Candida Variety in the Oral Cavity of Mexican Subjects with Type 2 Diabetes Mellitus and TLR2 Gene Expression.

BACKGROUND: The aim was to diagnose Candida in the oral cavity of subjects with type 2 diabetes mellitus (T2DM) using a genotyping technique and compare the results with those from conventional diagnosis by Papanicolaou (Pap) staining. METHODS: Palatal mucosa smears were performed on 18 dental care patients diagnosed with T2DM and grade I, II, and III prosthetic stomatitis who met the inclusion criteria; 18 healthy control subjects were also included in the study. Hemoglobin A1c (HbA1c) levels were determined from total blood. Using exfoliative cytology, the Pap staining technique was used to diagnose candidiasis. Exfoliative cytology was also used for molecular diagnosis; DNA was obtained for Candida genotyping, and RNA was used for gene expression studies. RESULTS: Clinical patterns indicated that all subjects were positive for Candida; however, Pap analysis revealed only three positive subjects, whereas end-point polymerase chain reaction (PCR) analysis revealed 15 subjects with some type of Candida. The most common Candida species found were Candida guilliermondii (38.8%), Candida krusei (33.3%), Candida tropicalis, and Candida lusitaniae (22.2%). Interestingly, the coexpression of different species of Candida was found in various patients. In all patients, HbA1c levels were increased. Gene expression analysis showed a significant decrease (p ≤ 0.05) in TLR2 expression in positive subjects, whereas TLR4 expression did not differ significantly among patients. CONCLUSIONS: The end-point PCR technique showed better sensitivity for the diagnosis of Candida when compared with the diagnosis by Pap staining. T2DM subjects showed an increased presence of C. guilliermondii that was correlated with decreased TLR2 expression.

A low-protein maternal diet during gestation affects the expression of key pancreatic ß-cell genes and the methylation status of the regulatory region of the MafA gene in the offspring of Wistar rats.

Maternal nutrition during gestation has important effects on gene expression-mediated metabolic programming in offspring. To evaluate the effect of a protein-restricted maternal diet during gestation, pancreatic islets from male progeny of Wistar rats were studied at postnatal days (PND) 36 (juveniles) and 90 (young adults). The expression of key genes involved in ß-cell function and the DNA methylation pattern of the regulatory regions of two such genes, Pdx1 (pancreatic and duodenal homeobox 1) and MafA (musculoaponeurotic fibrosarcoma oncogene family, protein A), were investigated. Gene expression analysis in the pancreatic islets of restricted offspring showed significant differences compared with the control group at PND 36 (P < 0.05). The insulin 1 and 2 (Ins1 and Ins2), Glut2 (glucose transporter 2), Pdx1, MafA, and Atf2 (activating transcription factor 2), genes were upregulated, while glucokinase (Gck) and NeuroD1 (neuronal differentiation 1) were downregulated. Additionally, we studied whether the gene expression differences in Pdx1 and MafA between control and restricted offspring were associated with differential DNA methylation status in their regulatory regions. A decrease in the DNA methylation levels was found in the 5' flanking region between nucleotides -8118 to -7750 of the MafA regulatory region in restricted offspring compared with control pancreatic islets. In conclusion, low protein availability during gestation causes the upregulation of MafA gene expression in pancreatic ß-cells in the male juvenile offspring at least in part through DNA hypomethylation. This process may contribute to developmental dysregulation of ß-cell function and influence the long-term health of the offspring.

Sex-differential RXRα gene methylation effects on mRNA and protein expression in umbilical cord of the offspring rat exposed to maternal obesity.

