Toll-like receptor inflammatory cascade and the development of diabetic kidney disease in children and adolescents with type 1 diabetes.

AIM: This study aimed to evaluate the association of toll-like receptor (TLR) inflammatory cascade with the development of diabetic kidney disease (DKD) in children and adolescents with type 1 diabetes (T1D). METHODS: A total of 49 T1D patients and 49 normoglycaemic (NG) subjects aged 5-20 years old were recruited. TLR2, TLR4, MYD88, NFKB, MCP1/CCL2 and IL18 mRNA expressions were measured in peripheral blood mononuclear cells by reverse transcription-quantitative polymerase chain reaction. Fasting glucose, glycated haemoglobin, serum urea, serum creatinine and urinary albumin-to-creatinine ratio (ACR) were determined. RESULTS: The mRNA expressions of TLR2, TLR4, MYD88 and NFKB were significantly increased in the T1D group compared with the NG group. The mRNA expression levels of MCP1/CCL2 and IL18 were higher in 21 T1D patients (42.9%) (average of MCP1/CCL2: 6.6-fold and IL18: 5.8-fold) than in NG patients. Furthermore, ACR was increased in the T1D group compared with the NG group. CONCLUSION: The increased mRNA expression of TLR2, TLR4, MYD88, NFKB, MCP1/CCL2 and IL18 favours the development of an inflammatory process that may lead to a decline in renal function and consequently DKD in children and adolescents with T1D. This suggests that these genes are early mediators of onset DKD since the beginning of the lives of the paediatric T1D patients.

MiR-3168, miR-6125, and miR-4718 as potential predictors of cisplatin-induced nephrotoxicity in patients with head and neck cancer.

BACKGROUND: No biomarker is available for identifying cancer patients at risk of developing nephrotoxicity when treated with cisplatin. METHODS: We performed microRNA (miRNA) sequencing using plasma collected 5 days after cisplatin treatment (D5) from twelve patients with head and neck cancer with and without nephrotoxicity (grade ≥ 2 increased serum creatinine). The most differentially expressed miRNAs between the two groups were selected for quantification at baseline and D5 in a larger cohort of patients. The association between miRNAs and nephrotoxicity was evaluated by calculating the odds ratio (OR) from univariate logistic regression. Receiver operating characteristic curves (ROC) were used to estimate the area under the curve (AUC), sensitivity, and specificity. RESULTS: MiR-3168 (p = 1.98 × 10- 8), miR-4718 (p = 4.24 × 10- 5), and miR-6125 (p = 6.60 × 10- 5) were the most differentially expressed miRNAs and were further quantified in 43, 48, and 53 patients, respectively. The baseline expression of miR-3168 (p = 0.0456, OR = 1.03, 95% CI: 1.00-1.06) and miR-4718 (p = 0.0388, OR = 1.56, 95% CI: 1.03-2.46) were associated with an increased risk of nephrotoxicity, whereas miR-6125 showed a trend (p = 0.0618, OR = 1.73, 95% CI: 0.98-3.29). MiR-4718 showed the highest AUC (0.77, 95% CI: 0.61-0.93) with sensitivity of 66.76 and specificity of 79.49. CONCLUSIONS: We have provided evidence of baseline plasmatic expression of miR-3168, miR-6125, and miR-4718 as potential predictors of cisplatin-induced nephrotoxicity.

Inhibiting Adenosine Receptor Signaling Promotes Accumulation of Effector CD4+ T Cells in the Lung Parenchyma During Severe Tuberculosis.

