Juliana Carvalho Santos

Relationship of IL-1 and TNF-α polymorphisms with Helicobacter pylori in gastric diseases in a Brazilian population

It is well known that the risk of development of gastric cancer (GC) in Helicobacter pylori-infected patients depends on several factors. Thus, the aim of this study was to investigate the effect of proinflammatory cytokine gene polymorphisms for IL-1β, IL-1RN and TNF-α on the development of GC in a Brazilian population. A total of 202 biopsies obtained from Brazilian patients with chronic gastritis and GC were included in the study. Infection with H. pylori cagA+ was determined by the polymerase chain reaction (PCR) as previously described. IL-1β, IL-1RN and TNF-α polymorphism genotyping was performed by restriction fragment length polymorphism PCR. Associations between gene polymorphisms, clinical diseases and virulence markers were evaluated using either the X2 test or the Fisher exact test. Our results demonstrated that the IL-1β -511 C/C and IL-1β -511 C/T alleles were associated with chronic gastritis in H. pylori-positive patients (P = 0.04 and P = 0.05, respectively) and the IL-1β -511 C/C genotype was associated with GC (P = 0.03). The frequency of IL-1RN alleles from patients with chronic gastritis and GC indicated that there was no difference between the genotypes of the groups studied. Similar results were found for TNF-α -308 gene polymorphisms. Our results indicate that the IL-1β -511 C/C and C/T gene polymorphisms are associated with chronic gastritis and GC development in H. pylori-infected individuals.

MGMT and MLH1 methylation in Helicobacter pylori-infected children and adults

AIM To evaluate the association between Helicobacter pylori (H. pylori) infection and MLH1 and MGMT methylation and its relationship with microsatellite instability (MSI). METHODS The methylation status of the MLH1 and MGMT promoter region was analysed by methylation specific methylation-polymerase chain reaction (MSP-PCR) in gastric biopsy samples from uninfected or H. pylori-infected children (n = 50), from adults with chronic gastritis (n = 97) and from adults with gastric cancer (n = 92). MLH1 and MGMT mRNA expression were measured by real-time PCR and normalised to a constitutive gene (β actin). MSI analysis was performed by screening MSI markers at 4 loci (Bat-25, Bat-26, D17S250 and D2S123) with PCR; PCR products were analysed by single strand conformation polymorphism followed by silver staining. Statistical analyses were performed with either the χ(2) test with Yates continuity correction or Fishers exact test, and statistical significance for expression analysis was assessed using an unpaired Students t-test. RESULTS Methylation was not detected in the promoter regions of MLH1 and MGMT in gastric biopsy samples from children, regardless of H. pylori infection status. The MGMT promoter was methylated in 51% of chronic gastritis adult patients and was associated with H. pylori infection (P < 0.05); this region was methylated in 66% of gastric cancer patients, and the difference in the percentage of methylated samples between these patients and those from H. pylori-infected chronic gastritis patients was statistically significant (P < 0.05). MLH1 methylation frequencies among H. pylori-infected and non-infected chronic gastritis adult patients were 13% and 7%, respectively. We observed methylation of the MLH1 promoter (39%) and increased MSI levels (68%) in samples from gastric cancer patients in comparison to samples from H. pylori-infected adult chronic gastritis patients (P < 0.001 and P < 0.01, respectively). The frequency of promoter methylation for both genes was higher in gastric cancer samples than in H. pylori-positive chronic gastritis samples (P < 0.05). The levels of MLH1 and MGMT mRNA were significantly reduced in chronic gastritis samples that were also hypermethylated (P < 0.01). CONCLUSION In summary, MGMT and MLH1 methylation did not occur in earlier-stage H. pylori infections and thus might depend on the duration of infection.

Redox markers and inflammation are differentially affected by atorvastatin, pravastatin or simvastatin administered before endotoxin-induced acute lung injury.

Statins are standard therapy for the treatment of lipid disorders, and the field of redox biology accepts that statins have antioxidant properties. Our aim in this report was to consider the pleiotropic effects of atorvastatin, pravastatin and simvastatin administered prior to endotoxin-induced acute lung injury. Male mice were divided into 5 groups and intraperitoneally injected with LPS (10 mg/kg), LPS plus atorvastatin (10 mg/kg/day; A + LPS group), LPS plus pravastatin (5 mg/kg/day; P + LPS group) or LPS plus simvastatin (20 mg/kg/day; S + LPS group). The control group received saline. All mice were sacrificed one day later. There were fewer leukocytes in the P + LPS and S + LPS groups than in the LPS group. MCP-1 cytokine levels were lower in the P + LPS group, while IL-6 levels were lower in the P + LPS and S + LPS groups. TNF-α was lower in all statin-treated groups. Levels of redox markers (superoxide dismutase and catalase) were lower in the A + LPS group (p < 0.01). The extent of lipid peroxidation (malondialdehyde and hydroperoxides) was reduced in all statin-treated groups (p < 0.05). Myeloperoxidase was lower in the P + LPS group (p < 0.01). Elastance levels were significantly greater in the LPS group compared to the statin groups. Our results suggest that atorvastatin and pravastatin but not simvastatin exhibit anti-inflammatory and antioxidant activity in endotoxin-induced acute lung injury.

