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Persistent molecules, such as pesticides, herbicides, and pharmaceuticals, pose significant threats to both the environment and human health. Advancements in developing efficient photocatalysts for degrading these substances can play a fundamental role in remediating contaminated environments, thereby enhancing safety for all forms of life. This study investigates the enhancement of photocatalytic efficiency achieved by incorporating La 3+ into Ag 3 PO 4 , using the co-precipitation method in an aqueous medium. These materials were utilized in the photocatalytic degradation of Rhodamine B (RhB) and Ciprofloxacin (CIP) under visible light irradiation, with monitoring conducted through high-performance liquid chromatography (HPLC). The synthesized materials exhibited improved stability and photodegradation levels for RhB. Particularly noteworthy was the 2% La 3+ -incorporated sample (APL2), which achieved a 32.6% mineralization of CIP, nearly three times higher than pure Ag 3 PO 4 . Toxicological analysis of the residue from CIP photodegradation using the microalga Raphidocelis subcapitata revealed high toxicity due to the leaching of Ag  +  ions from the catalyst. This underscores the necessity for cautious wastewater disposal after using the photocatalyst. The toxicity of the APL2 photocatalysts was thoroughly assessed through comprehensive toxicological tests involving embryo development in Danio rerio, revealing its potential to induce death and malformations in zebrafish embryos, even at low concentrations. This emphasizes the importance of meticulous management. Essentially, this study adeptly delineated a thorough toxicological profile intricately intertwined with the photocatalytic efficacy of newly developed catalysts and the resultant waste produced, prompting deliberations on the disposal of degraded materials post-exposure to photocatalysts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background/aims: Bladder cancer is considered one of the most aggressive neoplasms due to its recurrence and progression profile, and even with the improvement in diagnosis and treatment methods, the mortality rate has not shown a declining trend in recent decades. From this perspective, the search and development of more effective and safer therapeutic alternatives are necessary. Phytochemicals are excellent sources of active principles with therapeutic potential. [6]-Shogaol is a phenolic compound extracted from the ginger rhizomes that has shown antitumor effects in a wide variety of cancer models. However, there is no record in the literature of studies reporting these effects in models of bladder cancer. Thus, this study aimed to investigate the in vitro cytotoxic and pro-apoptotic potential of [6]-Shogaol against murine bladder cancer urothelial cells (MB49)., Methods: The cytotoxic effects of [6]-Shogaol on cell viability (MTT method), cell morphology (light microscopy), alteration of proliferative processes (clonogenic assay), oxidative stress pathway (levels of reactive oxygen species) and the induction of apoptotic events (flow cytometry and high-resolution epifluorescence imaging) were evaluated in murine urothelial bladder cancer cell lines (MB49), relative to non-tumor murine fibroblasts (L929)., Results: The results showed that [6]-Shogaol was able to induce concentration-dependent cytotoxic effects, which compromised cell viability, exhibiting an inhibitory concentration of 50% of cells (IC50) of 146.8 µM for MB49 tumor cells and 236.0 µM for L929 non-tumor fibroblasts. In addition to inhibiting and altering the proliferative processes if colony formation, it presented pro-apoptotic activity identified through a quantitative analysis and the observation of apoptotic phenotypes, events apparently mediated by the induction of nuclear fragmentation., Conclusion: The data presented suggest that [6]-Shogaol has a higher concentration-dependent cytotoxic and apoptosis-inducing potential in MB49 cells than in L929 fibroblasts. These results may contribute to the development of therapeutic alternatives for bladder cancer., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Inbred species are useful resources for a variety of biomedical research applications. To create isogenic zebrafish, it is feasible to stop meiosis II (repeatedly) or mitosis (two times) in a haploid embryo by applying pressure or by delivering a heat shock, respectively. In this study, to improve the repeatability, we suggest a less complicated approach based on sperm ultraviolet-C (UV-C) exposure for a shorter period followed by heat shock at various temperatures, eliminating the use of pressure in meiotic therapy since heat shock is more accessible to laboratories. In this study, the survivability rates of meiotic (Mei) and mitotic (Mit) gynogenesis offspring produced by various combinations of irradiation (28.5, 105, and 210 mJ/cm 2 ) and temperature (Mei: 40.40°C, 40.60°C, or 40.90°C; Mt: 41.40°C, 41.90°C, or 42.45°C) were compared with diploid (C) and haploid (H) controls. Our findings demonstrated that 40.60°C and 41.90°C were the most suitable temperatures to produce meiotic and mitotic gynogenesis, respectively, whereas 28.5 mJ/cm 2 was more successful in ensuring haploid embryos. As a result, we deduced that meiotic gynogenesis produces more viable offspring than the mitotic approach and requires a lower temperature to maintain the second polar body.