Maternal obesity (MO) induces negative consequences in the offspring development. Adiposity phenotype is associated with maternal diet at early pregnancy and DNA methylation marks in the RXRα promotor at birth. Glucocorticoids play an important role in the regulation of metabolism through the activation of nuclear hormone receptors such as the RXRα protein. The aim of the study was to analyze steroid hormone changes at the end of pregnancy in the obese mother and RXRα gene methylation in the umbilical cord. For this purpose, in a well-established MO model, female Wistar rats were fed either standard chow (controls: C) or high-fat obesogenic diet (MO) before and during pregnancy to evaluate at 19 days of gestation (19 dG): 1) maternal concentration of circulating steroid hormones in MO and C groups, 2) maternal and fetal weights, 3) analysis of correlation between hormones concentration and maternal and fetal weights, 4) DNA methylation status of a single locus of RXRα gene near the early growth response (EGR-1) protein DNA binding site, and 5) RXRα mRNA and protein expressions in umbilical cords. Our results demonstrate that at 19 dG, MO body weight before and during pregnancy was higher than C; MO progesterone and corticosterone serum concentrations were higher and estradiol lower than C. There were not differences in fetal weight between male and female per group, therefore averaged data was used; MO fetal weight was lower than C. Positive correlations were found between progesterone and corticosterone with maternal weight, and estradiol with fetal weight, while negative correlation was observed between corticosterone and fetal weight. Additionally, male umbilical cords from MO were hypermethylated in RXRα gene compared to male C group, without differences in the female groups; mRNA and protein expression of RXRα were decreased in F1 male but not in female MO compared to C. In conclusion, MO results in dysregulation of circulating steroid hormones of the obese mothers and low fetal weight in the F1, modifying DNA methylation of RXRα gene as well as RXRα mRNA and protein expression in the umbilical cord in a sex-dependent manner.

17ß-Estradiol transcriptionally modulates Nlrp1 and Nlrp3 inflammasomes in gonadectomized rats with inflammation.

CONTEXT: It has been reported that 17ß-estradiol (E2) reduces the expression of inflammatory molecules, but there are no data that show the effect of E2 on the transcriptional regulation of innate immunity-related molecules and inflammasomes. OBJECTIVE: To study the effect of 17ß-estradiol (E2) on the transcriptional expression of the NLR family, pyrin domain containing 1 (Nlrp1) and (Nlrp3) inflammasomes, which are mediators of inflammation. MATERIALS AND METHODS: Inflammation was induced in adult female gonadectomized (Gdx) rats by intramuscular injection of complete Freund's adjuvant (CFA). Measurements were taken at different times after the treatment. Gene expression determinations were done by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: CFA-induced inflammation increased the transcription of Nlrp3, IL-1ß (p < 0.05), vascular cell adhesion molecule 1 (VCAM1), E-selectin and estrogen receptor 1 alpha (ERα) (p < 0.001) and decreased the transcription of Nlrp1, Caspase-1, IL-33, NFKB1, ICAM1, ICAM2, GCRα, GCRß, UCP3 and PGC1α (p < 0.001) compared to the control. The administration of E2 to the inflamed tissue significantly increased the expression of Nlrp1, NFKB1, ERα, UCP3, Caspase-1, E-selectin (p < 0.001), IL-18 and ERα (p < 0.05) and decreased IL-1ß and VCAM1 (p < 0.005) compared to the control. DISCUSSION AND CONCLUSION: CFA differentially modulates the transcription of inflammasome-related genes and the administration of E2 increases the expression of ERα and Nlrp1 together with NFKB1, a key molecule in the activation of the inflammasomes. Finally, an analysis using the web interface GeneMANIA revealed an interaction between several genes, indicating a functional correlation in this model.

Expression of NLRP3 inflammasome, cytokines and vascular mediators in the skin of systemic sclerosis patients.

