5-Methyltetrahydrofolate-homocysteine methyltransferase gene polymorphism (MTR) and risk of head and neck cancer

The functional effect of the A>G transition at position 2756 on the MTR gene (5-methyltetrahydrofolate-homocysteine methyltransferase), involved in folate metabolism, may be a risk factor for head and neck squamous cell carcinoma (HNSCC). The frequency of MTR A2756G (rs1805087) polymorphism was compared between HNSCC patients and individuals without history of neoplasias. The association of this polymorphism with clinical histopathological parameters was evaluated. A total of 705 individuals were included in the study. The polymerase chain reaction-restriction fragment length polymorphism technique was used to genotype the polymorphism. For statistical analysis, the chi-square test (univariate analysis) was used for comparisons between groups and multiple logistic regression (multivariate analysis) was used for interactions between the polymorphism and risk factors and clinical histopathological parameters. Using univariate analysis, the results did not show significant differences in allelic or genotypic distributions. Multivariable analysis showed that tobacco and alcohol consumption (P < 0.05), AG genotype (P = 0.019) and G allele (P = 0.028) may be predictors of the disease and a higher frequency of the G polymorphic allele was detected in men with HNSCC compared to male controls (P = 0.008). The analysis of polymorphism regarding clinical histopathological parameters did not show any association with the primary site, aggressiveness, lymph node involvement or extension of the tumor. In conclusion, our data provide evidence that supports an association between the polymorphism and the risk of HNSCC.

Long-term social recognition memory in adult male rats: factor analysis of the social and non-social behaviors

A modified version of the intruder-resident paradigm was used to investigate if social recognition memory lasts at least 24 h. One hundred and forty-six adult male Wistar rats were used. Independent groups of rats were exposed to an intruder for 0.083, 0.5, 2, 24, or 168 h and tested 24 h after the first encounter with the familiar or a different conspecific. Factor analysis was employed to identify associations between behaviors and treatments. Resident rats exhibited a 24-h social recognition memory, as indicated by a 3- to 5-fold decrease in social behaviors in the second encounter with the same conspecific compared to those observed for a different conspecific, when the duration of the first encounter was 2 h or longer. It was possible to distinguish between two different categories of social behaviors and their expression depended on the duration of the first encounter. Sniffing the anogenital area (49.9% of the social behaviors), sniffing the body (17.9%), sniffing the head (3%), and following the conspecific (3.1%), exhibited mostly by resident rats, characterized social investigation and revealed long-term social recognition memory. However, dominance (23.8%) and mild aggression (2.3%), exhibited by both resident and intruders, characterized social agonistic behaviors and were not affected by memory. Differently, sniffing the environment (76.8% of the non-social behaviors) and rearing (14.3%), both exhibited mostly by adult intruder rats, characterized non-social behaviors. Together, these results show that social recognition memory in rats may last at least 24 h after a 2-h or longer exposure to the conspecific.

Antipyretic and antioxidant activities of 5-trifluoromethyl-4,5-dihydro-1H-pyrazoles in rats

The objective of this study was to determine the effect of eight 5-hydroxy-5-trifluoromethyl-4,5-dihydro-1H-1-carboxyamidepyrazoles (TFDPs) on rat body temperature and baker’s yeast-induced fever. TFDPs or vehicle (5% Tween 80 in 0.9% NaCl, 5 mL/kg) were injected subcutaneously and rectal temperature was measured as a function of time in 28-day-old male Wistar rats (N = 5-12 per group). Antipyretic activity was determined in feverish animals injected with baker’s yeast (Saccharomyces cerevisiae suspension, 0.135 mg/kg, 10 mL/kg, ip). 3-Ethyl- and 3-propyl-TFDP (140 and 200 56;mol/kg, respectively, 4 h after yeast injection) attenuated baker’s yeast-induced fever by 61 and 82%, respectively. These two effective antipyretics were selected for subsequent analysis of putative mechanisms of action. We then determined the effects on cyclooxygenase-1 and -2 (COX-1 and COX-2) activities on 1,1-diphenyl-2-picrylhydrazyl (DPPH) oxidation in vitro, on tumor necrosis factor-^5; (TNF-^5;) and interleukin-1β (IL-1β) levels and on leukocyte counts in the washes of peritoneal cavities of rats injected with baker’s yeast. While 3-ethyl- and 3-propyl-TFDP did not reduce baker’s yeast-induced increases of IL-1β or TNF-^5; levels, 3-ethyl-TFDP caused a 42% reduction in peritoneal leukocyte count. 3-Ethyl- and 3-propyl-TFDP did not alter COX-1 or COX-2 activities in vitro, but presented antioxidant activity in the DPPH assay with an IC50 of 39 mM (25-62) and 163 mM (136-196), respectively. The data indicate that mechanisms of action of these two novel antipyretic pyrazole derivatives do not involve the classic inhibition of the COX pathway or pyrogenic cytokine release.

