The Serum Levels of the Heavy Metals Cu, Zn, Cd, and Pb and Progression of COPD-A Preliminary Study.

There is evidence in previous studies that high levels of heavy metals may play a key role in the development of COPD due to the induction of chronic inflammation and oxidative stress. In this preliminary study, we used atomic absorption spectrophotometry to measure the levels of four heavy metals (Cu, Zn, Cd, and Pb) in blood serum of COPD patients and controls over 2 years. Clinical data on disease progression or absence were collected in patients living in the industrial region of Stara Zagora, Bulgaria. The mean values of Cu in the serum of patients with COPD and the control group were 374.29 ± 15.03 µg/L and 238.55 ± 175.31 µg/L, Zn­2010.435 ± 670.006 µg/L and 1672.78 ± 934.27 µg/L, Cd­0.334 ± 0.0216 µg/L and 0.395 ± 0.110 µg/L and Pb­0.0732 ± 0.009 µg/L and 0.075 ± 0.0153 µg/L. This is probably because these elements are biogenic and are used in the body for its anti-oxidant protection. In fact, it cannot be stated with certainty that elevated levels of Cu and Zn in the environment have a negative impact in COPD patients. There was a trend towards higher levels of the toxicants lead and cadmium in COPD patients compared to the control group of patients. There is a statistically unproven trend toward higher levels of lead and cadmium in COPD patients compared to controls, which to some extent supports our hypothesis that there is a relationship between environmental lead and cadmium levels and the COPD manifested. In COPD patients, a positive correlation was found between BMI and serum Cu levels (r = 0.413, p = 0.005). A higher concentration of serum Cu was found in men with BMI ≥ 30, compared to those with BMI < 30. There is also a positive correlation to a lesser extent between CRP and cadmium (r = 0.380; p = 0.019) and lead (r = 0.452; p = 0.004). The correlation of lead and cadmium with PSA also shows that these elements may also be associated with the presence of inflammatory processes. A significant negative correlation exists between Pb in the serum of patients with COPD and their blood hemoglobin (r = −356; p = 0.028). The results of our study suggest that higher doses of the trace elements Cu and Zn do not always have a negative effect in patients with COPD, while the toxicants Pb and Cd may be involved in COPD exacerbation and can be used as prognostic biomarkers for progression. Further studies are warranted to confirm these preliminary results.

Role of Single Nucleotide Polymorphism L55M in the Paraoxonase 1 Gene as a Risk and Prognostic Factor in Acute Coronary Syndrome.

The aim of the current study is to explore the possible role of L55M, (rs 854560, 163T > A) SNP as a predisposing factor for acute coronary syndrome (ACS) and to assess its potency as a prognostic biomarker for short (1 year) survival and for median (5 years) and 9-year long patients' outcome. Methods: The current work is a prospective case-control study with 77 patients with acute coronary syndrome (53 with ST-elevation myocardial infarction, STEMI, 14 with non-ST-elevation myocardial infarction, NSTEMI and 10 with unstable angina, UA) and 122 control individuals. Patients were followed-up for 9 years. The genotyping for PON1 L55M SNP was carried on by PCR-RFLP method. Results: The results of the genotyping for PON1 L55M SNP showed a statistically significant difference (p = 0.023) between the controls and the whole group of patients with acute coronary syndrome, as the individuals with genotype with at least one variant M allele had about 2.5-fold higher risk for developing ACS than those which are homozygous of the wild-type L allele (LL genotype). In patients with variant M allele genotypes (LM + MM) which suffer from non-ST-segment elevation ACS (NSTEACS, i.e., UA or NSTEMI), the serum levels of total cholesterol (TC) and triacylglycerols (TAG) are significantly higher than in NSTEACS patients with LL genotype (p = 0.022 for TC and p = 0.015 for TAG). There was no significant difference in the survival rate at the 1st, 5th and 9th year of follow-up between ACS patients with different genotypes, although it is worth to note that in the subgroup of NSTEACS, all patients (n = 13) with variant M allele genotypes (LM + MM) were alive at the end of the first year, while 2 of the patients with LL genotype (18.2%) were dead. Conclusions: The results of our current study suggest that the variant M allele and the M allele genotypes (LM + MM) of the PON1 L55M polymorphism are risk factors for acute coronary syndrome, especially for patients with STEMI, but do not support the possible effect of this polymorphism on the clinical progression and outcome of the patients with ACS either in short or long follow-up periods.