BACKGROUND: Tuberculous pneumonia, necrotic granulomatous lesions, and bacterial dissemination characterize severe forms of mycobacterial infection. METHODS: To evaluate the pulmonary CD4+ T-cell response during severe tuberculosis, C57BL/6 mice were infected with approximately 100 bacilli of 3 hypervirulent mycobacterial isolates (Mycobacterium tuberculosis strain Beijing 1471 and Mycobacterium bovis strains B2 and MP287/03) or the H37Rv M tuberculosis strain as reference for mycobacterial virulence. Because high expression of both CD39 and CD73 ectonucleotidases was detected on parenchymal CD4+ T cells, we investigated whether CD4+ T-cell suppression in the context of severe disease was due to the extracellular adenosine accumulation that resulted from tissue damage. RESULTS: Lowest expression of CD69, which is an activation marker implicated in maintaining cells in tissues, was observed in lungs from mice displaying the most severe pulmonary pathology. Reduced interferon (IFN)γ-producing CD4+ T cells were also found in the lung of these mice. Intranasal administration of the adenosine receptor antagonist caffeine substantially enhanced the frequency and number of parenchymal CD4+ T cells as well as both CD69 expression and IFNγ production. CONCLUSIONS: These results indicate that adenosine, which may be generated by extracellular adenosine triphosphate degradation, impairs the parenchymal CD4+ T-cell response and contributes to the development of severe tuberculosis.

Down regulation of protective genes is associated with cellular and antibody-mediated rejection.

Despite advances in immunosuppressive therapy, rejection still remains the main obstacle to a successful transplant. This study aims to explore the gene expression profile of the rejection process in order to decrease the number of unnecessary endomyocardial biopsies in stable patients. METHODS: A total of 300 formalin-fixed and paraffin-embedded (FFPE) endomyocardial biopsies sampled from 63 heart allograft recipients were included in this study. Acute cellular rejection (ACR) and antibody-mediated rejection (AMR) were diagnosed by histological analysis and immunohistochemical C4d staining, respectively. Analysis of gene expression was performed by quantitative real-time polymerase chain reaction. The samples were grouped according to the ISHLT rejection classification, aiming the statistical analysis. RESULTS: There was a significant decrease in the HMOX1, AIF1, and CCL2 transcript over the post-transplantation period in non-rejection group (P<.001). Furthermore, the ADIPOR1, ADIPOR2, BCL2L1, and VEGFA protective genes were significantly downregulated in the ACR group (P<.05). ADIPOR2, BCL2L1, IL6, and NOS2 genes were also significantly downregulated in the AMR group than in the non-rejection group (P<.05). CONCLUSION: The downregulations of the protective genes contribute to the allograft rejection, and the archived FFPE samples are useful for the gene expression analysis aiming the allograft rejection surveillance.

Influence of the CYP3A4/5 genetic score and ABCB1 polymorphisms on tacrolimus exposure and renal function in Brazilian kidney transplant patients.

BACKGROUND: Polymorphisms in genes encoding transport proteins and metabolizing enzymes involved in tacrolimus (TAC) disposition may be important sources of individual variability during treatment. OBJECTIVE: The aim of this study was to investigate the effect of combined CYP3A4 and CYP3A5 variants, using a CYP3A4/5 genetic score, and ABCB1 polymorphisms on therapeutic TAC monitoring and their relationship with clinical outcomes. MATERIAL AND METHODS: Brazilian kidney transplant recipients (n=151), who received TAC over 3 months after transplantation, were genotyped for CYP3A4 rs2242480 (g.20230G>A), CYP3A5 rs15524 (g.31611C>T) and rs776746 (g.6986A>G), ABCB1 rs1128503 (c.1236C>T), rs1045642 (c.3435C>T), and rs2032582 (c.2677G>T/A) polymorphisms. RESULTS: Frequencies of CYP3A4 g.20230A, CYP3A5 g.31611C, and g.6986A were 0.37, 0.26, and 0.28, respectively. These alleles were associated with TAC rapid metabolization and were used for CYP3A4/5 genetic score construction. A higher CYP3A4/5 genetic score was associated with higher TAC dose and lower concentrations for dose administered (Co/D, P<0.05). Ninety days after transplantation, the presence of two or more rapid metabolization alleles contributed toward 27.7% of Co/D variability and was associated with a lower estimated glomerular filtration rate values (P<0.05). For ABCB1, the frequencies of c.1236T, c.3435T, and c.2677T/A alleles were 0.42, 0.42, and 0.33/0.04. At 30 days after transplantation, patients carrying ABCB1 c.1236TT+c.3435TT+(c.2677TT+TA) genotypes had higher TAC Co/D than those with common or heterozygous genotypes (P<0.05). CONCLUSION: The results show the impact of the CYP3A4/5 genetic score on TAC exposure and renal function in Brazilian patients. Furthermore, ABCB1 polymorphisms, in a combined analysis, influenced TAC Co/D at 30 days after transplantation.