In vivo and in vitro anti-inflammatory and anti-nociceptive activities of lovastatin in rodents

Statins are among the most prescribed drugs in recent clinical practice. They are also known for their pleiotropic actions, which are independent of their lipid-lowering properties. The effect of lovastatin was investigated against carrageenan-induced paw edema in male Wistar rats (200-250 g) and on leukocyte migration, as measured by carrageenan-induced peritonitis in male Swiss mice (20-25 g), which are models of acute inflammation. Lovastatin (administered 1 h prior to carrageenan), at oral doses of 2, 5, and 10 mg/kg, markedly attenuated paw edema formation in rats at the 4th hour after carrageenan injection (25, 43, and 37% inhibition, respectively). Inhibitions of 20, 45 and 80% were observed in the leukocyte migration, as evaluated by carrageenan-induced peritonitis in mice with lovastatin doses of 0.5, 1 and 5 mg/kg, as compared to controls. Furthermore, lovastatin (administered 1 h before initiation) reduced the nociceptive effect of the formalin test in mice, at both phases, at doses of 2, 5, and 10 mg/kg: first phase (51, 65, and 70%, respectively) and second phase (73, 57, and 66% inhibition of licking time, respectively). The anti-nociceptive activity of lovastatin was inhibited by naloxone (3 mg/kg, sc). Lovastatin (0.01, 0.1, and 1 µg/mL) inhibited by 23, 79, and 86%, respectively, the release of myeloperoxidase from human neutrophils. Leukocyte (predominantly neutrophils) infiltration was almost completely reduced by lovastatin treatment, as observed in the model of acute paw edema with hematoxylin and eosin staining. In addition, lovastatin decreased the number of cells expressing tumor necrosis factor-^5; (TNF-^5;) and the inducible form of nitric oxide synthase (iNOS) activity. Therefore, the alterations in leukocyte activity and cytokine release could contribute to the anti-inflammatory activity of lovastatin.

Plasma concentrations and placental immunostaining of interleukin-10 and tumornecrosis factor-^5; as predictors of alterations in the embryo-fetal organism and the placental development of diabetic rats

Interleukin-10 (IL-10) appears to be the key cytokine for the maintenance of pregnancy and inhibits the secretion of inflammatory cytokines such as tumor necrosis factor-^5; (TNF-^5;). However, there are no studies evaluating the profile of these cytokines in diabetic rat models. Thus, our aim was to analyze IL-10 and TNF-^5; immunostaining in placental tissue and their respective concentrations in maternal plasma during pregnancy in diabetic rats in order to determine whether these cytokines can be used as predictors of alterations in the embryo-fetal organism and in placental development. These parameters were evaluated in non-diabetic (control; N = 15) and Wistar rats with streptozotocin (STZ)-induced diabetes (N = 15). At term, the dams (100 days of life) were killed under anesthesia and plasma and placental samples were collected for IL-10 and TNF-^5; determinations by ELISA and immunohistochemistry, respectively. The reproductive performance was analyzed. Plasma IL-10 concentrations were reduced in STZ rats compared to controls (7.6 ± 4.5 vs 20.9 ± 8.1 pg/mL). The placental scores of immunostaining intensity did not differ between groups (P > 0.05). Prevalence analysis showed that the IL-10 expression followed TNF-^5; expression, showing a balance between them. STZ rats also presented impaired reproductive performance and reduced plasma IL-10 levels related to damage during early embryonic development. However, the increased placental IL-10 as a compensatory mechanism for the deficit of maternal regulation permitted embryo development. Therefore, the data suggest that IL-10 can be used as a predictor of changes in the embryo-fetal organism and in placental development in pregnant diabetic rats.

