Development of novel real-time RT-qPCR methodologies for quantification of the COL11A1 mRNA general and C transcripts and evaluation in non-small cell lung cancer specimens.

PURPOSE: The purpose of this study was the development of new quantitative methodologies for the general (total) COL11A1 gene and the C transcript (RT-qPCR methods for A and E transcripts have already been developed by our group previously), the quantification of all COL11A1 transcripts and the investigation for the first time of their potential association with histopathological prognostic factors in lung cancer. METHODS: Real-time RT-qPCR methodologies with dual hybridization probes were developed on the Light Cycler 1.5 platform (Roche,Germany). All COL11A1 transcripts were measured in 27 cDNA lung tissue specimens in a blinded fashion (8 control and 19 non-small cell lung cancer (NSCLC) tissues with known histopathological data). Statistical analysis was performed with the IBM SPSS program. RESULTS: The novel real-time RT-qPCR methodologies were appropriately validated. All 19 NSCLC samples were positive for the general COL11A1 transcript (range 11.2-1198.0 copies/µg total RNA, while 5 out of 8 control samples were negative: mean values were also statistically significantly different (p<0.001). In 4 tumor samples (21%), no specific COL11A1 transcript was detected. Transcript C was detected in only 3 tumor samples. Regarding transcripts A and E, 13 out of 19 tumor samples were positive for either one (68%) and 11 for both (58%). CONCLUSIONS: No other statistically significant association of the specific transcripts with histopathological data was observed, most probably due to the limited number of samples. As the number of general COL11A1 transcripts/µg exceeds the sum of A+E+C transcripts in all samples, there is opportunity for discovery and identification of other transcripts as well.

Circulating resistin is a significant predictor of mortality independently from cardiovascular comorbidities in elderly, non-diabetic subjects with chronic kidney disease.

CONTEXT: Resistin is associated with inflammation, atherosclerosis and cardiovascular (CV) disease. OBJECTIVE: To associate circulating resistin with all-cause and CV mortality in chronic kidney disease (CKD) patients. METHODS: Serum resistin was determined in a cohort of 80 elderly, non-diabetic patients with stable CKD at different stages in a follow-up period of 5 years. RESULTS: Circulating resistin was significantly elevated in deceased compared to alive patients. Resistin emerged as an independent biomarker of all-cause and CV mortality after a 5-year follow-up period. CONCLUSION: Elevated circulating resistin was a significant independent predictor of CV and all-cause mortality in elderly, non-diabetic CKD patients.

Association of vitamin D receptor gene TaqI polymorphism with Alzheimer's disease in a Southeastern European Caucasian population.

The role of vitamin D in Alzheimer's Disease (AD) has been studied over the past years. The results from numerous studies have indicated that the molecular pathways involved in the development of AD are closely related to the molecular pathways of the mechanisms of action of vitamin D. However, only a limited number of studies have described the key role of vitamin D receptor (VDR) in the regulation of the functions of vitamin D and the potential effect of single nucleotide polymorphisms (SNPs) of the VDR gene. Thus, the aim of the present study was to investigate the VDR TaqI polymorphism in relation to AD in a Southeastern European Caucasian (SEC) cohort. Further, the present study aimed to compare the results obtained with those of other AD populations. For this purpose, blood samples from 90 confirmed patients with AD [median age, 74 years; median mini-mental state examination (MMSE) score of 21; median frontal assessment battery (FAB) score of 10] and 103 healthy controls (median age, 57 years) were analyzed to determine the genotypes of TaqI (rs731236) using quantitative PCR. The frequencies (%) of the TaqI TT, TC and CC genotypes in the controls/patients were 34/48.9, 47.6/41.1 and 18.4/10.0, respectively. Statistically significant differences were observed for the TaqI C allele [odds ratio (OR). 0.54; 95% confidence interval (CI), 0.30-0.96; P=0.035], the TaqI TT genotype (OR, 1.86; 95% CI, 1.04-3.32; P=0.035) and the TaqI CC genotype (OR, 0.119; 95% CI, 0.014-0.995; P=0.032,) in relation to the MMSE score <21 in the patient's group. The TaqI TT allele was found to increase the risk of developing AD by 1.86-fold in the SEC population, while the TaqI C allele may act protectively, with a 46% lower risk of developing the disease. Patients with the TaqI CC genotype were found to have an 88% less likelihood of developing severe cognitive impairment based on the MMSE score. On the whole, the present study did not confirm the results of previous studies on the VDR TaqI C allele in patients with AD.

Association of vitamin D receptor gene haplotypes with late-onset Alzheimer's disease in a Southeastern European Caucasian population.