Risperidone is an antipsychotic medication used in the treatment of conditions like autism and schizophrenia. The goal of the current study was to examine the effects of risperidone in zebrafish embryos ( Danio rerio ) with regard to survival, development, and cardiac and neural systems. The results showed that concentrations above 100 μM were associated with deaths, teratogenic effects, and cardiotoxic and neurotoxic effects. The findings support the utility of zebrafish for toxicological screening studies.

The systemic delivery of drugs employed by conventional methods has shown to be less effective than a localized delivery system. Many drugs have the effectiveness reduced by fast clearance, increasing the amount required for an efficient treatment. One way to overcome this drawback is through the use of thermoresponsive polymers that undergo a sol-gel transition at physiological temperature, allowing their injection directly in the desired site. In this work, thermosensitive nanocomposites based on poly( N -vinylcaprolactam) and silica particles with 80 and 330 nm were synthesized to be employed as delivery systems for hydrophobic (naringin) and hydrophilic (doxorubicin hydrochloride) drugs. The insertion of SiO 2 increased the rheological properties of the nanocomposite at 37 °C, which helps to prevent its diffusion away from the site of injection. The synthesized materials were also able to control the drug release for a period of 7 days under physiological conditions. Due to its higher hydrophobicity and better interaction with the PNVCL matrix, naringin presented a more controlled release. The Korsmeyer-Peppas model indicated different release mechanisms for each drug. At last, a preliminary in vitro study of DOX-loaded nanocomposites cultured with L929 and MB49 cells showed negligible toxic effects on healthy cells and better efficient inhibition of carcinoma cells.

The salt calcium chloride (CaCl₂) is widely used in industry as a food additive; levels for human consumption are regulated by international or governmental agencies. Generally, the food industry relies on toxicity studies conducted in mammals such as mice, rats, and rabbits for determining food safety. However, testing in mammals is time-consuming and expensive. Zebrafish have been used in a range of toxicological analyses and offer advantages with regard to sensitivity, time, and cost. However, information in not available with regard to whether the sensitivity of zebrafish to CaCl₂ is comparable to the concentrations of CaCl₂ used as food additives. The aim of this study was to compare the CaCl₂ tolerance of zebrafish embryos and larvae with concentrations currently approved as food additives. Acute toxicity, embryotoxicity, cardiotoxicity, and neurotoxicity assays were used to determine the threshold toxic concentration of CaCl₂ in zebrafish embryos and larvae. The data showed that doses above 0.4% had toxic effects on development and on the activity of the cardiac and neuronal systems. Furthermore, all embryos exposed to 0.8 and 1.6% of CaCl₂ died after 24 hpf. These findings are consistent with the limits of CaCl₂ concentrations approved by Codex Alimentarius . Therefore, zebrafish embryos could be suitable for screening food additives.