Variants of transcription factor 7-like 2 (TCF7L2) gene and incident glucose intolerance in Japanese-Brazilians

Common variants of the transcription factor 7-like 2 (TCF7L2) gene have been found to be associated with type 2 diabetes in different ethnic groups. The Japanese-Brazilian population has one of the highest prevalence rates of diabetes. Therefore, the aim of the present study was to assess whether two single-nucleotide polymorphisms (SNPs) of TCF7L2, rs7903146 and rs12255372, could predict the development of glucose intolerance in Japanese-Brazilians. In a population-based 7-year prospective study, we genotyped 222 individuals (72 males and 150 females, aged 56.2 ± 10.5 years) with normal glucose tolerance at baseline. In the study population, we found that the minor allele frequency was 0.05 for SNP rs7903146 and 0.03 for SNP rs12255372. No significant allele or genotype association with glucose intolerance incidence was found for either SNP. Haplotypes were constructed with these two SNPs and three haplotypes were defined: CG (frequency: 0.94), TT (frequency = 0.027) and TG (frequency = 0.026). None of the haplotypes provided evidence for association with the incidence of glucose intolerance. Despite no associations between incidence of glucose intolerance and SNPs of the TCF7L2 gene in Japanese-Brazilians, we found that carriers of the CT genotype for rs7903146 had significantly lower insulin levels 2 h after a 75-g glucose load than carriers of the CC genotype. In conclusion, in Japanese-Brazilians, a population with a high prevalence of type 2 diabetes, common TCF7L2 variants did not make major contributions to the incidence of glucose tolerance abnormalities.

Normal skin and hypertrophic scar fibroblasts differentially regulate collagen and fibronectin expression as well as mitochondrial membrane potential in response to basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) regulates skin wound healing; however, the underlying mechanism remains to be defined. In the present study, we determined the effects of bFGF on the regulation of cell growth as well as collagen and fibronectin expression in fibroblasts from normal human skin and from hypertrophic scars. We then explored the involvement of mitochondria in mediating bFGF-inducedeffects on the fibroblasts. We isolated and cultivated normal and hypertrophic scar fibroblasts from tissue biopsies of patients who underwent plastic surgery for repairing hypertrophic scars. The fibroblasts were then treated with different concentrations of bFGF (ranging from 0.1 to 1000 ng/mL). The growth of hypertrophic scar fibroblasts became slower with selective inhibition of type I collagen production after exposure to bFGF. However, type III collagen expression was affected in both normal and hypertrophic scar fibroblasts. Moreover, fibronectin expression in the normal fibroblasts was up-regulated after bFGF treatment. bFGF (1000 ng/mL) also induced mitochondrial depolarization in hypertrophic scar fibroblasts (P < 0.01). The cellular ATP level decreased in hypertrophic scar fibroblasts (P < 0.05), while it increased in the normal fibroblasts following treatment with bFGF (P < 0.01). These data suggest that bFGF has differential effects and mechanisms on fibroblasts of the normal skin and hypertrophic scars, indicating that bFGF may play a role in the early phase of skin wound healing and post-burn scar formation.

Plasma von Willebrand factor as a predictor of survival in pulmonary arterial hypertension associated with congenital heart disease

Biomarkers have been identified for pulmonary arterial hypertension, but are less well defined for specific etiologies such as congenital heart disease-associated pulmonary arterial hypertension (CHDPAH). We measured plasma levels of eight microvascular dysfunction markers in CHDPAH, and tested for associations with survival. A cohort of 46 inoperable CHDPAH patients (age 15.0 to 60.2 years, median 33.5 years, female:male 29:17) was prospectively followed for 0.7 to 4.0 years (median 3.6 years). Plasma levels of von Willebrand factor antigen (VWF:Ag), tissue plasminogen activator (t-PA) and its inhibitor (PAI-1), P-selectin, reactive C-protein, tumor necrosis factor alpha, and interleukin-6 and -10 were measured at baseline, and at 30, 90, and 180 days in all subjects. Levels of six of the eight proteins were significantly increased in patients versus controls (13 to 106% increase, P < 0.003). Interleukin-10 level was 2.06 times normal (P = 0.0003; Th2 cytokine response). Increased levels of four proteins (t-PA, PAI-1, P-selectin, and interleukin-6) correlated with disease severity indices (P < 0.05). Seven patients died during follow-up. An average VWF:Ag (mean of four determinations) above the level corresponding to the 95th percentile of controls (139 U/dL) was independently associated with a high risk of death (hazard ratio = 6.56, 95%CI = 1.46 to 29.4, P = 0.014). Thus, in CHDPAH, microvascular dysfunction appears to involve Th2 inflammatory response. Of the biomarkers studied, plasma vWF:Ag was independently associated with survival.

