The effect of filling technique on the cuspal strain, polymerization shrinkage stress, enamel crack formation and depth of cure of restored molars.

OBJECTIVE: Evaluate the effect of different restorative filling techniques on the residual shrinkage stress (ShrS), cuspal strain (CS), depth of cure (DC), and enamel crack formation (Ec) in molars with MOD restorations. METHODS: Post-gel shrinkage, elastic modulus, compressive and diametral tensile strength of the Filtek One Bulk Fill composite were calculated. Sixty molars with MOD preparations were restored using four filling techniques: Bulk; Horizontal; Oblique; Natural enamel and dentin substitution (NEDS) technique. CS was measured using a strain gauge (n = 10). The DC (n = 5) was measured using Knoop hardness. Shrinkage stress/strain was analyzed using 3D finite element analysis. The Ec analysis was carried out by transillumination. Two-way ANOVA with repeated measures and Tukey's HSD test (α = 0.05) was performed for the CS data. Two-Way ANOVA and Tukey's HSD test was performed for the DC data (α = 0.05). RESULTS: CS was higher at the lingual cusp for the horizontal and NEDS technique. No statistical difference was found between the buccal and lingual CS values for the Bulk (p = 0.367) or Oblique techniques (p = 0.192). CS values were lower for the Bulk. More enamel cracks were found for the Bulk. DC was lower at 4 mm regardless the filling technique. The Horizontal showed the highest ShrS values. The Bulk generated the lower ShrS values. SIGNIFICANCE: A Bulk technique caused the lowest shrinkage stress/strain. An Oblique technique yielded the best balance between stress, strain and crack formation. NEDS technique is a good alternative to decrease the number of increments while maintaining the stress levels nearby the Oblique technique.

Evaluation of α5ß1 integrin as a candidate marker for fertility in bull sperm samples.

With the progressive increase in the use of reproductive biotechnologies in the cattle industry, like artificial insemination and in vitro embryo production, the accurate determination of fertilizing competence of cryopreserved sperm samples is an essential issue. The routine methodology to assess bull sperm quality relies primarily on count, viability and motility of spermatozoa. However, these parameters do not tightly predict the reproductive success of samples. Therefore, identification of complementary markers of sperm functionality to strengthen the predictability of traditional spermogram is desirable to improve livestock reproduction practices. Previous results from our laboratory indicated that α5ß1 integrin plays a key role in bovine sperm function and mediates their interaction with the female reproductive tract. Thus, this study aimed to investigate whether the localization of α5ß1 held a correlation with fertilizing ability of bovine cryopreserved semen samples. Firstly, we assessed the quality of samples from six different bulls (A-F). We determined motility and viability of sperm samples after thawing and selection. Additionally, we measured the capacitation state of the samples by chlortetracycline (CTC) assay in the presence or absence of heparin, as an indicator of their responsiveness to a capacitating stimulus. Based on these assays, samples were classified being A the bull with the lowest quality and F the bull with the highest quality. Then, we studied the presence and localization of α5ß1 integrin. This protein showed a distribution pattern in the acrosomal (A), post-acrosomal (P) and acrosomal + post-acrosomal (A + P) regions with different localization percentages among the studied samples. Next, we determined the fertilizing ability of the samples in in vitro fertilization (IVF) assays and performed correlation analyses between IVF outcome and the routine spermogram parameters or α5ß1 integrin localization patterns. When the percentage of cells showing α5ß1 integrin was compared to fertilization rate, no correlation was observed. However, the presence of α5ß1 integrin in P and A + P regions (PA pattern), positively correlated with IVF rate (p < 0.05). These results suggest that while routine semen analyses failed to predict sperm reproductive competence, integrin localization in post-acrosomal region (PA pattern) showed a positive correlation with IVF outcome, thus posing an attractive marker to predict more accurately the reproductive performance of an individual.

Steroid hormones and first trimester vascular remodeling.