BACKGROUND: The activated NLRP3 inflammasome is associated with the etiology of fibrotic diseases. The role of inflammasomes in SSc is still poorly understood. OBJECTIVES: To determine the expression of NLRP3 (nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3) in the skin of patients with systemic sclerosis (SSc) and its relationship with pro-inflammatory cytokines and vascular mediators expression. METHODS: Skin biopsies were taken from 42 patients with either limited or diffuse SSc (21 lcSSc and 21 dcSSc), and from 13 healthy individuals. Using real-time polymerase chain reaction (PCR), the relative expression of caspase-1, IL-1ß, IL-18, IL-33, TGF-ß, ET-1, iNOS and eNOS genes, were measured. The location of NLRP3 and IL-1ß were also determined by immunohistochemistry. Clinical characteristics were evaluated. RESULTS: The mean age of the patients was 49.3 ± 12.9 (lcSSc), 44.6 ± 1 3.8 (dcSSc), and 45 ± 14.1 (healthy individuals). Compared to healthy individuals, the skin of both subtypes of SSc showed a significant increase (P < 0.05) in NLRP3, caspase-1, IL-1ß, IL-18 and ET-1. Samples of lcSSc also showed a significant increase of eNOS (P < 0.029), iNOS (P < 0.04) and TGF-ß (P < 0.05). Dermal fibrosis evaluated by modified Rodnan skin score (MRSS) had significant correlation with NLRP3, IL-1ß, IL-18, and ET-1. Immunohistochemical analysis showed stronger staining of NLRP3 and IL-1ß cytoplasmic expression in the keratinizing squamous epithelium of skin from SSc patients compared to controls. CONCLUSIONS: This study identified NLRP3 over-expression in skin of patients with SSc. Skin thickness correlates positively with the NLRP3 inflammasome gene expression and with the vascular mediator and pro-fibrotic ET-1, suggesting that NLRP3 inflammasome plays a role in the pathophysiology of skin fibrosis in human SSc.

Significance of CASP8AP2 and H2AFZ expression in survival and risk of relapse in children with acute lymphoblastic leukemia.

Novel biomarkers for risk refinement and stratification in childhood acute lymphoblastic leukemia (ALL) are needed to optimize treatment results. We studied the expression of CASP8AP2 and H2AFZ associated with relapse and survival in bone marrow samples from newly diagnosed children with ALL. We found: (a) an increased risk for early relapse in those patients with low expression of CASP8AP2 (odds ratio [OR] 3.93, 95% confidence interval [CI] 1.40-11.02, p < 0.05) confirming its usefulness as a predictive risk marker, although H2AFZ did not present the same effect; (b) patients with low expressions of CASP8AP2 and H2AFZ had inferior survival rates (p < 0.001); (c) the predictive values regarding low expressions of H2AFZ and CASP8AP2 and high white blood cell count suggest that these features could help to identify more accurately patients at greater risk of relapse.

Transcript levels of Toll-Like Receptors 5, 8 and 9 correlate with inflammatory activity in Ulcerative Colitis.

BACKGROUND: Dysregulation of innate immune response by Toll-Like Receptors (TLRs) is a key feature in Ulcerative Colitis (UC). Most studies have focused on TLR2, TLR3, and TLR4 participation in UC. However, few studies have explored other TLRs. Therefore, the aim of this study was to evaluate the mRNA profiles of TLR1 to 9 in colonic mucosa of UC patients, according to disease activity. METHODS: Colonic biopsies were taken from colon during colonoscopy in 51 patients with Ulcerative Colitis and 36 healthy controls. mRNA levels of TLR1 to 9, Tollip, inflammatory cytokines IL6 and TNF were assessed by RT-qPCR with hydrolysis probes. Characterization of TLR9 protein expression was performed by Immunohistochemistry. RESULTS: Toll-like receptors TLR8, TLR9, and IL6 mRNA levels were significantly higher in the colonic mucosa from UC patients (both quiescent and active) as compared to healthy individuals (p < 0.04). In the UC patients group the TLR2, TLR4, TLR8 and TLR9 mRNA levels were found to be significantly lower in patients with quiescent disease, as compared to those with active disease (p < 0.05), whereas TLR5 showed a trend (p = 0.06). IL6 and TNF mRNA levels were significantly higher in the presence of active disease and help to discriminate between quiescent and active disease (p < 0.05). Also, IL6 and TNF mRNA positively correlate with TLRs mRNA with the exception for TLR3, with stronger correlations for TLR5, TLR8, and TLR9 (p < 0.0001). TLR9 protein expression was mainly in the lamina propria infiltrate. CONCLUSIONS: This study demonstrates that TLR2, TLR4, TLR8, and TLR9 expression increases in active UC patients, and that the mRNA levels positively correlate with the severity of intestinal inflammation as well as with inflammatory cytokines.