Contrasting effects of nitric oxide and corticotropin- releasing factor within the dorsal periaqueductal gray on defensive behavior and nociception in mice

The anxiogenic and antinociceptive effects produced by glutamate N-methyl-D-aspartate receptor activation within the dorsal periaqueductal gray (dPAG) matter have been related to nitric oxide (NO) production, since injection of NO synthase (NOS) inhibitors reverses these effects. dPAG corticotropin-releasing factor receptor (CRFr) activation also induces anxiety-like behavior and antinociception, which, in turn, are selectively blocked by local infusion of the CRF type 1 receptor (CRFr1) antagonist, NBI 27914 [5-chloro-4-(N-(cyclopropyl)methyl-N-propylamino)-2-methyl-6-(2,4,6-trichlorophenyl)aminopyridine]. Here, we determined whether i) the blockade of the dPAG by CRFr1 attenuates the anxiogenic/antinociceptive effects induced by local infusion of the NO donor, NOC-9 [6-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine], and ii) the anxiogenic/antinociceptive effects induced by intra-dPAG CRF are prevented by local infusion of Nω-propyl-L-arginine (NPLA), a neuronal NOS inhibitor, in mice. Male Swiss mice (12 weeks old, 25-35 g, N = 8-14/group) were stereotaxically implanted with a 7-mm cannula aimed at the dPAG. Intra-dPAG NOC-9 (75 nmol) produced defensive-like behavior (jumping and running) and antinociception (assessed by the formalin test). Both effects were reversed by prior local infusion of NBI 27914 (2 nmol). Conversely, intra-dPAG NPLA (0.4 nmol) did not modify the anxiogenic/antinociceptive effects of CRF (150 pmol). These results suggest that CRFr1 plays an important role in the defensive behavior and antinociception produced by NO within the dPAG. In contrast, the anxiogenic and antinociceptive effects produced by intra-dPAG CRF are not related to NO synthesis in this limbic midbrain structure.

Reversal of the anticoagulant and anti-hemostatic effect of low molecular weight heparin by direct prothrombin activation

Lopap, found in the bristles of Lonomia obliqua caterpillar, is the first exogenous prothrombin activator that shows serine protease-like activity, independent of prothrombinase components and unique lipocalin reported to interfere with hemostasis mechanisms. To assess the action of an exogenous prothrombin activator reversing the anticoagulant and antihemostatic effect induced by low molecular weight heparin (LMWH), male New Zealand rabbits (N = 20, weighing 3.8-4.0 kg) allocated to 4 groups were anticoagulated with 1800 IU/kg LMWH (iv) over 2 min, followed by iv administration of saline (SG) or recombinant Lopap (rLopap) at 1 µg/kg (LG1) or 10 µg/kg (LG10), 10 min after the injection of LMWH, in a blind manner. Control animals (CG) were treated only with saline. The action of rLopap was assessed in terms of activated partial thromboplastin time (aPTT), prothrombin fragment F1+2, fibrinogen, and ear puncture bleeding time (BT) at 5, 10, 15, 17, 20, 30, 40, 60, and 90 min after initiation of LMWH infusion. LG10 animals showed a decrease of aPTT in more than 50% and BT near to normal baseline. The level of prothrombin fragment F1+2 measured by ELISA had a 6-fold increase with rLopap treatment (10 µg/kg) and was inversely proportional to BT in LMWH-treated animals. Thus, Lopap, obtained in recombinant form using E. coli expression system, was useful in antagonizing the effect of LMWH through direct prothrombin activation, which can be a possible strategy for the reversal of bleeding and anticoagulant events.