Pulmonary infection with hypervirulent Mycobacteria reveals a crucial role for the P2X7 receptor in aggressive forms of tuberculosis.

The purinergic P2X7 receptor (P2X7R) is a sensor of extracellular ATP, a damage-associated molecule that is released from necrotic cells and that induces pro-inflammatory cytokine production and cell death. To investigate whether the innate immune response to damage signals could contribute to the development of pulmonary necrotic lesions in severe forms of tuberculosis, disease progression was examined in C57BL/6 and P2X7R-/- mice that were intratracheally infected with highly virulent mycobacterial strains (Mycobacterium tuberculosis strain 1471 of the Beijing genotype family and Mycobacterium bovis strain MP287/03). The low-dose infection of C57BL/6 mice with bacteria of these strains caused the rapid development of extensive granulomatous pneumonia with necrotic areas, intense bacillus dissemination and anticipated animal death. In contrast, in P2X7R-/- mice, the lung pathology presented with moderate infiltrates of mononuclear leukocytes without visible signs of necrosis; the disease attenuation was accompanied by a delay in mortality. In vitro, the hypervirulent mycobacteria grew rapidly inside macrophages and induced death by a P2X7R-dependent mechanism that facilitated the release of bacilli. Furthermore, these bacteria were resistant to the protective mechanisms elicited in macrophages following extracellular ATP stimulation. Based on this study, we propose that the rapid intracellular growth of hypervirulent mycobacteria results in massive macrophage damage. The ATP released by damaged cells engages P2X7R and accelerates the necrotic death of infected macrophages and the release of bacilli. This vicious cycle exacerbates pneumonia and lung necrosis by promoting widespread cell destruction and bacillus dissemination. These findings suggest the use of drugs that have been designed to inhibit the P2X7R as a new therapeutic approach to treat the aggressive forms of tuberculosis.

Altered microRNome Profiling in Statin-Induced HepG2 Cells: A Pilot Study Identifying Potential new Biomarkers Involved in Lipid-Lowering Treatment.

PURPOSE: Statins are widely prescribed drugs to manage hypercholesterolemia. Despite they are considered effective lipid-lowering agents, significant inter-individual variability has been reported in relation to drug response. Among the reasons explaining this variation, genetic factors are known to partially contribute. Nonetheless, poor evidence exists regarding epigenetic factors involved. METHODS: We investigated if atorvastatin can modulate the cholesterol related miR-33 family. Furthermore, we analyzed the microRNA expression profiles in HepG2 cells treated for 24 hours with atorvastatin or simvastatin using a microarray platform. RESULTS: Our results indicate that atorvastatin does not influence the expression of the miR-33 family. In addition, microarray examination revealed that atorvastatin modulated thirteen miRs, whilst simvastatin only affected two miRs. All significantly modulated miRs after simvastastin therapy were also modulated by atorvastatin. In addition, four novel miRs with previously unreported functions were identified as statin-modulated. CONCLUSION: We identified several novel miRs affected by statin treatment. Additional research is needed to determine the biological significance of differentially expressed miRs identified in statins-induced HepG2 cells.

Antimicrobial photodynamic effect to treat residual pockets in periodontal patients: a randomized controlled clinical trial.