Successful implantation and placentation require neoangiogenesis and the remodeling of the uterine spiral arteries. Progesterone and estradiol control various of the placental functions, but their role in vascular remodeling remains controversial. Therefore, this chapter aims to summarize the current knowledge regarding the role of steroid hormones in the uteroplacental vascular remodeling during the first trimester of gestation.

Fibronectin induces capacitation-associated events through the endocannabinoid system in bull sperm.

Mammalian ejaculated spermatozoa must undergo a series of changes in the female reproductive tract, collectively called capacitation, in order to fertilize the oocyte. We reported that fibronectin (Fn), a glycoprotein from the extracellular matrix, and anandamide (AEA), one of the major members of the endocannabinoid family, are present in the bovine oviductal fluid and regulate bull sperm function. Also, AEA induces bovine sperm capacitation, through CB1 and TRPV1 receptors. In this work, we investigated if Fn induces bovine sperm capacitation thought the activation of the endocannabinoid system in this process. We incubated sperm with Fn (100 µg/ml) and/or capsazepine, a TRPV1 antagonist (0.1 µM) and some events related to sperm capacitation such as LPC-induced acrosome reaction, sperm-release from the oviduct, induction of PKA phosphorylated substrates (pPKAs) and protein tyrosine phosphorylation (pY) and nitric oxide (NO) production were assessed. Also, we studied the activity of fatty acid amide hydrolase (FAAH), the enzyme that degrades AEA. We found that Fn, via α5ß1 integrin, induced capacitation-associated events. Also, Fn stimulated signaling pathways associated to capacitation as cAMP/PKA and NO/NO synthase. Moreover, Fn decreased the FAAH activity and this correlated with sperm capacitation. Capsazepine reversed fibronectin-induced capacitation, and pPKAs and NO levels. The incubation of spermatozoa with R-methanandamide (1.4 nM), a stable analogue of AEA, increased cAMP and pPKAs levels. The presence of H89 (50 µM) or KT5720 (100 nM) (PKA inhibitors) prevented AEA-induced capacitation. In addition, R-methanandamide and capsaicin (0.01 µM), a TRPV1 agonist, increased NO production via the PKA pathway. These results indicate that Fn, through α5ß1, supports capacitation in bovine spermatozoa. This effect is dependent on the activation of TRPV1 through cAMP/PKA and NO signaling pathways. We propose that Fn could be considered as a new agent that promotes sperm capacitation in bull sperm. Our findings contribute to better understand the significance of Fn signaling in the capacitating events that lead to successful fertilization and embryo development in mammals including humans.

Artificial sweetener saccharin disrupts intestinal epithelial cells' barrier function in vitro.

SCOPE: Consumption of non-nutritive sweeteners (NNS) is a dietary practice used by those who wish to lose weight or by patients on a sugar-restricted diet such as those with DM2. Although these substances are safe, possible biological interactions with the digestive tract, particularly in relation to intestinal permeability, have not been studied. Thus, the current work sought to investigate the action of different NNS on intestinal permeability using an in vitro Caco-2 cell model. METHODS AND RESULTS: Caco-2 cells were incubated with acesulfame K, aspartame, saccharin, or sucralose at equimolar concentrations. Acesulfame K, aspartame, and sucralose did not disrupt monolayer integrity in the cells. However, saccharin increased paracellular permeability and decreased transepithelial electrical resistance (TEER) via a non-cytotoxic mechanism. The levels of the tight junction protein claudin-1 were reduced in Caco-2 cells that had previously been exposed to saccharin. The inhibition of nuclear factor-κB (NF-κB) was able to prevent the reduction in TEER induced by saccharin treatment. Thalidomide, as an inhibitor of ubiquitin ligase, was able to prevent the decrease in claudin-1 protein expression and the TEER reduction in Caco-2 cells. CONCLUSIONS: Saccharin disrupts monolayer integrity and alters paracellular permeability in a Caco-2 cell monolayer model, via a mechanism involving NF-κB activation, resulting in the ubiquitination of the tight junction protein claudin-1. Saccharin consumption may potentially alter the intestinal integrity in humans.