The Leucocyte Telomere Length, GSTM1 and GSTT1 Null Genotypes and the Risk of Chronic Obstructive Pulmonary Disease.

Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation and oxidative stress both in the airways and blood and other organs. Elevated oxidative stress and inflammation have been reported to affect leucocyte telomere length (LTL). Glutathione S-transferase (GST) enzymes are a large family of xenobiotic-metabolizing enzymes that utilize different ROS products. We aimed to explore the link between GSTM1 and GSTT1 gene polymorphisms, LTL and COPD risk. For GSTM1, we genotyped 152 COPD patients and 131 non-affected controls; for GSTT1, we genotyped 149 COPD patients and 130 controls. We were able to assess TL for 91 patients and 88 controls. There was a significant difference in the GSTM1 null genotype frequency between the patients and controls (0.59 vs. 0.38, p ≤ 0.000), but such was not found for GSTT1 (p = 0.192). When combining both polymorphisms, we obtained a significantly greater presence of at least one null genotype among patients (0.12 vs. 0.05, p = 0.027). An association between GSTT1 and LTL was not found. COPD patients carrying the GSTM1 null genotype had shorter telomeres compared to those carrying the non-null genotype (15,720 bp vs. 22,442 bp, p = 0.008); as for the controls, it was the opposite (31,354 bp vs. 17,800 bp, p = 0.020). The significance in both groups remained when combining GSTM1 and GSTT1 (COPD (at least one null) 16,409 bp vs. COPD (non-null) 22,092 bp, p = 0.029; control (at least one null) 29,666 bp vs. control (non-null) 16,370 bp, p = 0.027). The total glutathione level in GSTM1 non-null controls was higher compared to the null genotype (15.39 ng/mL vs. 5.53 ng/mL, p = 0.002). In COPD patients, we found no association (p = 0.301). In conclusion, according to our results, GSTM1, but not GSTT1, null genotypes might play a role in leucocyte telomere shortening, and thus be involved in the pathogenesis of COPD.

The leukocyte telomere length, single nucleotide polymorphisms near TERC gene and risk of COPD.

Chronic obstructive pulmonary disease (COPD) is characterized by irreversible airflow obstruction and is associated with chronic local and systemic inflammation and oxidative stress. The enhanced oxidative stress and inflammation have been reported to affect telomere length (TL). Furthermore, a number of SNPs at loci encoding the main components of the telomerase genes, TERT and TERC have been shown to correlate with TL. We aimed to explore the leukocyte TL and genotypes for single nucleotide polymorphisms, rs12696304 (C > G) and rs10936599 (C > T) near TERC in COPD cases and matched healthy controls using q-PCR technologies. Successful assessment of TL was performed for 91 patients and 88 controls. The patients had shorter TL (17919.36 ± 1203.01 bp) compared to controls (21 271.48 ± 1891.36 bp) although not significant (p = 0.137). The TL did not associate with the gender, age, spirometric indexes, smoking habits but tended to correlate negatively with BMI (Rho = - 0.215, p = 0.076) in the controls, but not in COPD patients. The genotype frequencies of the SNPs rs12696304 and rs10936599 were compared between patients and controls and the odds ratios (OR) for developing COPD were calculated. The carriers of the common homozygous (CC) genotypes of the SNPs had higher risk for COPD, compared to carriers of the variants alleles (rs12696304 CG+GG vs. CC; OR: 0.615, 95% CI [0.424-0.894], p = 0.011 and for rs10936599 CT+TT vs. CC OR = 0.668, 95% CI [0.457-0.976], p = 0.044). Analysis on the combined effects of the TERC rs12696304 (C > G) and rs10936599 (C > T) genotypes, CC/CC genotype combination was associated with higher risk for COPD (p < 0.0001) and marginally lower FEV1% pr. in patients with GOLD II (p = 0.052). There was no association between the SNP genotypes and TL. In summary, our results suggest that COPD patients may have shorter TL, and rs12696304 and rs10936599 near TERC may affect the risk of COPD independently of TL.