AIM: A randomized controlled clinical trial was designed to evaluate the efficacy of the photodynamic therapy (PDT) in the treatment of residual pockets of chronic periodontitis patients. MATERIAL AND METHODS: Thirty-four patients with at least four residual periodontal pockets undergoing maintenance care were included and randomly assigned to test group (PDT, n = 18) or control group (sham procedure, n = 16). The intervention was performed at baseline, 3, 6 and 12 months. Clinical parameters such as pocket probing depth (PPD), clinical attachment level (CAL), bleeding on probing (BoP) and plaque index (PI) were measured before intervention and after 3, 6 and 12 months. Subgingival samples were obtained at baseline, and after 7 days, 3, 6 and 12 months to quantify Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia by real-time polimerase chain reaction (PCR). RESULTS: All clinical variables showed significant improvement during the study, but there was no significant difference between test and control groups. The microbiological analyses showed no differences between groups at any time during the study. CONCLUSION: Within the limits of this clinical trial and considering the laser and photosensitizer protocol used, PDT failed to demonstrate additional clinical and bacteriological benefits in residual pockets treatment.

Association between Pro12Ala, Pvull, Avall, Sstl and ADIPOQ single-nucleotide polymorphisms with lipid and glycemic profiles of patients with Berardinelli-Seip syndrome.

BACKGROUND/AIMS: Berardinelli-Seip syndrome (BSS) is a recessive autosomal genetic disorder characterized by the near loss of adipose tissue with disturbance in lipid metabolism. METHODS: Biochemical and hormonal parameters and Pro12Ala, Pvull, Avall, Sstl and ADIPOQ polymorphisms in 22 patients with BSS were analyzed and examined for a possible association with lipid profiles. RESULTS: Parental consanguinity, insulin resistance and diabetes mellitus were observed in 63.6, 81.8 and 59.1% of patients, respectively. All individuals presented high triglyceride levels, and 68.1% of patients showed high cholesterol levels. The Pro/Pro genotype of the Pro12Ala polymorphism of the PPARγ2 gene was found in 86.3% of patients; the Ala/Ala variant was not observed in any patient. The PvuII polymorphism of the LPL gene showed a frequency of 50% for the P1P2 variant. The AvaII polymorphism of the LDLR gene showed a similar frequency of 40.9% for both CT and TT variants. The S1S1 genotype of the Sstl polymorphism of the APOC3 gene had a frequency of 86.3%. The CC allele of the ADIPOQ polymorphism of the adiponectin gene was found in 54.6% of patients. CONCLUSIONS: No association was found between lipid parameters and the relevant Pvull, Avall and Sstl polymorphisms. However, we did observe an association of the Pro12Ala and ADIPOQ polymorphisms with higher lipid levels, suggesting a close relationship between these factors.

PVC containing silver nanoparticles with antimicrobial properties effective against SARS-CoV-2.

Poly (vinyl chloride) (PVC) is commonly used to manufacture biomedical devices and hospital components, but it does not present antimicrobial activity enough to prevent biofouling. With the emergence of new microorganisms and viruses, such as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that was responsible for the global pandemic caused by Coronavirus Disease 2019 (COVID-19), it is evident the importance of the development of self-disinfectant PVC for hospital environments and medical clinics where infected people remain for a long time. In this contribution, PVC nanocomposites with silver nanoparticles (AgNPs) were prepared in the molten state. AgNPs are well-known as antimicrobial agents suitable for designing antimicrobial polymer nanocomposites. Adding 0.1 to 0.5 wt% AgNPs significantly reduced Young's modulus and ultimate tensile strength of PVC due to the emergence of microstructural defects in the PVC/AgNP nanocomposites, but the impact strength did not change significantly. Furthermore, nanocomposites have a higher yellowness index (YI) and lower optical bandgap values than PVC. The PVC/AgNP nanocomposites present virucidal activity against SARS-CoV-2 (B.1.1.28 strain) within 48 h when the AgNP content is at least 0.3 wt%, suitable for manufacturing furniture and hospital equipment with self-disinfectant capacity to avoid secondary routes of COVID-19 contagion.

