Predicted deleterious variants in ABCA1, LPL, LPA and KIF6 are associated with statin response and adverse events in patients with familial hypercholesterolemia and disturb protein structure and stability.

OBJECTIVES: This study explored the association of deleterious variants in pharmacodynamics (PD) genes with statin response and adverse effects in patients with familial hypercholesterolemia (FH) and analyzed their potential effects on protein structure and stability. METHODS: Clinical and laboratory data were obtained from 144 adult FH patients treated with statins. A panel of 32 PD genes was analyzed by exon-targeted gene sequencing. Deleterious variants were identified using prediction algorithms and their structural effects were analyzed by molecular modeling studies. RESULTS: A total of 102 variants were predicted as deleterious (83 missense, 8 stop-gain, 4 frameshift, 1 indel, 6 splicing). The variants ABCA1 rs769705621 (indel), LPA rs41267807 (p.Tyr2023Cys) and KIF6 rs20455 (p.Trp719Arg) were associated with reduced low-density lipoprotein cholesterol (LDLc) response to statins, and the LPL rs1801177 (p.Asp36Asn) with increased LDLc response (P < 0.05). LPA rs3124784 (p.Arg2016Cys) was predicted to increase statin response (P = 0.022), and ABCA1 rs769705621 to increase the risk of statin-related adverse events (SRAE) (P = 0.027). LPA p.Arg2016Cys and LPL p.Asn36Asp maintained interactions with solvent, LPA p.Tyr2023Cys reduced intramolecular interaction with Gln1987, and KIF6 p.Trp719Arg did not affect intramolecular interactions. DDMut analysis showed that LPA p.Arg2016Cys and p.Tyr2023Cys and LPL p.Asp36Asn caused energetically favorable changes, and KIF6 p.Trp719Arg resulted in unfavorable energetic changes, affecting protein stability. CONCLUSION: Deleterious variants in ABCA1, LPA, LPL and KIF6 are associated with variability in LDLc response to statins, and ABCA1 rs769705621 is associated with SRAE risk in FH patients. Molecular modeling studies suggest that LPA p.Tyr2023Cys and KIF6 p.Trp719Arg disturb protein conformational structure and stability.

Adenovirus 36 seropositivity is related to the expression of anti-adipogenic lncRNAs GAS5 and MEG3 in adipose tissue obtained from subjects with obesity.

BACKGROUND: Human Adenovirus D-36 (HAdV-D36) promotes adipogenesis in cellular and animal models and may contribute to the development of human obesity. Induction of PPARγ by HAdV-D36 seems to have a central role in the maintenance of adipogenic status. There is limited information about epigenetic mechanisms contributing to this process in human adipose tissue. This study evaluated the expression of lncRNAs (ADINR, GAS5 and MEG3) and miRNAs (miR-18a and miR-140) involved in the adipogenic process in visceral adipose tissue (VAT) of subjects with obesity with previous HAdV-D36 infection (seropositive) and unexposed (seronegative) subjects with obesity. METHODS: Individuals with obesity were grouped according to the presence of antibodies against HAdV-D36 (Seropositive: HAdV-D36[+], n = 29; and Seronegative: HAdV-D36[-], n = 28). Additionally, a group of individuals without obesity (n = 17) was selected as a control group. The HAdV-D36 serology was carried out by ELISA. Biopsies of VAT were obtained during an elective and clinically indicated surgery (bariatric or cholecystectomy). RNA extraction from VAT was performed and the expression of PPARG and non-coding RNAs was evaluated by qPCR. RESULTS: HAdV-D36[+] individuals had lower expression of anti-adipogenic lncRNAs GAS5 (p = 0.016) and MEG3 (p = 0.035) compared with HAdV-D36[-] subjects with obesity. HAdV-D36[+] subjects also presented increased expression of the adipogenic miRNA miR-18a (p = 0.042), which has been reported to be modulated by GAS5 through a RNA sponging mechanism during adipogenic differentiation. Additionally, an inverse correlation of GAS5 with PPARG expression was observed (r = -0.917, p = 0.01). CONCLUSION: Our results suggest that HAdV-D36 is related to non-coding RNAs implicated in adipogenesis, representing a potential mechanism by which previous HAdV-D36 infection could be associated with the long-term maintenance of adipogenic status, probably through the GAS5/miR-18a axis.