Serum and local IL-4, IL-5, IL-13 and immunoglobulin E in allergic rhinitis.

INTRODUCTION: Allergic rhinosinusitis (AR) is a clinical manifestation of a type 1 hypersensitive reaction. A complex of reactions involving components of the immune system - cells, mediators, cytokines, neuropeptides, adhesion molecules etc., are involved in the manifestation of the disease symptoms. AIM: To evaluate the role of some serum and local cytokines and IgE molecules in the pathogenesis of AR comparing results in patients and healthy controls. MATERIAL AND METHODS: The study was conducted at the Prof. Dr. St. Kirkovich University Hospital and Medical University, Stara Zagora, Bulgaria. RESULTS: A trend towards higher serum levels in patients with AR compared to controls was found for IL-4, but with no significant difference. In the group of AR patients, those with the intermittent form had higher, although with no significance, interleukin 4 (IL-4) levels in the lavage compared to those with the persistent form. In nasal lavage fluids a tendency towards higher IL-5 levels was found in intermittent AR patients compared to those with persistent AR. A slight trend towards significantly higher serum levels of IL-13 in overweight patients compared to those with normal weight was found. CONCLUSIONS: Regardless of the obvious differences of the concentrations of the cytokines studied in our groups, oftentimes no significant difference is observed. More studies should be conducted in order to show the role of IL-4, -5, -13, and IgE in the pathogenesis and severity of the disease.

Effects of the IL6 -174G>C promoter polymorphism and IL-6 serum levels on the progression of cutaneous malignant melanoma.

Cutaneous malignant melanoma (CMM) is one of the most immunogenic types of cancer, with a 6-fold higher rate of spontaneous regression than any other malignancy. In addition to responsiveness to different immunotherapies, the immunogenicity of CMM highlights the important role of the host immune system in the response to CMM. The present study aimed to explore the role of two functional promoter polymorphisms [IL6 -174G>C (rs1800785) and TNFA -308G>A (rs1800629)] in the regulation of the genes encoding the pro-inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor-α, specifically in patients with CMM. A total of 76 patients with CMM and 200 control subjects were genotyped using PCR-restriction fragment length polymorphism. The genotype frequencies for both single nucleotide polymorphisms (SNPs) did not differ significantly between the patients and controls (P=0.358 and P=0.810 for IL6 and TNFA, respectively). However, compared with carriers of C-allele genotypes (CG+CC), patients with the IL6 -174GG genotype exhibited more advanced melanoma (Clark scale ≥3; P=0.037) and shorter survival times, particularly those who worked outdoors (in conditions with increased sunlight exposure; P=0.016). Furthermore, the serum IL-6 levels of patients with CMM were significantly higher than those of the control subjects, which were associated with unfavorable blood and serum characteristics and tumor progression (development of new distant metastases; P=0.004), and with a shorter overall survival time (P=0.042). Using a Cox proportional hazard model, the IL6 -174GG genotype was found to be an independent prognostic factor for reduced survival time (P=0.030), together with sex (being male; P=0.004) and occupations with higher exposure to sunlight (P=0.047). In conclusion, the results of the present study indicated that the promoter polymorphisms IL6 -174G>C and TNFA -308G>A are not predisposing factors for CMM. However, the IL6 -174G>C SNP and IL-6 serum concentrations are likely to influence the progression of the disease, and the GG genotype and higher IL-6 serum levels may indicate shorter survival.

