Prognostic value of TP53 Pro47Ser and Arg72Pro single nucleotide polymorphisms and the susceptibility to gliomas in individuals from Southeast Brazil

The TP53 tumor suppressor gene codifies a protein responsible for preventing cells with genetic damage from growing and dividing by blocking cell growth or apoptosis pathways. A common single nucleotide polymorphism (SNP) in TP53 codon 72 (Arg72Pro) induces a 15-fold decrease of apoptosis-inducing ability and has been associated with susceptibility to human cancers. Recently, another TP53 SNP at codon 47 (Pro47Ser) was reported to have a low apoptosis-inducing ability; however, there are no association studies between this SNP and cancer. Aiming to study the role of TP53 Pro47Ser and Arg72Pro on glioma susceptibility and oncologic prognosis of patients, we investigated the genotype distribution of these SNPs in 94 gliomas (81 astrocytomas, 8 ependymomas and 5 oligodendrogliomas) and in 100 healthy subjects by the polymerase chain reaction-restriction fragment length polymorphism approach. Chi-square and Fisher exact test comparisons for genotype distributions and allele frequencies did not reveal any significant difference between patients and control groups. Overall and disease-free survivals were calculated by the Kaplan-Meier method, and the log-rank test was used for comparisons, but no significant statistical difference was observed between the two groups. Our data suggest that TP53 Pro47Ser and Arg72Pro SNPs are not involved either in susceptibility to developing gliomas or in patient survival, at least in the Brazilian population.

Mutational analysis of genes p14ARF, p15INK4b, p16INK4a, and PTEN in human nervous system tumors

The cancer is one of the most common and severe problems in clinical medicine, and nervous system tumors represent about 2% of the types of cancer. The central role of the nervous system in the maintenance of vital activities and the functional consequences of the loss of neurons can explain how severe brain cancers are. The cell cycle is a highly complex process, with a wide number of regulatory proteins involved, and such proteins can suffer alterations that transform normal cells into malignant ones. The INK4 family members (CDK inhibitors) are the cell cycle regulators that block the progression of the cycle through the R point, causing an arrest in G1 stage. The p14ARF (alternative reading frame) gene is a tumor suppressor that inhibits p53 degradation during the progression of the cell cycle. The PTEN gene is related to the induction of growth suppression through cell cycle arrest, to apoptosis and to the inhibition of cell adhesion and migration. The purpose of the present study was to assess the mutational state of the genes p14ARF, p15INK4b, p16INK4a, and PTEN in 64 human nervous system tumor samples. Homozygous deletions were found in exon 2 of the p15INK4b gene and exon 3 of the p16INK4a gene in two schwannomas. Three samples showed a guanine deletion (63 codon) which led to a loss of heterozygosity in the p15 gene, and no alterations could be seen in the PTEN gene. Although the group of patients was heterogeneous, our results are in accordance with other different studies that indicate that homozygous deletion and loss of heterozygosity in the INK4 family members are frequently observed in nervous system tumors.

Ultra-Deep Sequencing Reveals the microRNA Expression Pattern of the Human Stomach

Background: While microRNAs (miRNAs) play important roles in tissue differentiation and in maintaining basal physiology, little is known about the miRNA expression levels in stomach tissue. Alterations in the miRNA profile can lead to cell deregulation, which can induce neoplasia. Methodology/Principal Findings: A small RNA library of stomach tissue was sequenced using high-throughput SOLiD sequencing technology. We obtained 261,274 quality reads with perfect matches to the human miRnome, and 42% of known miRNAs were identified. Digital Gene Expression profiling (DGE) was performed based on read abundance and showed that fifteen miRNAs were highly expressed in gastric tissue. Subsequently, the expression of these miRNAs was validated in 10 healthy individuals by RT-PCR showed a significant correlation of 83.97% (P<0.05). Six miRNAs showed a low variable pattern of expression (miR-29b, miR-29c, miR-19b, miR-31, miR-148a, miR-451) and could be considered part of the expression pattern of the healthy gastric tissue. Conclusions/Significance: This study aimed to validate normal miRNA profiles of human gastric tissue to establish a reference profile for healthy individuals. Determining the regulatory processes acting in the stomach will be important in the fight against gastric cancer, which is the second-leading cause of cancer mortality worldwide.