The Impact of miR-155-5p on Myotube Differentiation: Elucidating Molecular Targets in Skeletal Muscle Disorders.

MicroRNAs are small regulatory molecules that control gene expression. An emerging property of muscle miRNAs is the cooperative regulation of transcriptional and epitranscriptional events controlling muscle phenotype. miR-155 has been related to muscular dystrophy and muscle cell atrophy. However, the function of miR-155 and its molecular targets in muscular dystrophies remain poorly understood. Through in silico and in vitro approaches, we identify distinct transcriptional profiles induced by miR-155-5p in muscle cells. The treated myotubes changed the expression of 359 genes (166 upregulated and 193 downregulated). We reanalyzed muscle transcriptomic data from dystrophin-deficient patients and detected overlap with gene expression patterns in miR-155-treated myotubes. Our analysis indicated that miR-155 regulates a set of transcripts, including Aldh1l, Nek2, Bub1b, Ramp3, Slc16a4, Plce1, Dync1i1, and Nr1h3. Enrichment analysis demonstrates 20 targets involved in metabolism, cell cycle regulation, muscle cell maintenance, and the immune system. Moreover, digital cytometry confirmed a significant increase in M2 macrophages, indicating miR-155's effects on immune response in dystrophic muscles. We highlight a critical miR-155 associated with disease-related pathways in skeletal muscle disorders.

Association of occupational exposure to pesticides with overweight in farmers in Southern Brazil.

INTRODUCTION: Exposure to pesticides may be related to overweight and associated comorbidities. The aim of this work was to evaluate occupational exposure to pesticides, overweight and associated comorbidities among farmers in Southern Brazil. METHODS: This cross-sectional study included a random sample of 257 farmers, living in the municipality of Mafra and Planalto, southern Brazil. Data on pesticide use and overweight prevalence from farmers were collected using an in-person interview questionnaire, followed by blood collection and biochemical analyses. RESULTS: Pesticide exposure was positively correlated with body mass index, waist circumference, waist-to-hip ratio, triglycerides and glucose levels, presence of hypertension and metabolic syndrome. Besides that, the fact of being exposed to pesticides represents a decrease of no protein thiol groups. Furthermore, the main pesticides used by farmers have hepatic toxicity. CONCLUSION: These findings suggest that exposure to pesticides may be associated with overweight and associated comorbidities. Further studies are required to validate our findings and elucidate the specific mechanisms by which these pollutants contribute to the development of overweight.

LDLR and PCSK9 3´UTR variants and their putative effects on microRNA molecular interactions in familial hypercholesterolemia: a computational approach.

BACKGROUND: Familial hypercholesterolemia (FH) is caused by pathogenic variants in low-density lipoprotein (LDL) receptor (LDLR) or its associated genes, including apolipoprotein B (APOB), proprotein convertase subtilisin/kexin type 9 (PCSK9), and LDLR adaptor protein 1 (LDLRAP1). However, approximately 40% of the FH patients clinically diagnosed (based on FH phenotypes) may not carry a causal variant in a FH-related gene. Variants located at 3' untranslated region (UTR) of FH-related genes could elucidate mechanisms involved in FH pathogenesis. This study used a computational approach to assess the effects of 3'UTR variants in FH-related genes on miRNAs molecular interactions and to explore the association of these variants with molecular diagnosis of FH. METHODS AND RESULTS: Exons and regulatory regions of FH-related genes were sequenced in 83 FH patients using an exon-target gene sequencing strategy. In silico prediction tools were used to study the effects of 3´UTR variants on interactions between miRNAs and target mRNAs. Pathogenic variants in FH-related genes (molecular diagnosis) were detected in 44.6% FH patients. Among 59 3'UTR variants identified, LDLR rs5742911 and PCSK9 rs17111557 were associated with molecular diagnosis of FH, whereas LDLR rs7258146 and rs7254521 and LDLRAP1 rs397860393 had an opposite effect (p < 0.05). 3´UTR variants in LDLR (rs5742911, rs7258146, rs7254521) and PCSK9 (rs17111557) disrupt interactions with several miRNAs, and more stable bindings were found with LDLR (miR-4435, miR-509-3 and miR-502) and PCSK9 (miR-4796). CONCLUSION: LDLR and PCSK9 3´UTR variants disturb miRNA:mRNA interactions that could affect gene expression and are potentially associated with molecular diagnosis of FH.