Vitamin D receptor (VDR) gene single nucleotide polymorphisms (SNPs) have been investigated over the past years with the aim of identifying any association with the development of Alzheimer's disease (AD). However, information regarding the potential association of VDR SNP haplotypes with AD is limited. The aim of the present study was to provide additional knowledge on the effects of VDR haplotypes on the development of late-onset AD in a cohort of Southeastern European Caucasians (SECs). The study sample included 78 patients with late-onset AD and 103 healthy subjects as the control group. VDR SNPs that were analyzed were TaqI (rs731236), BsmI (rs1544410) and FokI (rs2228570). The CAC (TaqI, BsmI and FokI) haplotype was found to be associated with a 53% lower risk of developing the disease (OR, 0.47; 95% CI, 0.23-0.96; P=0.04) and the TAC (TaqI, BsmI and FokI) haplotype was associated with an ~6-fold greater risk of developing AD (OR, 6.19; 95% CI, 1.91-20.13; P=0.0028). Female subjects carrying the TAC haplotype had a ~9-fold greater risk of developing AD in comparison to female control subjects (OR, 9.27; 95% CI, 1.86-46.28; P<0.05). The TaqI and BsmI polymorphisms were in high linkage disequilibrium (D'=0.9717, r=0.8467) and produced a haplotype with a statistically significant different frequency between the control and AD group. The TA (TaqI and BsmI) haplotype was associated with an ~8-fold greater risk of developing AD (OR, 8.27; 95% CI, 2.70-25.28; P<0.05). Female TA carriers had an ~14-fold greater risk of developing the disease in comparison to female control subjects (OR, 13.93; 95% CI, 2.95-65.87; P<0.05). On the whole, the present study demonstrates that in the SEC population, TAC and TA are risk haplotypes for AD, while the CAC haplotype may act protectively. SEC women carrying the TAC or TA haplotype are at a greater risk of developing AD, thus suggesting that women are markedly affected by the poor utilization of vitamin D induced by the VDR haplotype.

Impact of vitamin D receptor gene polymorphisms on vitiligo susceptibility and clinical features in a Southeastern European Caucasian population.

An association of vitamin D receptor (VDR) polymorphisms and vitiligo has been suggested. However, previous studies have reported contradictory results while including limited data among Caucasians. The aim of this single­center study was to evaluate the effect of three common VDR gene polymorphisms (FokI, TaqI and BsmI) on susceptibility and clinical aspects of vitiligo in a Southeastern European Caucasian population. A total of 110 unrelated vitiligo cases and 509 general population controls were enrolled from October 2018 to November 2019. Genomic DNA was extracted from whole blood after de­identification and anonymization of the samples and genotyped for the selected VDR polymorphisms by the qPCR (melting curve analysis). Subgroup analysis by clinical features among subsets of patients indicated that, compared to subjects with the FokI TT genotype or T allele, carriers of the FokI CC genotype or C allele exhibited significantly decreased risk of developing vitiligo before the age of 30 [TT vs. CC: odds ratio (OR)=0.286, 95% confidence interval (CI): 0.083­0.984, P=0.041; T vs. C: OR=0.545, 95% CI: 0.313­0.948, P=0.031]. Intra­patient analysis also revealed that, compared to T allele, the presence of TaqI C allele was adversely associated with the incidence of concurrent leukotrichia (T vs. C: OR=1.874, 95% CI: 1.018­3.451, P=0.042). Comparisons between the case and control groups showed no evidence to support an association between susceptibility to vitiligo and the VDR BsmI, TaqI, and FokI polymorphisms in this cohort. Thus, the studied VDR polymorphisms might indirectly impact the clinical course and treatment decision­making despite their lack of association with vitiligo per se. Further research with larger sample sizes, especially across Caucasian individuals, should be performed to confirm these findings.

Lazaroid (U-74389G) ameliorates lung injury due to lipid peroxidation and nitric oxide synthase-dependent reactive oxygen species generation caused by remote systematic ischemia-reperfusion following thoracoabdominal aortic occlusion.