ABCB1 haplotype is associated with major molecular response in chronic myeloid leukemia patients treated with standard-dose of imatinib.

BACKGROUND: Imatinib mesylate (IM) is a selective tyrosine kinase inhibitor used for treating chronic myeloid leukemia (CML). IM has high efficacy, however some individuals develop a resistance due to impaired bioavailability. Polymorphisms in genes encoding membrane transporters such as ABCB1 have been associated with differences in protein expression and function that influence the response to several drugs. AIM: To investigate the relationship of ABCB1 polymorphisms with markers of response to IM in patients with CML. METHODS: One hundred eighteen CML patients initially treated with a standard dose of IM (400mg/day) for 18months were selected at two health centers in Sao Paulo City, Brazil. The response criteria were based on the European LeukemiaNet recommendations. ABCB1 polymorphisms c.1236C>T (rs1128503), c.3435C>T (rs1045642) and c.2677G>T/A (rs2032582) were evaluated by PCR-RFLP. RESULTS: ABCB1 polymorphisms were not related with a risk for CML in this sample population (p<0.05). In the CML group, frequencies of ABCB1 SNPs were similar between responder and non-responder patients (p>0.05). In the responder group, the frequency of ABCB11236CT/2677GT/3435CT haplotype was higher in patients with major molecular response (MMR) (51.7%) than in patients without MMR (8.3%, p=0.010). Furthermore, carriers of this haplotype had increased the probability of reaching the MMR compared with the non-carriers (OR: 11.8; 95% CI: 1.43-97.3, p=0.022). CONCLUSIONS: The ABCB1 1236CT/2677GT/3435CT haplotype is positively associated with the major molecular response to IM in CML patients.

Blood DNA methylation marks discriminate Chagas cardiomyopathy disease clinical forms.

Chagas disease is a parasitic disease from South America, affecting around 7 million people worldwide. Decades after the infection, 30% of people develop chronic forms, including Chronic Chagas Cardiomyopathy (CCC), for which no treatment exists. Two stages characterized this form: the moderate form, characterized by a heart ejection fraction (EF) ≥ 0.4, and the severe form, associated to an EF < 0.4. We propose two sets of DNA methylation biomarkers which can predict in blood CCC occurrence, and CCC stage. This analysis, based on machine learning algorithms, makes predictions with more than 95% accuracy in a test cohort. Beyond their predictive capacity, these CpGs are located near genes involved in the immune response, the nervous system, ion transport or ATP synthesis, pathways known to be deregulated in CCCs. Among these genes, some are also differentially expressed in heart tissues. Interestingly, the CpGs of interest are tagged to genes mainly involved in nervous and ionic processes. Given the close link between methylation and gene expression, these lists of CpGs promise to be not only good biomarkers, but also good indicators of key elements in the development of this pathology.

Hereditary hemochromatosis: mutations in genes involved in iron homeostasis in Brazilian patients.