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.

Harmful Effects of Granulocytic Myeloid-Derived Suppressor Cells on Tuberculosis Caused by Hypervirulent Mycobacteria.

BACKGROUND: The role of myeloid-derived suppressor cells (MDSCs) in patients with severe tuberculosis who suffer from uncontrolled pulmonary inflammation caused by hypervirulent mycobacterial infection remains unclear. METHODS: This issue was addressed using C57BL/6 mice infected with highly virulent Mycobacterium bovis strain MP287/03. RESULTS: CD11b+GR1int population increased in the bone marrow, blood and lungs during advanced disease. Pulmonary CD11b+GR1int (Ly6GintLy6Cint) cells showed granularity similar to neutrophils and expressed immature myeloid cell markers. These immature neutrophils harbored intracellular bacilli and were preferentially located in the alveoli. T-cell suppression occurred concomitantly with CD11b+GR1int cell accumulation in the lungs. Furthermore, lung and bone marrow GR1+ cells suppressed both T-cell proliferation and interferon γ production in vitro. Anti-GR1 therapy given when MDSCs infiltrated the lungs prevented expansion and fusion of primary pulmonary lesions and the development of intragranulomatous caseous necrosis, along with increased mouse survival and partial recovery of T-cell function. Lung bacterial load was reduced by anti-GR1 treatment, but mycobacteria released from the depleted cells proliferated extracellularly in the alveoli, forming cords and clumps. CONCLUSIONS: Granulocytic MDSCs massively infiltrate the lungs during infection with hypervirulent mycobacteria, promoting bacterial growth and the development of inflammatory and necrotic lesions, and are promising targets for host-directed therapies.

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.

Minimum Alveolar Concentration of Sevoflurane as a Single Hypnotic Agent to Maintain BIS Below 50 in Patients During Normothermic Cardiopulmonary Bypass.

OBJECTIVES: Minimum alveolar concentration (MAC) of volatile anesthetic agents to maintain bispectral index (BIS) below 50 in 50% of patients was defined as MACBIS50. The primary objective of this study was to determine the minimum alveolar concentration of sevoflurane as a single hypnotic agent to maintain BIS below 50 in patients during normothermic cardiopulmonary bypass. DESIGN: Prospective and observational study. SETTING: Dante Pazzanese Institute of Cardiology, Brazil. PARTICIPANTS: Eighteen consecutive patients scheduled for elective coronary artery bypass grafting (CABG) with cardiopulmonary bypass (CPB) under general anesthesia, American Society of Anesthesiologists physical status classes III and IV, between the ages of 40 and 70, were included in the study. METHODS: All patients underwent inhalation induction with facial mask using sevoflurane (Cristália) in 100% oxygen, pancuronium (Cristália) 0.1 mg/kg, and sufentanil (Cristália) 0.5 µg/kg intravenously (IV) administered. A single bolus dose of sufentanil, 1.0 µg/kg IV, was administered before surgical incision. MACBIS50 was calculated using the midpoint concentration of patients involving a crossover (BIS < or ≥50) according to Dixon's Up-and-Down method. The Up-and-Down sequence also was analyzed by probit test that enabled the authors to obtain the effective dose 50 (ED50) and effective dose 95 (ED95) of sevoflurane to maintain a BIS value <50, with a 95% confidence interval (95% CI) of the mean. RESULTS: A total of 15 patients were analyzed in this study. MACBIS50 of sevoflurane as a single hypnotic agent was 0.82% (95% CI 0.47-1.16) in patients aged 40 to 70 undergoing CABG during normothermic CPB. The ED50 and ED95 of sevoflurane to maintain a BIS value <50 for the same context were 0.73% (95% CI 0.45-1.00) and 1.39 (95% CI 0.42-2.37) by means of probit analysis, respectively. CONCLUSION: MACBIS50 of sevoflurane as a single hypnotic agent was 0.82% in patients undergoing CABG during normothermic CPB.