Chemopreventive potential of the tannase-mediated biotransformation of green tea

Green tea (Camellia sinensis) is one of the most widely consumed beverages in the world. The cancer chemopreventive qualities of green tea have been well documented. Epigallocatechin gallate (EGCG) is often described as the most potently chemopreventive green tea catechin; however, the low bioavailability of EGCG is a limiting factor for its biological effect. Thus, the aim of this work was to test the chemopreventive potential of green tea extract and EGCG after tannase-mediated hydrolysis. The results showed that the biotransformed compounds retained most of the beneficial properties of the original compounds, and some beneficial properties were improved in the biotransformed compounds. Biotransformation of EGCG decreased its toxicity without affecting its antiproliferative effects. Furthermore, human cells gene expression profiling showed that the biotransformed compounds modulated the expression of several genes related to carcinogenesis. These results demonstrate the benefits of the biotechnological modification of natural food molecules, allowing the improvement of the nutraceutical potential of a beverage as green tea.

Effects of Yerba maté, a Plant Extract Formulation ("YGD") and Resveratrol in 3T3-L1 Adipogenesis

We aimed to evaluate the in vitro effects of yerba maté, YGD (a herbal preparation containing yerba maté, guarana and damiana), and resveratrol on adipogenesis. The anti-adipogenic effects of yerba mate, YGD, resveratrol and YGD + resveratrol and yerba mate + resveratrol combinations were evaluated in 3T3-L1 cells by Oil Red staining, cellular triglyceride content, and PCR quantitative array. The results demonstrated that all of the tested compounds inhibited adipogenesis. Yerba maté extract significantly down-regulated the expression of genes that play an important role in regulating adipogenesis, such as Adig, Axin, Cebpa, Fgf10, Lep, Lpl, and Pparγ2. In addition, these genes, YGD also repressed Bmp2, Ccnd1, Fasn, and Srebf1. Resveratrol also modulated the expression of Adig, Bmp2, Ccnd1, C/EBPα, Fasn, Fgf10, Lep, Lpl, and Pparγ2. Moreover, resveratrol repressed Cebpb, Cdk4, Fgf2, and Klf15. The yerba maté extract and YGD up-regulated the expression of genes involved in inhibiting adipogenesis, such as Dlk-1, Klf2, and Ucp1. Resveratrol also induced the expression of Klf2 and Ucp1. In addition resveratrol modulated the Ddit3, Foxo1, Sirt1, and Sirt2. The combined effects of these compounds on gene expression showed similar results observed from individual treatments. Our data indicates that the synergy between the compounds favors the inhibition of adipogenesis.

Endotoxin-induced acute lung injury is dependent upon oxidative response

The aim of the present study was to investigate the involvement of oxidative stress in acute lung injury (ALI) induced by lipopolysaccharide (LPS) and its effects upon cell structure, function and inflammation. In total, 108 male C57BL/6 mice were divided into seven groups: CTR Group (50 µL of saline) administered intratracheally (i.t.), LPS 6 h (10 µg of LPS − i.t.), LPS 12 h (10 µg of LPS − i.t.), LPS 24 h (10 µg of LPS − i.t.), LPS 48 h (10 µg of LPS − i.t.), LPS 24 h (10 µg − i.t.) + NAC 40 mg/kg (gavage) and 24 h LPS (10 µg − i.t.) + NAC 100 mg/kg (gavage). The antioxidant treatment protected the lungs from stress in the first 12 h, but significant oxidative stress induction was observed at the 24-hour time point, and, after 48 h, there was no protection exerted by the antioxidant treatment. NAC (N-acetylcysteine) reversed the elastance parameters, and ΔP1 and ΔP2 compared with 24 h LPS alone. NAC reduced the number of inflammatory cells in histology analysis when compared with the 24 h LPS alone-treated group. NAC also inhibited the transcription of NFκB, IL-6, TNF-α and COX2 usually induced by LPS. Our results suggest that oxidative stress plays an important role in structural, functional and inflammatory responses in the ALI model.

Correlation of MLH1 and MGMT expression and promoter methylation with genomic instability in patients with thyroid carcinoma

BackgroundGene silencing of the repair genes MLH1 and MGMT was shown to be a mechanism underlying the development of microsatellite instability (MSI), a phenotype frequently associated with various human malignancies. Recently, aberrant methylation of MLH1, MGMT and MSI were shown to be associated with mutations in genes such as BRAF, RAS and IDH1 in colon and brain tumours. Little is known about the methylation status of MLH1 and MGMT in thyroid tumours and its association with MSI and mutational status.MethodsIn a series of 96 thyroid tumours whose mutational profiles of BRAF, IDH1 and NRAS mutations and RET/PTC were previously determined, we investigated MLH1 and MGMT expression and methylation status by qPCR and methylation-specific PCR after bisulphite treatment, respectively. MSI was determined by PCR using seven standard microsatellite markers.ResultsSamples with point mutations (BRAF, IDH1 and NRAS) show a decrease in MLH1 expression when compared to negative samples. Additionally, malignant lesions show a higher MSI pattern than benign lesions. The MSI phenotype was also associated with down-regulation of MLH1.ConclusionsThe results of this study allow us to conclude that low expression of MLH1 is associated with BRAF V600E mutations, RET/PTC rearrangements and transitions (IDH1 and NRAS) in patients with thyroid carcinoma. In addition, a significant relationship between MSI status and histological subtypes was found.