Cost-effectiveness analysis of PCR for the rapid diagnosis of pulmonary tuberculosis

Background: Tuberculosis is one of the most prominent health problems in the world, causing 1.75 million deaths each year. Rapid clinical diagnosis is important in patients who have comorbidities such as Human Immunodeficiency Virus (HIV) infection. Direct microscopy has low sensitivity and culture takes 3 to 6 weeks [1-3]. Therefore, new tools for TB diagnosis are necessary, especially in health settings with a high prevalence of HIV/TB co-infection. Methods: In a public reference TB/HIV hospital in Brazil, we compared the cost-effectiveness of diagnostic strategies for diagnosis of pulmonary TB: Acid fast bacilli smear microscopy by Ziehl-Neelsen staining (AFB smear) plus culture and AFB smear plus colorimetric test (PCR dot-blot). From May 2003 to May 2004, sputum was collected consecutively from PTB suspects attending the Parthenon Reference Hospital. Sputum samples were examined by AFB smear, culture, and PCR dot-blot. The gold standard was a positive culture combined with the definition of clinical PTB. Cost analysis included health services and patient costs. Results: The AFB smear plus PCR dot-blot require the lowest laboratory investment for equipment (US$ 20,000). The total screening costs are 3.8 times for AFB smear plus culture versus for AFB smear plus PCR dot blot costs (US$ 5,635,760 versus US$ 1,498, 660). Costs per correctly diagnosed case were US$ 50,773 and US$ 13,749 for AFB smear plus culture and AFB smear plus PCR dot-blot, respectively. AFB smear plus PCR dot-blot was more cost-effective than AFB smear plus culture, when the cost of treating all correctly diagnosed cases was considered. The cost of returning patients, which are not treated due to a negative result, to the health service, was higher in AFB smear plus culture than for AFB smear plus PCR dot-blot, US$ 374,778,045 and US$ 110,849,055, respectively. Conclusion: AFB smear associated with PCR dot-blot associated has the potential to be a cost-effective tool in the fight against PTB for patients attended in the TB/HIV reference hospital.

Differential expression of E-cadherin gene in human neuroepithelial tumors

Cadherins are cell-to-cell adhesion molecules that play an important role in the establishment of adherent-type junctions by mediating calcium-dependent cellular interactions. The CDH1 gene encodes the transmembrane glycoprotein E-cadherin which is important in maintaining homophilic cell-cell adhesion in epithelial tissues. E-cadherin interacts with catenin proteins to maintain tissue architecture. Structural defects or loss of expression of E-cadherin have been reported as a common feature in several human cancer types. This study aimed to evaluate the expression of E-cadherin and their correlation with clinical features in microdissected brain tumor samples from 81 patients, divided into 62 astrocytic tumors grades I to IV and 19 medulloblastomas, and from 5 white matter non-neoplasic brain tissue samples. E-cadherin (CDH1) gene expression was analyzed by quantitative real-time polymerase chain reaction. Mann-Whitney, Kruskal-Wallis, Kaplan-Meir, and log-rank tests were performed for statistical analyses. We observed a decrease in expression among pathological grades of neuroepithelial tumors. Non-neoplasic brain tissue showed a higher expression level of CDH1 gene than did neuroepithelial tumors. Expression of E-cadherin gene was higher in astrocytic than embryonal tumors (P = 0.0168). Low-grade malignancy astrocytomas (grades I-II) showed higher CDH1 expression than did high-grade malignancy astrocytomas (grades III-IV) and medulloblastomas (P < 0.0001). Non-neoplasic brain tissue showed a higher expression level of CDH1 gene than grade I malignancy astrocytomas, considered as benign tumors (P = 0.0473). These results suggest that a decrease in E-cadherin gene expression level in high-grade neuroepithelial tumors may be a hallmark of malignancy in dedifferentiated tumors and that it may be possibly correlated with their progression and dissemination.