Molecular basis of pyruvate kinase deficiency among Tunisians: description of new mutations affecting coding and noncoding regions in the PKLR gene.

INTRODUCTION: Pyruvate kinase (PK) deficiency is one of the most common hereditary nonspherocytic hemolytic anemias worldwide with clinical manifestations ranging from mild to severe hemolysis. However, investigation of this enzymopathy is lacking in Tunisia. We report here a pioneer investigation of PK deficiency among Tunisian cases referred to our laboratory for biological analysis of unknown cause of hemolytic anemia. METHODS: Two hundred and fifty-three patients with unknown cause of hemolytic anemia have been addressed to our laboratory in order to investigate for red blood cells genetic disorders. Red cell enzyme activities were measured by standard methods, and molecular analysis was performed by DNA sequencing. The interpretation of mutation effect and the molecular modeling were performed by using specific software. RESULTS: Six different PKLR mutations were found (c.966-1G>T; c.965+1G>A; c.721G>T; c.1163C>A; c.1456C>T; c.1537T>A), among which four are described for the first time. Genotype-phenotype correlations for the novel missense mutations were investigated by three-dimensional structure analysis. CONCLUSION: This study provides important data of PK deficiency among Tunisians. It might be followed by a large neonatal screening to determine the spectrum of PK mutations and identify potential deficient patients for an early medical follow-up.

Detection of new pathogenic mutations in patients with congenital haemolytic anaemia using next-generation sequencing.

INTRODUCTION: Congenital haemolytic anaemia (CHA) refers to a group of genetically heterogeneous disorders, mainly caused by changes in genes encoding globin chains, cytoskeletal proteins and red cell enzymes, in which accurate diagnosis can be challenging with conventional techniques. METHODS: To set-up a comprehensive assay for detecting mutations that could improve aetiological diagnosis, we designed a custom panel for sequencing coding regions from 40 genes known to be involved in the pathogenesis of CHA, using the Ion Torrent™ (Thermo Fisher Scientific, S.L. Waltham, MA, USA) Personal Genome Machine (PGM) Sequencer. A control group of 16 samples with previously known mutations and a test group of 10 patients with unknown mutations were included for assay validation and application, respectively. RESULTS: In the test group, we identified pathogenic mutations in all cases: four patients had novel mutations in genes related to membrane defects (SPTB, ANK1, SLC4A1 and EPB41), four were homozygous or compound heterozygous for mutations in genes related to enzyme deficiencies (GPI, TPI1 and GSS), one had a mutation in the HBB gene and another presented a homozygous mutation in the ADAMTS13 gene. CONCLUSIONS: Ion PGM sequencing with our custom panel is a highly efficient way to detect mutations causing haemolytic anaemia, including new variations. It is a high-throughput detection method that is ready for application in clinical laboratories.

Lysophosphatidic acid increases the production of pivotal mediators of decidualization and vascularization in the rat uterus.

INTRODUCTION: The decidual reaction and the formation of new vessels in the uterus are two crucial processes during embryo implantation. Previously, we observed that lysophosphatidic acid (LPA) increases cyclooxygenase-2 derived - prostaglandin E2 production during implantation in the rat uterus and that it augments the expression of decidualization (IGFBP-1) and vascularization (IL-10) markers. Both cyclooxygenase and nitric oxide synthase (NOS) are known enzymes involved in these processes. Thus, we became interested in studying which factors contribute to LPA receptor-specific role during the decidual and the vascular reaction at implantation. METHODS: We adopted a pharmacological approach in vitro incubating the uterus from rats on day 5 of gestation (day of implantation) with LPA, DGPP (a highly selective antagonist of LPA3, an LPA receptor) and cyclooxygenase and NOS selective and non-selective inhibitors. We determined NOS activity, prostaglandin E2 production and IGFBP-1 and IL-10 expression to evaluate decidualization and vascularization. RESULTS: We observed that LPA augmented the activity of the inducible NOS isoform through LPA1/LPA3. Inducible NOS activity participated in the induction of cyclooxygenase-2/prostaglandin E2 increase stimulated by LPA. Also, cyclooxygenase-2 derived prostaglandins mediated LPA-stimulatory action on NOS activity. Both cyclooxygenase-2 and inducible NOS mediated LPA effect on IGFBP-1 and IL-10 expression. CONCLUSIONS: These results suggest the participation of LPA/LPA3 in the production of crucial molecules involved in vascularization and decidualization, two main processes that prepare the uterine milieu for embryo invasion during implantation.