MMP12 -82 A>G Promoter Polymorphism in Bronchial Asthma in a Population of Central Bulgaria.

A characteristic feature of inflamed lungs in bronchial asthma (BA) is airway remodeling. Due to limited information on this topic in the literature, we aimed to explore the possible role of polymorphisms in the promoter region of the macrophage elastase gene MMP12 82A>G (rs2276109) as a predisposing factor for BA in an ethnic Bulgarian population. Using restriction fragment length polymorphism analysis of polymerase chain reaction-amplified fragments (PCR-RFLP), we performed genotype analysis of 58 patients and 119 control individuals. We found statistically significant differences in the distribution of genotypes (P = .008) and alleles (P = .004) between patients and nonaffected controls. In the dominant model, carriers of the G allele genotypes had 3.6-fold lower risk for BA, compared with those with the AA genotype, after adjustment for age and sex (odds ratio [OR], -0.277; 95% confidence interval [CI], .12-.65; P = .003). The results of our study suggest that the variant G allele of the MMP12 -82 A>G promoter polymorphism might be considered protective for development of BA in ethnic Bulgarian adults residing in central Bulgaria.

The G allele of MMP12 -82 A > G promoter polymorphism as a protective factor for COPD in Bulgarian population.

Chronic inflammation and remodelling of the small airways are features related to chronic obstructive pulmonary disease (COPD). In the current study, we aimed to explore the possible role of MMP12 -82 A > G (rs2276109) promoter polymorphism in the development of COPD in a population from Bulgaria (167 patients with COPD and 119 control individuals). The genotype and allele distributions differed significantly between COPD patients and controls (p = .010 and p = .043, respectively, χ2 test). The genotypes containing at least one variant G allele (AA + GG) were more frequent in the control group than in patients (36.1% vs. 22.2%) determining 2.96-fold lower risk for COPD after adjustment for age, sex and smoking habits (OR = 0.338, 95%CI: 0.168-0.682, p = .002). Our results suggest that carriers of genotypes with at least one copy of minor G allele of rs2276109 might have lower risk for COPD development, with no marked effect on the lung function and severity of the disease.

Effects of common functional MMP12 gene polymorphisms on PD in a Polish population.

The present study investigated associations of two functional MMP12 polymorphisms with PD risk and cognitive impairment in PD. A total of 478 study subjects (241 PD and 237 age and sex matched controls) were included in the study. UPDRS score, Hoehn-Yahr staging and Schwab-England scale were used to assess motor abilities and activity during daily life. All patients were classified into groups with dementia (PDD, n=72) and without dementia (nPDD, n=159) based on the neuropsychological assessment. The two most common functional single nucleotide polymorphisms (SNPs) in MMP12 gene were determined using TaqMan real-time PCR assays. Frequencies of evaluated MMP12 rs2276109 alleles and genotypes were similar in PD and the controls, whereas rs652438G allele genotypes were significantly more frequent among healthy individuals (p=0.013, OR 0.47 (0.26-0.85). The rs2276109 and rs652438 allele and genotype frequencies were not associated with dementia in PD patients. The current results suggest that MMP12 rs652438 but not MMP12 rs2276109 may affect the risk for PD, as the minor G allele genotypes might be a protective factor.

Frequency of the common promoter polymorphism MMP2 -1306 C>T in a population from central Bulgaria.