Peripheral Blood miRome Identified miR-155 as Potential Biomarker of MetS and Cardiometabolic Risk in Obese Patients.

This study explored circulating miRNAs and target genes associated with metabolic syndrome (MetS) and cardiometabolic risk in obese patients. Small-RNA sequencing was used to assess the peripheral blood miRNome of 12 obese subjects (6 MetS and 6 non-MetS). Differentially expressed miRNAs and target genes were further analyzed by qPCR in a larger sample of obese patients (48 MetS and 32 non-MetS). miRNA:mRNA interactions were studied using in silico tools. miRNome analysis identified 10 downregulated miRNAs in MetS compared to non-Met patients (p < 0.05). In silico studies revealed three miRNAs (miR-155, miR-181a, and let-7a) and their predictive targets (CCAAT/enhancer-binding protein beta-CEBPB, KRAS proto-oncogene, GTPase-KRAS and suppressor of cytokine signaling 1-SOCS1) with a potential role in the insulin receptor signaling pathway. miR-155 expression was reduced and CEBPB mRNA levels were increased in MetS patients (p < 0.05), and these effects were correlated with the number of MetS diagnostic criteria (p < 0.05). Increased HOMA-IR (>7.6) was associated with low miR-155 levels, high CEBPB expression, and serum hsCRP (p < 0.05). miR-155 was negatively correlated with CEBPB, HOMA-IR, and plasma fibrinogen, and positively correlated with serum adiponectin (p < 0.05). Downregulation of circulating miR-155 is associated with insulin resistance, poor glycemic control, and increased MetS-related cardiometabolic risk, and these effects are potentially mediated by interaction with CEBPB.

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.

Circulating miRNAs in acute new-onset atrial fibrillation and their target mRNA network.

BACKGROUND: MicroRNAs (miRNAs) are involved in the pathogenesis of atrial fibrillation (AF), acting on development and progression. Our pilot study investigated the expression of six miRNAs and their miRNA-mRNA interactions in patients with acute new-onset AF, well-controlled AF, and normal sinus rhythm (controls). METHODS AND RESULTS: Plasma of acute new-onset AF patients (n = 5) was collected in the emergency room when patients presented with irregular and fast-atrial fibrillation rhythm. Samples from well-controlled AF (n = 16) and control (n =  15) patients were collected during medical appointments following an ECG. Expression of miR-21, miR-133a, miR-133b, miR-150, miR-328, and miR-499 was analyzed by real-time PCR. Ingenuity Pathway Analysis and the TargetScan database identified the top 30 mRNA targets of these miRNA, seeking the miRNA-mRNA interactions in cardiovascular process. Increased expression of miR-133b (1.4-fold), miR-328 (2.0-fold), and miR-499 (2.3-fold) was observed in patients with acute new-onset AF, compared with well-controlled AF and control patients. Decreased expression of miR-21 was seen in patients with well-controlled AF compared to those with acute new-onset AF and controls (0.6-fold). The miRNA-mRNA interaction demonstrated that SMAD7 and FASLG genes were the targets of miR-21, miR-133b, and miR-499 and were directly related to AF, being involved in apoptosis and fibrosis. CONCLUSION: The miRNAs had different expression profiles dependent on the AF condition, with higher expression in the acute new-onset AF than well-controlled AF. Clinically, this may contribute to an effective assessment for patients, leading to early detection of AF and monitoring to reduce the risk of other serious cardiovascular events.