Research Smad2 and Smad6 as predictors of overall survival in oral squamous cell carcinoma patients

Background: To test if the expression of Smad1-8 mRNAs were predictive of survival in patients with oral squamous cell carcinoma (SCC). Patients and Methods: We analyzed, prospectively, the expression of Smad1-8, by means of Ribonuclease Protection Assay in 48 primary, operable, oral SCC. In addition, 21 larynx, 10 oropharynx and 4 hypopharynx SCC and 65 matched adjacent mucosa, available for study, were also included. For survival analysis, patients were categorized as positive or negative for each Smad, according to median mRNA expression. We also performed real-time quantitative PCR (QRTPCR) to asses the pattern of TGF beta 1, TGF beta 2, TGF beta 3 in oral SCC. Results: Our results showed that Smad2 and Smad6 mRNA expression were both associated with survival in Oral SCC patients. Cox Multivariate analysis revealed that Smad6 positivity and Smad2 negativity were both predictive of good prognosis for oral SCC patients, independent of lymph nodal status (P = 0.003 and P = 0.029, respectively). In addition, simultaneously Smad2(-) and Smad6(+) oral SCC group of patients did not reach median overall survival (mOS) whereas the mOS of Smad2(+)/Smad6(-) subgroup was 11.6 months (P = 0.004, univariate analysis). Regarding to TGF beta isoforms, we found that Smad2 mRNA and TGF beta 1 mRNA were inversely correlated (p = 0.05, R = -0.33), and that seven of the eight TGF beta 1(+) patients were Smad2(-). In larynx SCC, Smad7(-) patients did not reach mOS whereas mOS of Smad7(+) patients were only 7.0 months (P = 0.04). No other correlations were found among Smad expression, clinico-pathological characteristics and survival in oral, larynx, hypopharynx, oropharynx or the entire head and neck SCC population. Conclusion: Smad6 together with Smad2 may be prognostic factors, independent of nodal status in oral SCC after curative resection. The underlying mechanism which involves aberrant TGF beta signaling should be better clarified in the future.

Tumor slices as a model to evaluate doxorubicin in vitro treatment and expression of trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 in canine mammary gland cancer

Background: In women with breast cancer submitted to neoadjuvant chemotherapy based in doxorubicin, tumor expression of groups of three genes (PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2) have classified them as responsive or resistant. We have investigated whether expression of these trios of genes could predict mammary carcinoma response in dogs and whether tumor slices, which maintain epithelial-mesenchymal interactions, could be used to evaluate drug response in vitro. Methods: Tumors from 38 dogs were sliced and cultured with or without doxorubicin 1 mu M for 24 h. Tumor cells were counted by two observers to establish a percentage variation in cell number, between slices. Based on these results, a reduction in cell number between treated and control samples >= 21.7%, arbitrarily classified samples, as drug responsive. Tumor expression of PRSS11, MTSS1, CLPTM1 and SMYD2, was evaluated by real time PCR. Relative expression results were then transformed to their natural logarithm values, which were spatially disposed according to the expression of trios of genes, comprising PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. Fisher linear discrimination test was used to generate a separation plane between responsive and non-responsive tumors. Results: Culture of tumor slices for 24 h was feasible. Nine samples were considered responsive and 29 non-responsive to doxorubicin, considering the pre-established cut-off value of cell number reduction = 21.7%, between doxorubicin treated and control samples. Relative gene expression was evaluated and tumor samples were then spatially distributed according to the expression of the trios of genes: PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2. A separation plane was generated. However, no clear separation between responsive and non-responsive samples could be observed. Conclusion: Three-dimensional distribution of samples according to the expression of the trios of genes PRSS11, MTSS1, CLPTM1 and PRSS11, MTSS1, SMYD2 could not predict doxorubicin in vitro responsiveness. Short term culture of mammary gland cancer slices may be an interesting model to evaluate chemotherapy activity.