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 χ² 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.

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-1ß, 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-1ß, 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 χ² test or the Fisher exact test. Our results demonstrated that the IL-1ß -511 C/C and IL-1ß -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-1ß -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-1ß -511 C/C and C/T gene polymorphisms are associated with chronic gastritis and GC development in H. pylori-infected individuals.

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.

Evaluation of miltefosine for the treatment of dogs naturally infected with L. infantum (=L. chagasi) in Brazil.

Dogs naturally infected with Leishmania Infantum (=L. chagasi) were treated with miltefosine using different therapeutic regimens. The animals were evaluated for clinical evolution, biochemical parameters, parasite load (by real-time PCR), cytokine levels and humoral response. After treatment and during the following 24 months, there was progressive clinical improvement and complete recovery in 50% (7/14) of the treated animals. There was a decrease in the smear positivity of the bone marrow after treatment, and there was also a gradual and constant decrease in positive cultures at the end of the follow-up period. However, the PCR detection of parasite DNA remained positive. In general, all animals presented a significant increase in parasite load 6 months after treatment. The IFN-γ levels in all the groups tended to increase during follow-up period, regardless of the miltefosine dose administered. The IL-4 and IL-10 levels of the animals tended to decrease during follow-up, except after 300 days when only IL-10 increased. The serum antibodies identified antigens that ranged from 116 kDa to less than 29 kDa in the Western blot assay. Furthermore, 300 days after treatment, qualitative and quantitative differences in the antigen profiles were observed. Antigens of 97 and 46 kDa were the most intensely recognized. Higher levels of antigen-specific Leishmania IgG were detected before and 300 days after treatment in all groups. Taking together, the improvement in the clinical symptoms was not followed by parasitological clearance, suggesting that treatment with miltefosine is not recommended, especially in endemic areas like Brazil, where children are the major victims and dogs are involved in the maintenance of the parasite cycle.

Kawasaki disease and human bocavirus--potential association?

Kawasaki disease (KD) is an acute febrile multisystem vasculitic syndrome of unknown etiology, occurring mostly in infants and children younger than 5 years of age. We present a 13-month-old male with KD from whom was found human bocavirus DNA in nasopharyngeal secretions. Human bocavirus DNA in a patient with KD raised question about the coincidental or possible etiological association.

Dual effect of nitric oxide on uterine prostaglandin synthesis in a murine model of preterm labour.