IFN-stimulated gene expression is a useful potential molecular marker of response to antiviral treatment with Peg-IFNα 2b and ribavirin in patients with hepatitis C virus genotype 1.

BACKGROUND AND AIMS: We undertook this study to determine the baseline gene expression of IFI27, IFIT1, IFI6, ISG15, IRF-1, IRF-3, OAS-2 and CXCL10 and its usefulness as molecular markers of response to antiviral treatment with peg-IFNα 2b/RBV in patients with hepatitis C virus genotype 1 (HCV-1). METHODS: Gene expression was analyzed by RT-PCR in baseline liver biopsies from 42 HCV-1 patients who were treated with Peg-IFNα 2b/RBV for 48 weeks. In addition, we investigated gene expression of these genes in a second liver biopsy obtained 24 weeks post-treatment in sustained viral response (SVR) and relapser patients. RESULTS: Thirteen patients achieved SVR, four were relapsers, four patients with viral response (VR) discontinued the following for 24 weeks post-treatment and 21 patients did not respond to antiviral therapy (NR). All patients with HCV-1 showed gene overexpression in baseline liver tissue, but only IFI27, IFIT1, IFI6, ISG15, and CXCL10 showed differential gene expression, which is inversely related to the response to antiviral therapy. Thus, liver tissue of NR patients showed upregulation of these genes, whereas patients with SVR gene expression level was significantly lower. Furthermore, 24 weeks afterwards treatment, SVR patients showed a significant downregulation of such genes, which was consistent with the RNA-HCV suppression. ISGs (IFI27, IFIT1, IFI6) and chemokine CXCL10 showed the best positive and negative predictive values on SVR to IFN/RBV therapy (range: 70.8-75% and 71.43-82.35%), respectively. CONCLUSIONS: IFI27, IFIT1, IFI6, ISG15, and CXCL10 genes are potential biological markers useful for predicting response to Peg-IFNα 2b/RBV therapy in HCV-1 patients.

Identificación de distintos loci de susceptibilidad relacionados al desarrollo de diabetes de inicio temprano y enfermedad cardiovascular en familias mexicanas / Identifying different susceptibility loci associated with early onset diabetes and cardiovascular disease in mexican families

La enfermedad arterial coronaria y la diabetes mellitus figuran entre las primeras causas de mortalidad y morbilidad en México. Factores genéticos juegan un papel fundamental en el desarrollo de estas entidades. A partir del reconocimiento y estudio de familias con formas monogénicas de diabetes y distintas dislipidemias asociadas al desarrollo de ateroesclerosis, se han identificado en los últimos años distintos genes y loci relacionados con estos padecimientos a través de estudios de mapeo genético. Estos estudios han evidenciado la heterogeneidad genética que existe en cuanto al tipo de genes involucrados en los distintos grupos étnicos. El estudio de familias mexicanas con diabetes de inicio temprano e hiperlipidemia familiar combinada mostró la participación de distintos loci génicos asociados a estas entidades en la población mexicana. Esto muestra la utilidad de las estrategias de mapeo para la identificación del componente genético de estas entidades en nuestra población.

Coronary artery disease and diabetes mellitus are among the primary mortality and morbidity causes in Mexico. Genetic factors play a fundamental role in the development of these entities. In the past few years due to the recognition and study of families with monogenic forms of diabetes and dislipidemias associated with development of atherosclerosis, several genes and loci have been associated with these conditions through genetic linkage studies. These studies have provided evidence of the genetic heterogeneity that exists and the type of genes involved in different ethnic groups. The study of Mexican families with early onset diabetes and combined familial hyperlipidemia showed the participation of different genetic loci associated with these conditions in the Mexican population. These findings show the value of gene mapping strategies in the identification of the genetic component in these entities in our population.