Critical role for CCR2 and HMGB1 in induction of experimental endotoxic shock

Our aim was to investigate CCR2 and HMGB1 involvement in a murine model of endotoxic shock. We used C57BL/6 CCR2 knockout (KO) mice and wild-type (WT) littermates to establish an optimal dose of LPS. CCR2 KO mice survived more frequently than WT mice after 80, 40 and 20 mg/kg of LPS i.p. Inflammation and redox markers were high in WT mice than in CCR2 KO mice. HMGB1 expression was reduced in CCR2 KO mice in parallel to ERK 1/2 activation. Therefore, we used glycyrrhizic acid (50 mg/kg), an HMGB1 inhibitor in WT mice injected with LPS, and mortality was fully abolished. Thus, drugs targeting CCR2 and HMGB1 could represent future resources for sepsis treatment.

Thiopurine-methyltransferase variants in inflammatory bowel disease: prevalence and toxicity in Brazilian patients.

AIM To analyze the prevalence of thiopurine-methyltransferase (TPMT) genotypes and their association with drug toxicity in inflammatory bowel disease (IBD) patients from southeastern Brazil. METHODS A total of 219 consecutive patients with IBD, of which 146 had Crohns disease and 73 had ulcerative colitis, regularly seen at the outpatient unit of the Division of Gastroenterology at the University Hospital Pedro Ernesto of the State University of Rio de Janeiro, a tertiary referral center, were enrolled in this study from February 2009 to January 2011. We analyzed the presence of major TPMT genetic variants (TPMT 2, 3A, 3C) in IBD patients by means of a specific allele and RFLP-PCR. Genomic DNA was isolated from peripheral blood leukocytes by proteinase-K/Sodium Dodecyl Sulfate digestion and phenol-chloroform extraction. TPMT 2 (C238G), TPMT 3A (G460A/A719G), and TPMT 3C (A719G) genotypes were detected by real-time polymerase chain reaction followed by direct sequencing with specific primers. Clinical data were systematically recorded, and correlated with the genotype results. RESULTS The distribution of the selected TPMT gene polymorphism TPMT 2 (C238G), TPMT 3A (G460A/A719G), and TPMT 3C (A719G) genotypes was 3.6%, 5.4%, and 7.7% of the patients, respectively. Among the side effects recorded from patients taking azathioprine, 14 patients presented with pancreatitis and/or an elevation of pancreatic enzymes, while 6 patients had liver toxicity, and 2 patients exhibited myelosuppression/neutropenia. TPMT polymorphisms were detected in 37/219 patients (8 heterozygous for 2, 11 heterozygous for 3A, and 18 heterozygous for 3C). No homozygotic polymorphisms were found. Despite the prevalence of the TPMT 3C genotype, no differences among the genotype frequencies were significant. Although no association was detected regarding myelotoxicity or hepatotoxicity, a trend towards the elevation of pancreatic enzymes was observed for TPMT 2 and TPMT 3C genotypes. CONCLUSION The prevalence of TPMT genotypes was high among Brazilian patients. Variants genes 2 and 3C may be associated with azathioprine pancreatic toxicity in a IBD southeastern Brazilian population.

Effect of APE1 T2197G (Asp148Glu) Polymorphism on APE1, XRCC1, PARP1 and OGG1 Expression in Patients with Colorectal Cancer

It has been hypothesized that genetic variation in base excision repair (BER) might modify colorectal adenoma risk. Thus, we evaluated the influence of APE1 T2197G (Asp148Glu) polymorphism on APE1, XRCC1, PARP1 and OGG1 expression in normal and tumor samples from patients with colorectal cancer. The results indicate a downregulation of OGG1 and an upregulation of XRCC1 expression in tumor tissue. Regarding the anatomical location of APE1, OGG1 and PARP-1, a decrease in gene expression was observed among patients with cancer in the rectum. In patients with or without some degree of tumor invasion, a significant downregulation in OGG1 was observed in tumor tissue. Interestingly, when taking into account the tumor stage, patients with more advanced grades (III and IV) showed a significant repression for APE1, OGG1 and PARP-1. XRCC1 expression levels were significantly enhanced in tumor samples and were correlated with all clinical and histopathological data. Concerning the polymorphism T2197G, GG genotype carriers exhibited a significantly reduced expression of genes of the BER repair system (APE1, XRCC1 and PARP1). In summary, our data show that patients with colorectal cancer present expression changes in several BER genes, suggesting a role for APE1, XRCC1, PARP1 and OGG1 and APE1 polymorphism in colorectal carcinogenesis.