with inflammation. Serum levels of various substances and their combinations have been associated with the presence of the disease (psoriasis) and have shown the potential to reflect its activity. The aim of the present study is to contribute to the elucidation of pathophysiological links between psoriasis, its pro-inflammatory comorbidity metabolic syndrome (MetS), and the expression of clusterin and elafin, which are reflected in the pathophysiological “portfolio” of both diseases. Material and methods: Clinical examinations (PASI score), ELISA (clusterin, elafin), and biochemical analyses (parameters of MetS) were performed. Results: We found that patients with psoriasis were more often afflicted by MetS, compared to the healthy controls. Clusterin and elafin levels were higher in the patients than in the controls but did not correlate to the severity of psoriasis. Conclusion: Our data suggest that patients with psoriasis are more susceptible to developing other systemic inflammatory diseases, such as MetS. The levels of clusterin and elafin, which are tightly linked to inflammation, were significantly increased in the patients, compared to the controls, but the presence of MetS in patients did not further increase these levels.

Goeckerman therapy (GT) of psoriasis vulgaris is based on the application of crude coal tar and ultraviolet radiation. We investigated DNA damage by the number of micronucleated binucleated cells (MNBC) in lymphocytes, serum homocysteine, vitamin B12, folic acid, and two polymorphisms (C677T and A1298C) in the MTHFR gene in 35 patients with exacerbated psoriasis vulgaris classified according to the psoriasis area and severity index (PASI) score and treated by GT. The median of PASI score decreased from nineteen to five, and MNBC increased from 10 to 18‰ after GT (p < 0.001 in both cases). Correlations of MNBC with homocysteine (Spearman’s rho = 0.420, p = 0.012) and vitamin B12 (rho = −0.389, p = 0.021) before the therapy were observed. Hyperhomocysteinemia was an independent predictor of genotoxicity (OR 9.91; 95% CI, 2.09–55.67; p = 0.003). Homocysteine was higher in females than in males (13 vs. 12 µmol/L, p = 0.045). In contrast, vitamin B12 levels in the females were lower than in the males (160 vs. 192 pmol/L, p = 0.047). Vitamin B12 in the females were negatively influenced by smoking status (160 pmol/L in smokers vs. 192 pmol/L in non-smokers, p = 0.025). A significantly higher MNBC was found in CC homozygous patients (A1298C polymorphism) than in AC heterozygotes (32 vs. 16‰, p = 0.005) and AA homozygotes (32 vs. 18‰, p = 0.036). Our data showed that homocysteine participates in the pathogenesis of psoriasis. Its serum levels correlated with MNBC and allowed the prediction of DNA damage to appear within GT. Both micronutrients status and homocysteine metabolic pathway contribute to the genotoxicity of GT.

BRAF mutation detection in progressive RR-TC could play a role in decision-making of targeted therapies in the near future. So far, only multi-kinase inhibitors (sorafenib and lenvatinib) are legally accepted. On the other hand, for patients with disseminated BRAF mutant malignant melanoma or lung cancer, selective treatments with RAF and MEK inhibitors (vemurafenib, dabrafenib, and trametinib) are available. A crucial advantage of these inhibitors in the treatment of thyroid cancer is their ability to restore expression of the genes involved in iodine metabolism in cancer cells that have lost this ability, thus opening the door for radioiodine treatment again. Key words thyroid cancer - BRAF mutation - biological therapy - tyrosine kinase inhibitor - MEK inhibitor The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers. Submitted: 4. 6. 2018 Accepted: 1. 8. 2018.

The aryl hydrocarbon receptor (AhR) is highly expressed in psoriasis skin lesions. The aim of this study was to investigate serum concentrations of AhR, cytochromes P450 (CYP) 1A1 and 1B1 in patients with exacerbated psoriasis vulgaris treated with combined therapy of ultraviolet radiation (UVR) and crude coal tar. The analyses were performed by using enzyme-linked immunosorbent assays. Before the treatment, the patients had significantly higher serum levels of AhR and CYP1A1 than healthy controls. AhR median noticeably decreased after the therapy; nevertheless, it remained significantly higher compared to the controls. CYP1A1 levels measured before and after the therapy did not differ significantly. Serum CYP1A1 positively correlated with AhR values before and after the treatment. The serum values of CYP1B1 were very low and we did not see any differences between the study group and the control group. The study demonstrated that serum levels of AhR and CYP1A1 could indicate their immunopathological and metabolic roles in exacerbated psoriasis.

Goeckerman therapy (GT) represents an effective treatment of psoriasis including a combination of pharmaceutical grade crude coal tar (CCT) and ultraviolet irradiation (UV-R). Coal tar contains a mixture of polycyclic aromatic hydrocarbons. The best known carcinogenic polyaromate – benzo[a]pyrene is metabolized into a highly reactive benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE). Glutathione S-transferase M1 (GSTM1) catalyses the conjugation of drugs, toxins and products of oxidative stress with glutathione. The aim of the study is to found possible associations between GSTM1 genotypes and the level of BPDE-DNA adducts in 46 psoriatic patients treated with GT. For genotyping, droplet digital PCR was applied. The GSTM1 copy number was normalized to β-globin reference gene. In five GSTM1*1/*1 subjects, the GSTM1 to β-globin ratio moved from 0.99 to 1.03 with a median of 1.01. GSTM1*0/*1 heterozygotes (n = 20) contained only one GSTM1 function allele which conditioned the ratio 0.47–0.53 (median 0.50). GSTM1*0/*0 individuals (n = 21) showed no amplification of the null variants because of the large deletion in GSTM1 . BPDE-DNA concentrations ranged from 1.8 to 66.3 ng/µg with a median of 12.3 ng/µg. GSTM1*0/*0 and GSTM1*0/*1 genotypes showed non-significantly higher concentrations of BPDE-DNA adducts than the GSTM1*1/*1 one (12.3 and 12.4 vs 7.8 ng/µg). The non-significant relationship between BPDE-DNA adducts and GSTM1 genotypes in psoriatic patients could be associated with relatively low doses of CCT and short-term UV-R exposures used in GT.