Intragraft vasculitis and gene expression analysis: Association with acute rejection and prediction of mortality in long-term heart transplantation.

INTRODUCTION: Vasculitis entails heterogeneous origins; it starts with an inflammatory process that leads to small vessels' necrosis, hemorrhage, and ischemic lesion, and may further result in occlusion of the vascular lumen. Vasculitis' contribution to allograft rejection is still unclear. This study aims to investigate the incidence of vasculitis in the early stages of heart transplantation as well as to assess the intragraft genes' expression associated with vascular function and subsequently to verify the way in which it affects the outcome of the allograft. METHODS: In this retrospective study, 300 archive paraffin-embedded endomyocardial biopsies from 63 heart allograft recipients were assessed. Cellular rejection and vasculitis were diagnosed through histological analysis, and antibody-mediated rejection was performed with immunohistochemical C4d staining. The transcripts of ICAM, VCAM, VEGF, CCL2, IFNG, TGFB, TNF, ADIPOR1, and ADIPOR2 genes were examined through quantitative polymerase chain reaction using B2M for normalization. RESULTS: We observed a higher prevalence of severe vasculitis in the early period of post-transplant, and recovery was observed to take place around 1 year post-transplant. Additionally, vasculitis was found to be directly associated with acute cellular rejection and antibody-mediated rejection. The intense C4d capillary positivity predicts higher long-term cardiovascular disease mortality. In comparison with the vasculitis-free group, the group with severe vasculitis displayed reduced left ventricular ejection fraction and an upregulation of VCAM and IFNG associated with the downregulation of VEGF, ADIPOR1, and ADIPOR2. CONCLUSION: The vasculitis associated with the presence of C4d and the change in intragraft gene expression profile may contribute to poor allograft outcomes.

Application of aqueous two-phase micellar system to improve extraction of adenoviral particles from cell lysate.

Viral vectors are important in medical approaches, such as disease prevention and gene therapy, and their production depends on efficient prepurification steps. In the present study, an aqueous two-phase micellar system (ATPMS) was evaluated to extract human adenovirus type 5 particles from a cell lysate. Adenovirus was cultured in human embryonic kidney 293 (HEK-293) cells to a concentration of 1.4 × 1010 particles/mL. Cells were lysed, and the system formed by direct addition of Triton X-114 in a 23 full factorial design with center points. The systems were formed with Triton X-114 at a final concentration of 1.0, 6.0, and 11.0% (w/w), cell lysate pH of 6.0, 6.5, and 7.0, and incubation temperatures at 33, 35, and 37 °C. Adenovirus particles recovered from partition phases were measured by qPCR. The best system condition was with 11.0% (w/w) of Triton X-114, a cell lysate pH of 7.0, and an incubation temperature at 33 °C, yielding 3.51 × 1010 adenovirus particles/mL, which increased the initial adenovirus particles concentration by 2.3-fold, purifying it by 2.2-fold from the cell lysate, and removing cell debris. In conclusion, these results demonstrated that the use of an aqueous two-phase micellar system in the early steps of downstream processing could improve viral particle extraction from cultured cells while integrating clarification, concentration, and prepurification steps.

Analysis of Circulating miR-1, miR-23a, and miR-26a in Atrial Fibrillation Patients Undergoing Coronary Bypass Artery Grafting Surgery.