Regulation of inflammation is a critical process for maintaining physiological homeostasis. The λ-carrageenan (λ-CGN) is a mucopolysaccharide extracted from the cell wall of red algae ( Chondrus crispus ) capable of inducing acute intestinal inflammation, which is translated into the production of acute phase reactants secreted into the blood circulation. However, the associated mechanisms in vertebrates are not well understood. Here, we investigated the crucial factors behind the inflammatory milieu of λ-CGN-mediated inflammation administered at 0, 1.75, and 3.5% (v/w) by i.p. injection into the peritoneal cavity of adult zebrafish (ZF) ( Danio rerio ). We found that polymorphonuclear leukocytes (neutrophils) and lymphocytes infiltrating the ZF peritoneal cavity had short-term persistence. Nevertheless, they generate a strong pattern of inflammation that affects systemically and is enough to produce edema in the cavity. Consistent with these findings, cell infiltration, which causes notable tissue changes, resulted in the overexpression of several acute inflammatory markers at the protein level. Using reversed-phase high-performance liquid chromatography followed by a hybrid linear ion-trap mass spectrometry shotgun proteomic approach, we identified 2938 plasma proteins among the animals injected with PBS and 3.5% λ-CGN. First, the bioinformatic analysis revealed the composition of the plasma proteome. Interestingly, 72 commonly expressed proteins were recorded among the treated and control groups, but, surprisingly, 2830 novel proteins were differentially expressed exclusively in the λ-CGN-induced group. Furthermore, from the commonly expressed proteins, compared to the control group 62 proteins got a significant ( p < 0.05) upregulation in the λ-CGN-treated group, while the remaining ten proteins were downregulated. Next, we obtained the major protein-protein interaction networks between hub protein clusters in the blood plasma of the λ-CGN induced group. Moreover, to understand the molecular underpinnings of these effects based on the unveiled protein sets, we performed a bioinformatic structural similarity analysis and generated overlapping 3D reconstructions between ZF and humans during acute inflammation. Biological pathway analysis pointed to the activation and abundance of diverse classical immune and acute phase reactants, several catalytic enzymes, and varied proteins supporting the immune response. Together, this information can be used for testing and finding novel pharmacological targets to treat human intestinal inflammatory diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Charlie-Silva, Feitosa, Pontes, Fernandes, Nóbrega, Gomes, Prata, Ferraris, Melo, Conde, Rodrigues, Aracati, Corrêa-Junior, Manrique, Superio, Garcez, Conceição, Yoshimura, Núñez, Eto, Fernandes, Freitas, Ribeiro, Nedoluzhko, Lopes-Ferreira, Borra, Barcellos, Perez, Malafaia, Cunha, Belo and Galindo-Villegas.)

Low oxygen concentration inside the tumor microenvironment represents a major barrier for photodynamic therapy of many malignant tumors, especially urothelial bladder cancer. In this context, titanium dioxide, which has a low cost and can generate high ROS levels regardless of local O 2 concentrations, could be a potential type of photosensitizer for treating this type of cancer. However, the use of UV can be a major disadvantage, since it promotes breakage of the chemical bonds of the DNA molecule on normal tissues. In the present study, we focused on the cytotoxic activities of a new material (Ti(OH) 4 ) capable of absorbing visible light and producing high amounts of ROS. We used the malignant bladder cell line MB49 to evaluate the effects of multiple concentrations of Ti(OH) 4 on the cytotoxicity, proliferation, migration, and production of ROS. In addition, the mechanisms of cell death were investigated using FACS, accumulation of lysosomal acid vacuoles, caspase-3 activity, and mitochondrial electrical potential assays. The results showed that exposure of Ti(OH) 4 to visible light stimulates the production of ROS and causes dose-dependent necrosis in tumor cells. Also, Ti(OH) 4 was capable of inhibiting the proliferation and migration of MB49 in low concentrations. An increase in the mitochondrial membrane potential associated with the accumulation of acid lysosomes and low caspase-3 activity suggests that type II cell death could be initiated by autophagic dysfunction mechanisms associated with high ROS production. In conclusion, the characteristics of Ti(OH) 4 make it a potential photosensitizer against bladder cancer., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Background: Activating the immune system for therapeutic benefit has long been a goal in immunology, especially in cancer treatment, but the low immunogenicity of antitumor vaccines remains a limiting factor in the fight against malignant neoplasms. The increase in the immunogenicity of weak antigens using biodegradable polymers, such as chitosan, has been observed in the field of cancer immunotherapy. However, the effects of the vaccine using a combination of tumor cells and a thermoreversible delivery system based on chitosan in bladder cancer models, mainly using the intravesical route to stimulate the antitumor immune response, are unknown. We propose to evaluate the efficacy of a polymeric gel matrix (TPG) formed by poloxamer 407 and chitosan, associated with MB49 cells, as an intravesical antitumor vaccine using a C57BL/6 murine model of bladder urothelial carcinoma. The effectiveness of immunization was analyzed with the formation of three experimental groups: Control, TPG and TPG + MB49. In the vaccination phase, the TPG + MB49 group underwent a traumatic injury to the bladder wall with immediate intravesical instillation of the vaccine compound containing MB49 cells embedded in TPG. The TPG group was subjected to the same procedures using the compound containing the gel diluted in medium, and the control group using only the medium. After 21 days, the animals were challenged with tumor induction., Results: In vitro tests showed loss of viability and inability to proliferate after exposure to TPG. In vivo tests showed that animals previously immunized with TPG + MB49 had higher cumulative survival, as well as significantly lower bladder weight and size in contrast to the other two groups that did not show a statistically different tumor evolution. In addition, the splenocytes of these animals also showed a higher rate of antitumor cytotoxicity in relation to the TPG and control groups., Conclusions: We can conclude that MB49 cells embedded in a polymeric thermoreversible gel matrix with chitosan used in the form of an intravesical vaccine are able to stimulate the immune response and affect the development of the bladder tumor in an orthotopic and syngeneic C57BL/6 murine model., (© 2022. The Author(s).)