Exaggerated blood pressure response during the exercise treadmill test as a risk factor for hypertension

Exaggerated blood pressure response (EBPR) during the exercise treadmill test (ETT) has been considered to be a risk factor for hypertension. The relationship of polymorphisms of the renin-angiotensin system gene with hypertension has not been established. Our objective was to evaluate whether EBPR during exercise is a clinical marker for hypertension. The study concerned a historical cohort of normotensive individuals. The exposed individuals were those who presented EBPR. At the end of the observation period (41.7 months = 3.5 years), the development of hypertension was analyzed within the two groups. Genetic polymorphisms and blood pressure behavior were assessed as independent variables, together with the classical risk factors for hypertension. The I/D gene polymorphism of the angiotensin-converting enzyme and M235T of angiotensinogen were ruled out as risk factors for hypertension. EBPR during ETT is not an independent influence on the chances of developing hypertension. No differences were observed between the hypertensive and normotensive individuals regarding gender (P = 0.655), skin color (P = 0.636), family history of hypertension (P = 0.225), diabetes mellitus (P = 0.285), or hypertriglyceridemia (P = 0.734). The risk of developing hypertension increased with increasing body mass index (BMI) and advancing age. The risk factors, which independently influenced the development of hypertension, were age and BMI. EBPR did not constitute an independent risk factor for hypertension and is probably a preclinical phase in the spectrum of normotension and hypertension.

The epigenetic modifiers 5-aza-2'-deoxycytidine and trichostatin A influence adipocyte differentiation in human mesenchymal stem cells

Epigenetic mechanisms such as DNA methylation and histone modification are important in stem cell differentiation. Methylation is principally associated with transcriptional repression, and histone acetylation is correlated with an active chromatin state. We determined the effects of these epigenetic mechanisms on adipocyte differentiation in mesenchymal stem cells (MSCs) derived from bone marrow (BM-MSCs) and adipose tissue (ADSCs) using the chromatin-modifying agents trichostatin A (TSA), a histone deacetylase inhibitor, and 5-aza-2′-deoxycytidine (5azadC), a demethylating agent. Subconfluent MSC cultures were treated with 5, 50, or 500 nM TSA or with 1, 10, or 100 µM 5azadC for 2 days before the initiation of adipogenesis. The differentiation was quantified and expression of the adipocyte genes PPARG and FABP4 and of the anti-adipocyte gene GATA2 was evaluated. TSA decreased adipogenesis, except in BM-MSCs treated with 5 nM TSA. Only treatment with 500 nM TSA decreased cell proliferation. 5azadC treatment decreased proliferation and adipocyte differentiation in all conditions evaluated, resulting in the downregulation of PPARG and FABP4 and the upregulation of GATA2. The response to treatment was stronger in ADSCs than in BM-MSCs, suggesting that epigenetic memories may differ between cells of different origins. As epigenetic signatures affect differentiation, it should be possible to direct the use of MSCs in cell therapies to improve process efficiency by considering the various sources available.

Relationship of macrophage migration inhibitory factor levels in PBMCs, lesional skin and serum with disease severity and activity in vitiligo vulgaris

Melanocyte loss in vitiligo vulgaris is believed to be an autoimmune process. Macrophage migration inhibitory factor (MIF) is involved in many autoimmune skin diseases. We determined the possible role of MIF in the pathogenesis of vitiligo vulgaris, and describe the relationship between MIF expressions and disease severity and activity. Serum MIF concentrations and mRNA levels in PBMCs were measured in 44 vitiligo vulgaris patients and 32 normal controls, using ELISA and real-time RT-PCR. Skin biopsies from 15 patients and 6 controls were analyzed by real-time RT-PCR. Values are reported as median (25th-75th percentile). Serum MIF concentrations were significantly increased in patients [35.81 (10.98-43.66) ng/mL] compared to controls [7.69 (6.01-9.03) ng/mL]. MIF mRNA levels were significantly higher in PBMCs from patients [7.17 (3.59-8.87)] than controls [1.67 (1.23-2.42)]. There was also a significant difference in MIF mRNA levels in PBMCs between progressive and stable patients [7.86 (5.85-9.13)vs 4.33 (2.23-8.39)] and in serum MIF concentrations [40.47 (27.71-46.79) vs 26.80 (10.55-36.07) ng/mL]. In addition, the vitiligo area severity index scores of patients correlated positively with changes of both serum MIF concentrations (r = 0.488) and MIF mRNA levels in PBMCs (r = 0.426). MIF mRNA levels were significantly higher in lesional than in normal skin [2.43 (2.13-7.59)vs 1.18 (0.94-1.83)] and in patients in the progressive stage than in the stable stage [7.52 (2.43-8.84)vs 2.13 (1.98-2.64)]. These correlations suggest that MIF participates in the pathogenesis of vitiligo vulgaris and may be useful as an index of disease severity and activity.