Atrial fibrillation (AF) is the most common arrhythmia after cardiac surgery. From a pathophysiological point of view, a myriad of factors such as trauma, atrial dilation, ischemia, mechanical myopericarditis, autonomic imbalance, loss of connexins, AF nest remodeling, inflammation, sutures, and dysfunction caused by postextracorporeal circulation can contribute to postoperative atrial fibrillation (POAF) resulting in a longer hospital stay and consequently higher cost. Recent studies showed that short fragments of RNA, called microRNA (miRNA), can contribute to the development of several cardiovascular diseases, including AF. The aim of this study was to evaluate the levels of circulating miRNAs (miR-1, -23a, and -26a) that can be involved in POAF. Patients submitted to coronary artery bypass graft surgery were grouped in POAF (24 patients) and without POAF (24 patients). Results showed older age, longer clamp-time, and more days in the intensive care unit as well as a longer total hospital stay in the POAF group. Preoperative levels of circulating miRNAs were similar. Analysis of miRNAs revealed significantly lower circulating levels of miRNA-23a (P = 0.02) and -26a (P = 0.01) in the POAF group during the postoperative period. Receiver operating characteristic (ROC) analysis showed the area under the ROC curve of miR-23a and miR-26a for predicting FA was 0.63 (95% confidence interval [CI]: 0.51-0.74; P = 0.02) and 0.66 (95% CI: 0.55-0.77; P = 0.01), respectively. Our data suggests that circulating miRNA-23a and -26a may be involved in the underlying biology of postoperative AF development.

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.

Low bone mineral density in patients with type 1 diabetes: association with reduced expression of IGF1, IGF1R and TGF B 1 in peripheral blood mononuclear cells.

BACKGROUND: The negative effects of type 1 diabetes (T1D) on growth factors of bone metabolism lead to a reduction in bone mineral density. This study aimed to evaluate the association between bone mineral density and insulin-like growth factor 1 (IGF1), insulin-like growth factor 1 receptor (IGF1R) and transforming growth factor beta 1 (TGFB1) expressions in children and adolescents with T1D. Moreover, the influences of age at diagnosis, time since diagnosis, glycaemic control and albuminuria on bone mineral density were investigated. METHODS: Eighty-six T1D children/adolescents (T1D group) and ninety normoglycaemic controls (normoglycaemic group) were included. T1D patients were analysed as a whole and also in subsets of patients with good glycaemic control (glycated hemoglobin concentration ≤7.5%) and with poor glycaemic control (glycated hemoglobin concentration >7.5%). Bone mineral density was assessed by dual energy x-ray absorptiometry. Glycaemic control, renal function and bone markers were also assessed. IGF1, IGF1R and TGFB1 expressions were determined in peripheral blood mononuclear cells by real-time polymerase chain reaction. RESULTS: Patients with T1D showed low bone mineral density and poor glycaemic control. Serum total calcium and urinary albumin-to-creatinine ratio were higher in patients with poor glycaemic control compared to those with good glycemic control (p = 0.003 and p = 0.035, respectively). There was a reduction of IGF1, IGF1R and TGFB1 expressions in the T1D patients and in the subset with poor glycaemic control compared to normoglycaemic controls (p < 0.05). CONCLUSIONS: The decreased IGF1, IGF1R and TGFB1 expressions in the T1D patients, who presented with T1D at an early age, had been diagnosed with T1D for a longer time, had poor glycaemic control and albuminuria may contribute to low bone mineral density. Copyright © 2016 John Wiley & Sons, Ltd.

Use of transgenic Aedes aegypti in Brazil: risk perception and assessment.

The OX513A strain of Aedes aegypti, which was developed by the British company Oxitec, expresses a self-limiting transgene that prevents larvae from developing to adulthood. In April 2014, the Brazilian National Technical Commission on Biosafety completed a risk assessment of OX513A and concluded that the strain did not present new biological risks to humans or the environment and could be released in Brazil. At that point, Brazil became the first country to approve the unconstrained release of a genetically modified mosquito. During the assessment, the commission produced a comprehensive list of - and systematically analysed - the perceived hazards. Such hazards included the potential survival to adulthood of immature stages carrying the transgene - should the transgene fail to be expressed or be turned off by exposure to sufficient environmental tetracycline. Other perceived hazards included the potential allergenicity and/or toxicity of the proteins expressed by the gene, the potential for gene flow or increased transmission of human pathogens and the occupation of vacant breeding sites by other vector species. The Zika epidemic both elevated the perceived importance of Ae. aegypti as a vector - among policy-makers and regulators as well as the general public - and increased concerns over the release of males of the OX513A strain. We have therefore reassessed the potential hazards. We found that release of the transgenic mosquitoes would still be both safe and of great potential value in the control of diseases spread by Ae. aegypti, such as chikungunya, dengue and Zika.