BACKGROUND: p.C282Y mutation and rare variants in the HFE gene have been associated with hereditary hemochromatosis (HH). HH is also caused by mutations in other genes, such as the hemojuvelin (HJV), hepcidin (HAMP), transferrin receptor 2 (TFR2) and ferroportin (SLC40A1). The low rate homozygous p.C282Y mutation in Brazil is suggestive that mutations in non-HFE genes may be linked to HH phenotype. AIM: To screen exon-by-exon DNA sequences of HFE, HJV, HAMP, TFR2 and SLC40A1 genes to characterize the molecular basis of HH in a sample of the Brazilian population. MATERIALS AND METHODS: Fifty-one patients with primary iron overload (transferrin saturation ≥50% in females and ≥60% in males) were selected. Subsequent bidirectional DNA sequencing of HFE, HJV, HAMP, TFR2 and SLC40A1 exons was performed. RESULTS: Thirty-seven (72.5%) out of the 51 patients presented at least one HFE mutation. The most frequent genotype associated with HH was the homozygous p.C282Y mutation (n=11, 21.6%). In addition, heterozygous HFE p.S65C mutation was found in combination with p.H63D in two patients and homozygous HFE p.H63D was found in two patients as well. Sequencing in the HJV and HAMP genes revealed HJV p.E302K, HJV p.A310G, HJV p.G320V and HAMP p.R59G alterations. Molecular and clinical diagnosis of juvenile hemochromatosis (homozygous form for the HJV p.G320V) was described for the first time in Brazil. Three TFR2 polymorphisms (p.A75V, p.A617A and p.R752H) and six SLC40A1 polymorphisms (rs13008848, rs11568351, rs11568345, rs11568344, rs2304704, rs11568346) and the novel mutation SLC40A1 p.G204S were also found. CONCLUSIONS: The HFE p.C282Y in homozygosity or in heterozygosity with p.H63D was the most frequent mutation associated with HH in this sample. The HJV p.E302K and HAMP p.R59G variants, and the novel SLC40A1 p.G204S mutation may also be linked to primary iron overload but their role in the pathophysiology of HH remain to be elucidated.

Apolipoprotein E mRNA expression in mononuclear cells from normolipidemic and hypercholesterolemic individuals treated with atorvastatin.

BACKGROUND: Apolipoprotein E (apoE) is a key component of the lipid metabolism. Polymorphisms at the apoE gene (APOE) have been associated with cardiovascular disease, lipid levels and lipid-lowering response to statins. We evaluated the effects on APOE expression of hypercholesterolemia, APOE ε2/ε3/ε4 genotypes and atorvastatin treatment in Brazilian individuals. The relationship of APOE genotypes and plasma lipids and atorvastatin response was also tested in this population. METHODS: APOE ε2/ε3/ε4 and plasma lipids were evaluated in 181 normolipidemic (NL) and 181 hypercholesterolemic (HC) subjects. HC individuals with indication for lowering-cholesterol treatment (n = 141) were treated with atorvastatin (10 mg/day/4-weeks). APOE genotypes and APOE mRNA in peripheral blood mononuclear cells (PBMC) were analyzed by TaqMan real time PCR. RESULTS: HC had lower APOE expression than NL group (p < 0.05) and individuals with low APOE expression showed higher plasma total and LDL cholesterol and apoB, as well as higher apoAI (p < 0.05). Individuals carrying ε2 allele have reduced risk for hypercholesterolemia (OR: 0.27, 95% I.C.: 0.08-0.85, p < 0.05) and NL ε2 carriers had lower total and LDL cholesterol and apoB levels, and higher HDL cholesterol than non-carriers (p < 0.05). APOE genotypes did not affect APOE expression and atorvastatin response. Atorvastatin treatment do not modify APOE expression, however those individuals without LDL cholesterol goal achievement after atorvastatin treatment according to the IV Brazilian Guidelines for Dyslipidemia and Atherosclerosis Prevention had lower APOE expression than patients with desirable response after the treatment (p < 0.05). CONCLUSIONS: APOE expression in PBMC is modulated by hypercholesterolemia and the APOE mRNA level regulates the plasma lipid profile. Moreover the expression profile is not modulated neither by atorvastatin nor APOE genotypes. In our population, APOE ε2 allele confers protection against hypercholesterolemia and a less atherogenic lipid profile. Moreover, low APOE expression after treatment of patients with poor response suggests a possible role of APOE level in atorvastatin response.

ABCA1 and ABCG1 expressions are regulated by statins and ezetimibe in Caco-2 cells.