Zebrafish is considered an unprecedented animal model in drug discovery. A review of the literature presents highlights and elucidates the biological effects of chemical components found in Cannabis sativa. Particular attention is paid to endocannabinoid system (eCB) and its main receptors (CB1 and CB2). The zebrafish model is a promising one for the study of cannabinoids because of the many similarities to the human system. Despite the recent advances on the eCB system, there is still the need to elucidate some of the interactions and, thus, the zebrafish model can be used for that purpose as it respects the 3Rs concept and reduced time and costs. In view of the relevance of cannabinoids in the treatment and prevention of diseases, as well as the importance of the zebrafish animal model in elucidating the biological effects of new drugs, the aim of this study was to bring to light information on the use of the zebrafish animal model in testing C. sativa-based medicines., (© 2022. The Author(s).)

Background: Recently a screen from a library of 1.8 million compounds identified in vitro a potent activity of the 2-aminobenzimidazoles series against Leishmania infantum, the etiological agent responsible by over 20.000 deaths each year. Several analogs were synthesized and in vitro tested through an optimization program, leading to a promising 2-aminobenzimidazoles derived compound (2amnbzl-d) that was progressed to in vivo mice studies. However, the not expected toxic effects prevented its progression to more advanced preclinical and clinical phases of drug development. Due to limitations of cell models in detecting whole organism complex interactions, 90% of the compounds submitted to pre-clinical tests are reproved. The use of Zebrafish embryo models could improve this rate, saving mammals, time and costs in the development of new drugs. To test this hypothesis, we compared 2amnbzl-d with two compounds with already established safety profile: carbamazepine and benznidazole, using an embryo Zebrafish platform based on acute toxicity, hepatotoxicity, neurotoxicity and cardiotoxicity assays (Pltf-AcHpNrCd)., Results: Tests were performed blindly, and the results demonstrated the presence of lethal and teratogenic effects (CL50%: 14.8 µM; EC50%: 8.6 µM), hepatotoxic in concentrations above 7.5 µM and neurotoxic in embryos exposed to 15 µM of 2amnbzl-d. Nevertheless, benznidazole exposition showed no toxicity and only the 100 µM of carbamazepine induced a bradycardia., Conclusions: Results using Pltf-AcHpNrCd with zebrafish reproduced that found in the toxicological tests with mammals to a portion of the costs and time of experimentation., (© 2021. The Author(s).)