Serological Survey for Antibodies to Encephalitozoon cuniculi in Ownerless Dogs From Urban Areas of Brazil and Colombia

There are 3 strains of Encephalitozoon cuniculi that occur in mammals. Strain III is associated with clinical disease in dogs, although some can be asymptomatic carriers and excrete spores in their urine. Several cases of human E. cuniculi infection caused by strain III have been observed in immunocompromised patients, indicating that E. cuniculi should be considered a zoonotic agent. Encephalitozoon cuniculi can cause fatal disease in maternally-infected or young dogs. Clinical signs in these animals included blindness, encephalitis, retarded growth rate, and nephritis. Encephalitozoon cuniculi has also been associated with primary renal failure in adult dogs. The present study used the direct agglutination test (DAT, cut-off 1:50) and the indirect fluorescent antibody test (IFAT, cut-off 1:10) to examine the prevalence of antibodies to E. cuniculi in dogs from Brazil and Colombia. Using the DAG, 31 (27.4%) of 113 dogs from Brazil and 47 (18.5%) of 254 dogs from Colombia were seropositive. Nine (14.3%) of 63 dogs from Brazil and IS (35.3%) of the 51 dogs from Colombia were seropositive by indirect immunofluorescent antibody test. These results indicate that dogs from Brazil and Colombia are exposed to E. cuniculi.

Nd:YAG Laser Improves Biocompatibility of Human Dental Root Surfaces

Objective: Our goal was to compare the in vivo biocompatibility of dental root surfaces submitted to four different treatments after tooth avulsion followed by implantation into rat subcutaneous tissue. Background Data: Dental root surface preparation prior to replanting teeth remains a challenge for endodontists. Root surface changes made by Nd:YAG irradiation could be an alternative preparation. Methods: Forty-eight freshly extracted human dental roots were randomly divided into four treatment groups prior to implantation into rat subcutaneous tissue: G1, dry root, left in the environment up to 3 h; G2, the same treatment as G1, followed by a soaking treatment in a 2.4% sodium fluoride solution (pH 5.5); G3, root soaked in physiologic saline after avulsion for 72 h; G4, the same treatment as G1, followed by Nd:YAG laser irradiation (2.0 W, 20 Hz, 100 mJ, and 124.34 J/cm(2)). The animals were sacrificed 1, 7, and 45 d later. Histological and scanning electron microscopy analyses were done. Results: All dental roots were involved and in intimate contact with connective tissue capsules of variable thicknesses. Differences were observed in the degree of inflammation and in connective tissue maturation. In G3 the inflammatory infiltrate was maintained for 45 d, whereas the Nd:YAG laser irradiation (G4) led to milder responses. The overall aspects of the root surfaces were similar, except by the irradiated roots, where fusion and resolidification of the root surface covering the dentinal tubules were observed. Conclusion: Nd:YAG laser irradiation improves the biocompatibility of dental root and thus could be an alternative treatment of dental root prior to replantation.

Cavity Preparation and Influence of Restorative Materials on the Prevention of Secondary Caries

Objective: This in vitro study evaluated the influence of cavity preparation using the Er:YAG laser and restorative materials containing fluoride on preventing caries lesions. Background: It has been suggested that cavity preparation using the Er:YAG laser has a potential for improving resistance to secondary caries on enamel. Methods: Forty unerupted human third molars teeth were sectioned into 72 blocks of dental enamel and distributed into two groups to prepare cavities measuring (1.6 mm diameter) with diamond burs (DB) or Er:YAG laser (LA; 6 Hz, 300 mJ, 47 J/cm(2)). After that, each group was divided into three subgroups and restored with a glass-ionomer cement (GI), a resin-modified glass-ionomer (RM), or a composite resin (CR). Blocks were thermal cycled and submitted to a pH challenge to develop artificial caries-like lesions. Lesions were evaluated by Knoop microhardness test. An average of four indentations was used. Statistical analyses were performed by ANOVA followed by Tukey's test. Results: The results (in Knoop hardness number) for DB cavity preparation were GI, 235.5 (+/- 75.5); RM, 137.1 (+/- 64.1); and CR, 39.3 (+/- 26.5). For LA cavity preparation, the results were GI, 410.0 (+/- 129.7); RM, 310.3 (+/- 119.5); and CR, 96.4 (+/- 57.4). Conclusions: There was less development of caries lesion around LA-prepared cavities than around the DB-prepared cavities; however, no synergistic cariostatic effect was observed between the Er:YAG laser and glass ionomer cement.