Elevated levels of plasma osteoprotegerin are associated with all-cause mortality risk and atherosclerosis in patients with stages 3 to 5 chronic kidney disease

Osteoprotegerin (OPG) regulates bone mass by inhibiting osteoclast differentiation and activation, and plays a role in vascular calcification. We evaluated the relationship between osteoprotegerin levels and inflammatory markers, atherosclerosis, and mortality in patients with stages 3-5 chronic kidney disease. A total of 145 subjects (median age 61 years, 61% men; 36 patients on hemodialysis, 55 patients on peritoneal dialysis, and 54 patients with stages 3-5 chronic kidney disease) were studied. Clinical characteristics, markers of mineral metabolism (including fibroblast growth factor-23 [FGF-23]) and inflammation (high-sensitivity C-reactive protein [hsCRP] and interleukin-6 [IL-6]), and the intima-media thickness (IMT) in the common carotid arteries were measured at baseline. Cardiac function was assessed by color tissue Doppler echocardiography. After 36 months follow-up, the survival rate by Kaplan-Meier analysis was significantly different according to OPG levels (χ2=14.33; P=0.002). Increased OPG levels were positively associated with IL-6 (r=0.38, P<0.001), FGF-23 (r=0.26, P<0.001) and hsCRP (r=0.0.24, P=0.003). In addition, OPG was positively associated with troponin I (r=0.54, P<0.001) and IMT (r=0.39, P<0.0001). Finally, in Cox analysis, only OPG (HR=1.07, 95%CI=1.02-1.13) and hsCRP (HR=1.02, 95%CI=1.01-1.04) were independently associated with increased risk of death. These results suggested that elevated levels of serum OPG might be associated with atherosclerosis and all-cause mortality in patients with chronic kidney disease.

Promoting inflammatory lymphangiogenesis by vascular endothelial growth factor-C (VEGF-C) aggravated intestinal inflammation in mice with experimental acute colitis

Angiogenesis and lymphangiogenesis are thought to play a role in the pathogenesis of inflammatory bowel diseases (IBD). However, it is not understood if inflammatory lymphangiogenesis is a pathological consequence or a productive attempt to resolve the inflammation. This study investigated the effect of lymphangiogenesis on intestinal inflammation by overexpressing a lymphangiogenesis factor, vascular endothelial growth factor-C (VEGF-C), in a mouse model of acute colitis. Forty eight-week-old female C57BL/6 mice were treated with recombinant adenovirus overexpressing VEGF-C or with recombinant VEGF-C156S protein. Acute colitis was then established by exposing the mice to 5% dextran sodium sulfate (DSS) for 7 days. Mice were evaluated for disease activity index (DAI), colonic inflammatory changes, colon edema, microvessel density, lymphatic vessel density (LVD), and VEGFR-3mRNA expression in colon tissue. When acute colitis was induced in mice overexpressing VEGF-C, there was a significant increase in colonic epithelial damage, inflammatory edema, microvessel density, and neutrophil infiltration compared to control mice. These mice also exhibited increased lymphatic vessel density (73.0±3.9 vs 38.2±1.9, P<0.001) and lymphatic vessel size (1974.6±104.3 vs 1639.0±91.5, P<0.001) compared to control mice. Additionally, the expression of VEGFR-3 mRNA was significantly upregulated in VEGF-C156S mice compared to DSS-treated mice after induction of colitis (42.0±1.4 vs 3.5±0.4, P<0.001). Stimulation of lymphangiogenesis by VEGF-C during acute colitis promoted inflammatory lymphangiogenesis in the colon and aggravated intestinal inflammation. Inflammatory lymphangiogenesis may have pleiotropic effects at different stages of IBD.