La souche OX513A d'Aedes aegypti, qui a été créée par la société britannique Oxitec, exprime un transgène autolimitant qui empêche les larves de se développer et de devenir adultes. En avril 2014, la Commission technique nationale de biosécurité du Brésil a procédé à une évaluation des risques liés à la souche OX513A et conclu qu'elle ne présentait pas de nouveaux risques biologiques pour les êtres humains ou l'environnement et pouvait être lâchée au Brésil. Le Brésil est donc devenu le premier pays à approuver le lâcher non contraint d'un moustique génétiquement modifié. Au cours de l'évaluation, la commission a établi une liste exhaustive des risques perçus, qu'elle a par ailleurs systématiquement analysés. Ces risques incluaient la survie potentielle à l'âge adulte des larves immatures porteuses du transgène ­ si le transgène ne s'exprime pas ou est désactivé par une exposition à la tétracycline suffisante dans l'environnement. Les autres risques perçus incluaient les potentielles propriétés allergisantes et/ou la toxicité des protéines exprimées par le gène, l'éventualité d'un flux de gènes ou d'une transmission accrue d'agents pathogènes pour l'homme et l'occupation de sites de reproduction vacants par d'autres espèces vectrices. L'épidémie d'infections à virus Zika a accentué l'importance accordée par les responsables politiques, les organismes de réglementation ainsi que le grand public à Ae. aegypti en tant que moustique vecteur, et a accru l'inquiétude relative au lâcher de mâles de la souche OX513A. Nous avons donc réévalué les risques potentiels. Nous estimons que le lâcher de moustiques transgéniques serait à la fois sans danger et extrêmement utile pour lutter contre les maladies transmises par Ae. aegypti, telles que le chikungunya, la dengue et le virus Zika.

La cepa OX513A de Aedes aegypti, que desarrolló la empresa británica Oxitec, expresa un transgén autolimitado que impide que las larvas se desarrollen hasta la edad adulta. En abril de 2014, la Comisión Nacional Técnica de Bioseguridad de Brasil realizó una evaluación de riesgos de OX513A y concluyó que la cepa no presentaba nuevos riesgos biológicos para los humanos o el medioambiente y que podría liberarse en Brasil. En ese momento, Brasil se convirtió en el primer país en aprobar la liberación ilimitada de un mosquito modificado genéticamente. A lo largo de la evaluación, la comisión redactó una lista completa, y analizada sistemáticamente, de las posibles contingencias. Entre dichos peligros se encontraba la posible supervivencia hasta la edad adulta de etapas inmaduras que portan el transgén, en caso de que éste no consiga expresarse o se inutilice debido a la exposición a la suficiente tetraciclina medioambiental. Otras posibles contingencias eran la alergia y/o toxicidad de las proteínas expresadas por el gen, la posibilidad de un flujo genético o el aumento de la transmisión de patógenos humanos y la ocupación de lugares de cría desocupados por parte de otras especies vectores. La epidemia por el virus de Zika aumentó la importancia de Ae. aegypti como vector, entre los responsables y reguladores políticos, así como entre el público general, y aumentó las preocupaciones acerca de la liberación de machos de la cepa OX513A. Por lo tanto, se han vuelto a evaluar los posibles riesgos. Se ha descubierto que la liberación de mosquitos transgénicos sería segura y tendría un gran valor potencial en el control de la propagación de enfermedades por Ae. aegypti, como la fiebre chikungunya, el dengue y la enfermedad por el virus de Zika.