Apical Leakage of Three Resin-Based Endodontic Sealers after 810-nm-Diode Laser Irradiation

Objective: To evaluate the influence of 810-nm-diode laser irradiation, applied before root canal filling, on apical sealing ability of three different resin-based sealers (AH Plus, EndoRez, and RealSeal). Background: Lasers have been widely used in endodontics. The dentin wall changes caused by laser irradiation could improve the sealing ability of endodontic cements. Methods: Sixty single-rooted human teeth were divided into six groups, according to the endodontic sealer used and previous 810-nm-diode laser irradiation. The protocol for laser irradiation was 2.5W in a continuous wave, in scanning mode, with four irradiations per tooth. After sample preparation, they were analyzed according to apical leakage with silver nitrate impregnation. Results: The RealSeal sealer achieved minimum leakage rates (1.24 mm), with significant differences at the 1% level (Tukey's test, p < 0.01) from AH Plus (1.84 mm) in nonirradiated groups. When the laser was used, there were also significant differences at the 5% level (p < 0.05) between irradiated groups (1.31 and 1.78 mm, respectively). Conclusion: The 810-nm-diode laser irradiation did not promote significant differences in apical leakage.

Effect of Nd:YAG Laser and Acidulated Phosphate Fluoride on Bovine and Human Enamel Submitted to Erosion/Abrasion or Erosion Only: An in Vitro Preliminary Study

Objective: The aim of the present in vitro study was to evaluate, using two different methodologies, the effectiveness of pulsed Nd:YAG laser irradiation associated with topical acidulated phosphate fluoride (APF) for preventing enamel erosion and structure loss under regimes of erosion and abrasion or erosion only. Background Data: An increased incidence of noncarious lesions (erosion and abrasion) has been observed, consequently new preventative therapies have been proposed. Materials and Methods: Two different methodologies were performed. For the first, 100 bovine crowns were submitted to four different treatments (n = 25): no treatment (control), 4 min application of APF, Nd:YAG laser irradiation (1 W, 100 mJ, 10 Hz, 141.5 J/cm(2)), and Nd:YAG laser irradiation+4 min of APF. After the specimens were exposed to citric acid (2% w/v; 30 min), they were submitted to 5000 brushing cycles. Specimen mass was measured before and after the treatments. For the second methodology, 20 human crowns were embedded in acrylic resin and cut surfaces were exposed and polished. The specimens were divided into four groups (n = 10): no treatment (control), APF for 4 min, Nd:YAG laser irradiation (1 W, 100 mJ, 10 Hz, 125 J/cm(2)), and Nd:YAG laser irradiation+APF. The samples were then immersed in citric acid (2% w/v; 90 min). Vickers hardness was obtained before and after the treatments. Results: The Nd:YAG laser irradiation+APF (bovine and human enamel) was more effective and yielded statistically significant results for surface microhardness and enamel wear. Conclusion: Nd:YAG laser irradiation associated with APF reduced bovine enamel wear and human enamel softening when samples were submitted to a regime of erosion and abrasion or erosion only in vitro.

Investigation of mast cells in human gingiva following low-intensity laser irradiation

Objective: The aims of the present study were to investigate the effect of low-intensity laser irradiation on the total number of mast cells as well as the percentage of degranulation in human gingiva. Blood vessel dilation was also evaluated. Background Data: It has been proposed that low-intensity laser irradiation can ameliorate pain, swelling, and inflammation. In periodontal tissue, mast cells may influence either the destructive events or the defense mechanism against periodontal disease via secretion of cytokines and through cellular migration to improve the healing process. Mast cells play an important role in the inflammatory process. Methods: Twenty patients with gingival enlargement indicated for gingivectomy were selected. Gingival fragments were obtained from each patient and divided into three different groups before surgery. One fragment was removed without any irradiation. The two others were submitted to punctual irradiation with an energy density of 8 J/cm(2) at an output power of 50 mW at 36 Hz for 36 sec before gingivectomy. Nondegranulated and degranulated mast cells were counted in five areas of the gingival fragment connective tissue. Major and minor diameters of the blood vessels were also measured. Results: Both red and infrared radiation promoted a significant increase in mast cell degranulation compared to controls; however, no statistically significant differences (p > 0.05) were observed between the irradiated groups. No significant differences among the groups were observed regarding blood vessel size. Conclusion: The results suggests that red and infrared wavelengths promote mast cell degranulation in human gingival tissue, although no dilation of blood vessels was observed. The effects of premature degranulation of mast cells in human tissue and the laser radiation protocol applied in this study encourage further investigations to extend these results into clinical practice.