BACKGROUND: Enterocytes play a crucial role in high-density lipoprotein (HDL) biogenesis. Statins and ezetimibe are potent lowering-cholesterol drugs, which can also influence HDL plasma concentrations. We hypothesized that these drugs could modulate the expression of intestinal ABCA1 and ABCG1, two genes involved in HDL metabolism. METHODS: Caco-2 cells were used as a model of the human intestinal cells and were treated with statins (0.01-1 µmol/L) and/or ezetimibe (0.5-5.0 µmol/L) for 12 h or 24 h. Gene expression was examined using real-time PCR. RESULTS: ABCA1 level was more abundant than ABCG1 in Caco-2 cells. ABCA1 was downregulated after 12-h and 24-h treatment with atorvastatin (0.1 and 1.0 µmol/L) or simvastatin (0.01, 0.1 and 1 µmol/L) (p<0.05). In statin-treated cells, ABCG1 levels remained unaltered. Ezetimibe alone did not induce change of ABCA1 or ABCG1 mRNA levels (p>0.05) but 24-h ezetimibe (2.5 or 5.0 µmol/L) plus simvastatin (1 µmol/L) treatment decreased the transcription of ABCA1 and ABCG1 (p<0.05). CONCLUSIONS: Our findings reveal that, at the concentrations studied, statins isolated or combined with ezetimibe, but not ezetimibe alone, downregulate ABCA1 mRNA expression in Caco-2 cells. Moreover, simvastatin combined with ezetimibe treatment also decrease the ABCG1 levels in these cells.

Tumor necrosis factor-α and interleukin-6 expression in leukocytes and their association with polymorphisms and bone markers in diabetic individuals treated with pioglitazone.

BACKGROUND: Pioglitazone is a peroxisome proliferator-activated receptor gamma (PPARγ) activator used in the treatment of type 2 diabetes (DM2) patients and it has been suggested that can induce bone loss. Tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) mRNA expression in blood leukocytes and the relationship with polymorphisms and bone markers in DM2 treated with pioglitazone were investigated. METHODS: DM2 (n=53) and normoglycemic (NG, n=52) individuals were included. DM2 patients were treated with pioglitazone (45 mg/day/16 weeks). mRNA expression was evaluated by real-time polymerase chain reaction (PCR). TNFA -308G>A and IL6 -174G>C polymorphisms were detected by PCR-RFLP and high resolution melting polymerase chain reaction (HRM-PCR). RESULTS: Pioglitazone reduced bone specific alkaline phosphatase (bALP) and increased TNFα in DM2 group (p<0.001). DM2 or pioglitazone did not influence TNFα and IL-6 expression (p>0.05). TNFA -308A allele was associated with reduced basal TNFα mRNA levels in NG and DM2 and reduced alkaline phosphatase (tALP) after treatment (p<0.05). IL6 -174C allele was associated with decreased oral glucose tolerance test (OGTT)-2 h in DM2 individuals (p<0.05). CONCLUSIONS: TNFA -308G >A polymorphism appear to be involved in regulation of gene expression independently of hyperglycemia and its interaction with pioglitazone may modify tALP, a important bone marker. IL6 -174G>C variant is related with reduced risk of postprandial hyperglycemia but not with mRNA expression or bone markers.

Pharmacogenetics of OATP transporters reveals that SLCO1B1 c.388A>G variant is determinant of increased atorvastatin response.

AIMS: The relationship between variants in SLCO1B1 and SLCO2B1 genes and lipid-lowering response to atorvastatin was investigated. MATERIAL AND METHODS: One-hundred-thirty-six unrelated individuals with hypercholesterolemia were selected and treated with atorvastatin (10 mg/day/4 weeks). They were genotyped with a panel of ancestry informative markers for individual African component of ancestry (ACA) estimation by SNaPshot(®) and SLCO1B1 (c.388A>G, c.463C>A and c.521T>C) and SLCO2B1 (-71T>C) gene polymorphisms were identified by TaqMan(®) Real-time PCR. RESULTS: Subjects carrying SLCO1B1 c.388GG genotype exhibited significantly high low-density lipoprotein (LDL) cholesterol reduction relative to c.388AA+c.388AG carriers (41 vs. 37%, p = 0.034). Haplotype analysis revealed that homozygous of SLCO1B1*15 (c.521C and c.388G) variant had similar response to statin relative to heterozygous and non-carriers. A multivariate logistic regression analysis confirmed that c.388GG genotype was associated with higher LDL cholesterol reduction in the study population (OR: 3.2, CI95%:1.3-8.0, p < 0.05). CONCLUSION: SLCO1B1 c.388A>G polymorphism causes significant increase in atorvastatin response and may be an important marker for predicting efficacy of lipid-lowering therapy.