BACKGROUND AND PURPOSE: Maternal infections are one of the main causes of adverse developmental outcomes including embryonic resorption and preterm labour. In this study a mouse model of inflammation-associated preterm delivery was developed, and used to study the relationship between nitric oxide (NO) and prostaglandins (PGs). EXPERIMENTAL APPROACH: The murine model of preterm labour was achieved by assaying different doses of bacterial lipopolysaccharides (LPS). Once established, it was used to analyse uterine levels of prostaglandins E(2) and F(2α) (by radioimmunoassay), cyclooxygenases (COX) and NOS proteins (by Western blot) and NO synthase (NOS) activity. Effects of inhibitors of COX and NOS on LPS-induced preterm labour were also studied. In vitro assays with a nitric oxide donor (SNAP) were performed to analyse the modulation of prostaglandin production by NO. KEY RESULTS: Lipopolysaccharide increased uterine NO and PG synthesis and induced preterm delivery. Co-administration of meloxicam, a cyclooxygenase-2 inhibitor, or aminoguanidine, an inducible NOS inhibitor, prevented LPS-induced preterm delivery and blocked the increase in PGs and NO. Notably, the levels of NO were found to determine its effect on PG synthesis; low concentrations of NO reduced PG synthesis whereas high concentrations augmented them. CONCLUSIONS AND IMPLICATIONS: An infection-associated model of preterm labour showed that preterm delivery can be prevented by decreasing PG or NO production. NO was found to have a dual effect on PG synthesis depending on its concentration. These data contribute to the understanding of the interaction between NO and PGs in pregnancy and parturition, and could help to improve neonatal outcomes.

Real-time PCR in clinical practice: a powerful tool for evaluating Leishmania chagasi loads in naturally infected dogs.

The performance of the less expensive SYBR-Green-based PCR assay, for quantifying Leishmania chagasi in smears of bone-marrow aspirates from naturally infected, mongrel dogs, was recently compared with that of a similar PCR based on TaqMan chemistry. Aspirates were obtained from 36 infected dogs and examined for parasites by direct examination, culture, and quantitative PCR (qPCR) using specific primers (based on the parasite's kinetoplast DNA), DNA extracted from a smear, and either the SYBR-Green or TaqMan chemistries. Every aspirate smear was found PCR-positive for L. chagasi (whether the assay employed SYBR Green or TaqMan) but only 74% of the aspirates were found positive by culture and only 33% by direct, microscopical examination. There was no evidence of PCR inhibition when the DNA was collected from smears, and the parasite loads estimated using the SYBR-Green PCR were almost identical to those estimated using the TaqMan PCR (r=0.99). As a method for quantifying parasite loads in dogs infected with L. chagasi (and, probably, other mammals infected with other leishmanial parasites), PCR based on SYBR Green may therefore be an appropriate and inexpensive alternative to PCR based on TaqMan, and a reliable clinical tool.

Nitric oxide synthase 1 and cyclooxygenase-2 enzymes are targets of muscarinic activation in normal and inflamed NIH3T3 cells.

OBJECTIVE: Fibroblasts are sentinel cells that could serve as intermediaries in the immune reaction in the inflammatory process. In this work, we investigate the action of the muscarinic agonist carbachol (CARB) on the expression and function of nitric oxide synthase (NOS) and cyclooxygenase (COX) in fibroblasts under normal or inflammatory conditions. METHODS: The normal fibroblast cell line, 3T3, from NIH swiss mouse embryo, was used. The inflammatory milieu was mimicked with lipopolysaccharide (LPS) (10 ng/ml) plus interferon gamma (IFNgamma) (0.5 ng/ml). Nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production were measured by Griess reagent and radioimmunoassay, respectively. NOS, COX, and nuclear transcription factor kappa B (NF-kappaB) were studied by Western blot. RESULTS: CARB increased NO synthesis by 57 +/- 5%, while a 150 +/- 10% increase in NO liberation was triggered by LPS plus IFNgamma treatment. CARB added to LPS plus IFNgamma potentiated NO synthesis by 227 +/- 19%. CARB also upregulated NOS1 protein expression via NF-kappaB activation. In addition CARB and LPS plus IFNgamma stimulated PGE(2) synthesis by 72 +/- 9 and 42 +/- 4%, respectively, while CARB added to LPS plus IFNgamma treated cells produced a synergism in PGE(2) liberation (130 +/- 12%) via COX-2. CONCLUSION: Activation of muscarinic acetylcholine receptors can mimic mild inflammatory conditions or can deepen pre-existing inflammation, establishing a fine-tuned set-up on fibroblasts that in turn could be alerting the immune system.