Mechanism of Heparin Acceleration of Tissue Inhibitor of Metalloproteases-1 (TIMP-1) Degradation by the Human Neutrophil Elastase

Heparin has been shown to regulate human neutrophil elastase (HNE) activity. We have assessed the regulatory effect of heparin on Tissue Inhibitor of Metalloproteases-1 [TIMP-1] hydrolysis by HNE employing the recombinant form of TIMP-1 and correlated FRET-peptides comprising the TIMP-1 cleavage site. Heparin accelerates 2.5-fold TIMP-1 hydrolysis by HNE. The kinetic parameters of this reaction were monitored with the aid of a FRET-peptide substrate that mimics the TIMP-1 cleavage site in pre-steady-state conditionsby using a stopped-flow fluorescence system. The hydrolysis of the FRET-peptide substrate by HNE exhibits a pre-steady-state burst phase followed by a linear, steady-state pseudo-first-order reaction. The HNE acylation step (k(2)=21 +/- 1 s(-1)) was much higher than the HNE deacylation step (k(3)=0.57 +/- 0.05 s(-1)). The presence of heparin induces a dramatic effect in the pre-steady-state behavior of HNE. Heparin induces transient lag phase kinetics in HNE cleavage of the FRET-peptide substrate. The pre-steady-state analysis revealed that heparin affects all steps of the reaction through enhancing the ES complex concentration, increasing k(1) 2.4-fold and reducing k(-1) 3.1-fold. Heparin also promotes a 7.8-fold decrease in the k(2) value, whereas the k(3) value in the presence of heparin was increased 58-fold. These results clearly show that heparin binding accelerates deacylation and slows down acylation. Heparin shifts the HNE pH activity profile to the right, allowing HNE to be active at alkaline pH. Molecular docking and kinetic analysis suggest that heparin induces conformational changes in HNE structure. Here, we are showing for the first time that heparin is able to accelerate the hydrolysis of TIMP-1 by HNE. The degradation of TIMP-1is associated to important physiopathological states involving excessive activation of MMPs.

In Vivo Effects on the Expression of Vascular Endothelial Growth Factor-A(165) Messenger Ribonucleic Acid of an Infrared Diode Laser Associated or Not with a Visible Red Diode Laser

Objective: This study investigated and correlated the kinetic expression of vascular endothelial growth factor (VEGF)-A(165) messenger ribonucleic acid (mRNA) with the associated use or not of an infrared laser and a visible red laser during the wound healing in rats. Background Data: There is a lack of scientific evidence demonstrating the influence of low-level laser therapy (LLLT) on the expression of VEGF mRNA in vivo. Materials and Methods: Forty-five Wistar rats were randomly allocated to one of three groups: I (n = 5, nonoperated animals), II (n = 25, operated animals), and III (n = 25, animals operated and subjected to laser irradiation). A surgical wound was performed using a scalpel in the right side of the tongue of operated animals. In group III, two sessions of laser irradiation were performed, one right after the surgical procedure (infrared laser, 780 nm, 70mW, 35 J/cm(2)) and the other 48 h later (visible red laser, 660 nm, 40mW, 5J/cm(2)). Five animals each were sacrificed 1, 3, 5, and 7 days postoperatively in groups II and III, and samples of tongue tissue were obtained. The animals of group I were sacrificed on day 7. Total RNA was extracted using guanidine-isothiocyanate-phenol-chloroform method. The results of horizontal electrophoresis after reverse transcription polymerase chain reaction permitted the ratio of VEGF-A(165) mRNA and glyceraldehyde 3-phosphate dehydrogenase mRNA expression for groups I, II, and III to be assessed (two-way analysis of variance and Tukey test, p<0.05). Results: The expression of VEGF-A(165) mRNA in group II (0.770 +/- 0.098) was statistically greater than that observed in groups I (0.523 +/- 0.164) and III (0.504 +/- 0.069) in the first day after surgery (p<0.05). Significant differences between the groups were not observed in other time periods. Conclusion: LLLT influenced the expression of VEGF-A(165) mRNA during wound healing after a surgical procedure on the tongue of Wistar rats.