Functional analysis of PCSK9 3'UTR variants and mRNA-miRNA interactions in patients with familial hypercholesterolemia.

Aim: Functional analysis of PCSK9 3'UTR variants and mRNA-miRNA interactions were explored in patients with familial hypercholesterolemia (FH). Materials & methods:PCSK9 3'UTR variants were identified by exon-targeted gene sequencing. Functional effects of 3'UTR variants and mRNA-miRNA interactions were analyzed using in silico and in vitro studies in HEK293FT and HepG2 cells. Results: Twelve PCSK9 3'UTR variants were detected in 88 FH patients. c.*75C >T and c.*345C >T disrupted interactions with miR-6875, miR-4721 and miR-564. Transient transfection of the c.*345C >T decreased luciferase activity in HEK293FT cells. miR-4721 and miR-564 mimics reduced PCSK9 expression in HepG2 cells. Conclusion:PCSK9 c.*345C >T has a possible role as loss-of-function variant. miR-4721 and miR-564 downregulate PCSK9 and may be useful to improve lipid profile in FH patients.

High serum miR-421 is associated with metabolic dysregulation and inflammation in patients with metabolic syndrome.

Aim: To explore the association of circulating miRNAs with adiposity, metabolic status and inflammatory biomarkers in patients with metabolic syndrome (MetS). Methods: Serum levels of 372 miRNAs were measured in patients with (n = 6) and without MetS (n = 6) by quantitative PCR array, and dysregulated miRNAs were validated in a larger cohort (MetS, n = 89; non-MetS, n = 144). Results: In the screening study, seven miRNAs were dysregulated in patients with MetS, and miR-421 remained increased in the validation study. miR-421 was associated with a high risk of MetS and insulin resistance and hypertension and correlated with glycated hemoglobin, triacylglycerols, high-sensitivity CRP, IL-6, resistin and adiponectin (p < 0.05). Conclusion: Circulating miR-421 is a potential biomarker for insulin resistance, metabolic dysregulation and inflammatory status in patients with MetS.

Profiling plasma-extracellular vesicle proteins and microRNAs in diabetes onset in middle-aged male participants in the ELSA-Brasil study.

We measured plasma-derived extracellular vesicle (EV) proteins and their microRNA (miRNA) cargos in normoglycemic (NG), glucose intolerant (GI), and newly diagnosed diabetes mellitus (DM) in middle-aged male participants of the Brazilian Longitudinal Study of Adult Health (ELSA-Brazil). Mass spectrometry revealed decreased IGHG-1 and increased ITIH2 protein levels in the GI group compared with that in the NG group and higher serotransferrin in EVs in the DM group than in those in the NG and GI groups. The GI group also showed increased serum ferritin levels, as evaluated by biochemical analysis, compared with those in both groups. Seventeen miRNAs were differentially expressed (DEMiRs) in the plasma EVs of the three groups. DM patients showed upregulation of miR-141-3p and downregulation of miR-324-5p and -376c-3p compared with the NG and GI groups. The DM and GI groups showed increased miR-26b-5p expression compared with that in the NG group. The DM group showed decreased miR-374b-5p levels compared with those in the GI group and higher concentrations than those in the NG group. Thus, three EV proteins and five DEMiR cargos have potential prognostic importance for diabetic complications mainly associated with the immune function and iron status of GI and DM patients.