Matrix metalloproteinases (MMPs) are a family of highly homologous extracellular Zn2+-dependent endopeptidases, also known as matrixins. MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are considered to play a key role in a variety of physiological processes as well as in the development and progression of a vast majority of pathological conditions. Most of the genes encoding MMPs, including MMP-2, are highly polymorphic. One of the single nucleotide polymorphisms with functional activity in the promoter region of MMP2 is the transition MMP2 -1306C>T (rs243865). The aim of the present study was to evaluate the genotype and allele frequencies of the common promoter polymorphism -1306C>T in MMP2 in 75 individuals from central Bulgaria and to compare our results with those of other population studies. We found that 76.0% of the randomly enrolled individuals are carriers of the CC genotype, 17.3% of CT, and 6.7% of the TT genotype. The minor allele frequency (MAF) was 15.3%. Interestingly, the obtained genotype frequencies appeared to differ from those of some other Caucasian populations (USA - 55/38/7, MAF 26%; The Netherlands - 52.8/40.5/6.7, MAF 26.9%; Austria - 55.6/35.5/8.9, MAF 27.2%), but were closer to the values of the reported global genotype distribution (75.3/21.3/3.4, MAF 14%).

Investigation of the role of MMP3 -1171insA polymorphism in cutaneous malignant melanoma - a preliminary study.

Coetaneous malignant melanoma is the most aggressive cancer of the skin with a high rate of mortality worldwide. Degradation of basement membranes and extracellular matrix is an essential step in cancer invasion and metastasis. Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play key roles in this step. MMP-3 also called stromelysin-1 was one of the first proteinases found to be associated with cancer. In the gene of MMP-3 (MMP3), an insertion/deletion of an A nucleotide at position -1171 in promoter region has been identified and shown to effect the expression activity of the gene. The present study was conducted to investigate the relation of MMP3 -1171insA polymorphism with skin malignant melanoma risk in a pilot case-control study of Bulgarian patients (n = 26) and unaffected controls (n = 172). The genotypes of controls and melanoma patients were in Hardy-Weinberg equilibrium. The results showed no statistically significant difference both in genotype and allele frequencies of MMP3 -1171insA polymorphism between melanoma patients and healthy controls either in crude analyses (p = 0.360 and 0.790, c2-test) or after adjustment for age and sex. The comparison of some clinical characteristics between the patients with different genotypes showed a trend for longer survival of patients with 6A/6A genotype compared to the carriers of 5A allele (5A/5A+5A/6A genotypes, p = 0.118, Log rank test). The results of our current preliminary study do not provide evidence for the role of the promoter polymorphism -1171insA in MMP3 as a risk factor for development of coetaneous melanoma, but suggest its implication in progression of the diseases.

Obesity in Bulgarian patients with chronic obstructive pulmonary disease.

It has been well defined that obesity is strongly linked with several respiratory symptoms and diseases, but no convincing evidence has been provided for chronic obstructive pulmonary disease (COPD). In the current study, we aim to assess the possible prevalence of obesity in patients with COPD in a cross-sectional case-control study of individuals from the region of Stara Zagora, Bulgaria, and to explore whether the body mass has some effect on the lung function of COPD patients. The study included 158 patients with COPD (Global Initiative for Chronic Obstructive Lung Disease (GOLD) II, III, and IV stages) and 123 individuals unaffected by the disease (control). A higher frequency of obesity compared to the controls (20.3%) was observed in patients with COPD (29.1%, p=0.093), especially in those with GOLD II stage (37.7%, p=0.009). Prevalence of obesity was highest in COPD GOLD II, followed by GOLD III and IV stages (p=0.068). When diabetes was considered as confounding factor, we found a significant prevalence of obesity in COPD patients than the controls with diabetes (p=0.031). Interestingly, there was a statistically significant moderate positive correlation between the body mass index and forced expiratory volume in one second as a percentage of predicted value in the whole patients' group (R=0.295, p=0.0002) as well as in the subgroups of GOLD II (R=0.257, p=0.024) and GOLD III COPD (R=0.259, p=0.031).The results of our study propose that the increased body mass, particularly obesity is frequent comorbidity to COPD, especially to less severe diseases. Moreover, the results suggest that the higher body weight may provide some protection against the impairment of lung functions in patients with stable COPD.