INTRODUCTION: Lung ischemia-reperfusion injury after thoracoabdominal aortic occlusion represents a major complication, which increases morbidity and mortality. In the present study we hypothesized that lazaroid U-74389G intravenous administration protects from lung ischemia-reperfusion injury through lipid peroxidation inhibition. MATERIALS AND METHODS: A total of 24 pigs were randomized in three groups. Group I (n = 8) underwent sham operation, group II (n = 8) underwent thoracoabdominal aortic occlusion for 45min and received placebo and group III (n = 8) received 3 doses of lazaroid (3 mg/kg) 60 and 30min before thoracoabdominal aortic occlusion and at 30min during thoracoabdominal aortic occlusion (duration 45min). Aortic occlusion was performed with aortic balloon-catheters under fluoroscopic guidance. All animals were sacrificed at the 7 t h postoperative day and lung specimens were harvested for molecular analysis. RESULTS: mRNA levels of leukotrienes LB4 (LTB4R2), LC4 (LTC4S) and nitric oxide synthase (NOS) isoforms including iNOS, nNOS and eNOS were determined with real-time RT-qPCR. Nitric oxide can either induce (iNOS) or inhibit (nNOS and eNOS) lipid peroxidation based on its specific isoform origin. Group III showed significantly reduced mRNA levels of LTB4R2 (-63.7%), LTC4S (-35.9%) and iNOS (-60.2%) when compared with group II (P < 0.05, for all). The mRNA levels of nNOS was significantly increased (+37.4%), while eNOS was slightly increased (+2.1%) in group III when compared with group II (P < 0.05 and P = 0.467 respectively). CONCLUSION: Lazaroid U-74389G may represent an effective pharmacologic intervention in reducing lung ischemia-reperfusion injury following thoracoabdominal aortic occlusion.

Beta 1, Beta 2 and Beta 3 Adrenergic Receptor Gene Polymorphisms in a Southeastern European Population.

Genetic polymorphisms in ß1-, ß2- and ß3-adrenergic receptors (ß-ARs) have been associated with chronic non-communicable disorders, such as cardiovascular diseases, asthma, chronic obstructive pulmonary disease (COPD) and obesity, as well as ß-agonists and antagonists response and toxicity. The purpose of this study was to determine the frequency distribution of ADRB1 genetic variants Ser49Gly and Arg389Gly, ADRB2 variants Gly16Arg and Gln27Glu, ADRB3 variant Trp64Arg in a Southeastern European Caucasian (SEC) population sample and to establish a comparison with existing data from other human populations. A sample of 431 men and 590 women volunteered to participate in this genotyping analysis after anonymization and de-identification. Real Time PCR (Melting Curve Analysis) followed DNA extraction from buccal swabs and statistical analysis of the results was performed. The allele frequencies in the SEC population were Ser49 (90.3%), Arg389 (69.49%), Gly16 (61.61%), Gln27 (65.72%), and Trp64 (94.52%), while a Hardy-Weinberg Equilibrium (HWE) was detected in the population studied. Comparisons for the Ser49Gly, Gln27Glu, and Trp64Arg allele distributions demonstrated significant differences between SEC and the European group. European subgroups comparisons showed that allele distributions were similar for four of the five SNPs between SEC and Southwestern European Caucasians (SWC), while they were quite distinct from the Northwestern European Caucasians (NWC). These data underline the importance of interethnic variability of ß-ARs genetic polymorphisms.

Evaluation of a particle enhanced turbidimetric assay for the measurement of neutrophil gelatinase-associated lipocalin in plasma and urine on Architect-8000: Analytical performance and establishment of reference values.

OBJECTIVES: Neutrophil gelatinase-associated lipocalin (NGAL) is a promising biomarker for acute kidney injury. NGAL can be measured in both blood and urine. Apart from kidney injury, NGAL levels in both plasma and urine can be influenced by various pathological situations. Accurate evaluation and comparison of results deriving from clinical studies require robust assays, appropriate specimen handling and reference intervals that will reflect its levels in a healthy population for both biological matrices. METHODS: We report the analytical validation of a latex particle-enhanced turbidimetric immunoassay (PETIA) aimed to measure NGAL in plasma and urine on an automated biochemistry analyzer (ABBOTT-Architect-8000). Assay performance characteristics were evaluated using standard protocols. Urine and plasma specimen storage requirements were determined and reference ranges for blood and urine were determined using healthy controls. RESULTS: The assay is precise (total CV%<4.8%), and sensitive (limit of quantification: 8.4 ng/mL for plasma and 9.0 ng/mL for urine), showing no hook effect. Calibration is stable for at least 30 days. The assay showed excellent linearity over the studied interval (20-4450 ng/mL). The analyte is stable at 4 °C for at least 5 days, and at 20 °C for 4h. Gender specific reference ranges for plasma (male: 38.7-157.6 ng/mL, female: 24.4-142.5 ng/mL) and unisex for urine (<9.0-49.41 ng/mL) are proposed. CONCLUSION: Our data indicate that NGAL can be measured with adequate precision and sensitivity on automated biochemistry analyzers and its measurement could easily be added to a standard panel to screen kidney diseases.