Obesity is an epidemic disease and the expansion of adipose tissue, especially visceral fat, promotes the secretion of factors that lead to comorbidities such as diabetes and cardiovascular diseases. Thus, diet and exercise have been proposed as an intervention to reverse these complications. An adipocytokine, known as irisin, mediates the beneficial effects of exercise. It has been proposed as a therapeutic potential in controlling obesity. In view of the above, this paper attempts to determine the modulation of irisin, visceral adiposity and biochemical markers in response to dietary intervention and aerobic exercise. To do this, 52 diet-induced obese male Wistar rats were divided into the following four groups: high-fat diet and exercise (HFD-Ex); HFD-Sedentary (HFD-Sed); chow-diet and exercise (CD-Exercise); and CD-Sed. The exercise-trained group performed a treadmill protocol for 60 min/day, 3 days/week for 8 weeks. Body mass (BM), body fat (BF), fat mass (FM), and fat-free mass (FFM) were analyzed. Mesenteric (MES), epididymal (EPI), and retroperitoneal (RET) adipose tissue was collected and histological analysis was performed. Biochemical irisin, triglycerides, glucose, insulin and inflammatory markers were determined and, FNDC5 protein expression was analyzed. In this study, the diet was the most important factor in reducing visceral adiposity in the short and long term. Exercise was an important factor in preserving muscle mass and reducing visceral depots after a long term. Moreover, the combination of diet and exercise can enhance these effects. Diet and exercise exclusively were the factors capable of increasing the values of irisin/FNDC5, however it did not bring cumulative effects of both interventions. Prescriptions to enhance the obesity treatments should involve reducing visceral adiposity by reducing the fat content in the diet associated with aerobic exercise., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Furino, Alves, Marine, Sene-Fiorese, Rodrigues, Arrais-Lima, Mattiello, Castro, Borra, Rocha, Malavazi and Duarte.)

Background/aims: Cancer is the second most deadly disease in the world. The bladder cancer is one of the most aggressive types and shows a continuous increase in the number of cases. The use of bacteria as live vectors to deliver molecules directly to the tumor is a promising tool and has been used as an adjuvant treatment against several types of cancer. The aim of this study was to investigate the antitumor effect of Interleukin 2 (IL-2), TNF-related apoptosis-inducing ligand (TRAIL) and protein MIX against murine bladder cancer cells, lineage MB49., Methods: The attenuated Salmonella strain SL3261 was transformed by inserting the IL-2 and TRAIL genes. The effects of proteins on cell viability (MTT method), cell morphology (optical microscopy), cell recovery (clonogenic assay), cell membrane (lactate dehydrogenase release - LDH), on oxidative stress pathway (levels of nitric oxide, NO) and apoptosis (flow cytometry and high resolution epifluorescence images) were evaluated at intervals of 24 and 48 hours of action., Results: The results showed that there was a decrease in cell viability via damage to the cell membrane, alteration of cell morphology, non-recovery of cells, increase in the production of NO and incubate for of cells in the state of apoptosis in the two periods analyzed., Conclusion: The data presented suggest that IL-2, TRAIL and their MIX proteins in MB49 cells have cytotoxic potential and that this is associated with oxidative stress and apoptosis pathways. These results may contribute to the development of new therapeutic strategies for bladder cancer., Competing Interests: The authors have no conflicts of interest to declare., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Background: Treatments based on production of reactive oxygen species for bladder cancer such as photodynamic therapy (PDT) have been marginalized due to low specificity and the existence of resistance mainly associated with the up-regulation of Heat Shock Proteins (HSPs). To overcome these barriers, the establishment of strategies combining PDTs with HSP inhibitors may be promising and the identification of HSPs involved with oxidative stress from bladder tumors in animal models represents a key step in this direction., Materials: Thus, the present study aims to identify cytosolic and mitochondrial HSPs up expressed in murine bladder tumors and in the urothelial carcinoma cell line MB49 by qRT-PCR screening, and to analyze the importance of the activity of the HSPs associated with oxidative stress protection in the survival of the MB49 using strategy of inhibition in vitro., Results: Results showed that both tumor tissues and MB49 cells in culture had significant overexpression of the mitochondrial HSPA9 (mortalin) and HSP60 mRNAs, while the cytosolic HSP90 was overexpressed only in the tumor. The effect of mortalin in the MB49 cells survival under oxidative stress was evaluated in vitro in presence of the specific inhibitor MKT-077 and H 2 O 2 . The findings showed that MB49 viability was permanently reduced by the MKT-077 in a dose-dependent manner by inducing apoptosis or necrosis, mainly under oxidative stress conditions., Conclusion: Results suggest that mortalin is preferentially expressed in the MB49 cancer model and plays a key role in tumoral survival, especially under oxidative stress, making this HSP a potential target for an alternative treatment combining PDT with HSP inhibitors., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Artemisinin extracted from Artemisia annua L. plants has a range of properties that qualifies it to treat several diseases, such as malaria and cancer. However, it has short half-life, which requires making continuous use of it, which has motivated the association of artemisinin (ART) with polymeric nanoparticles to increase its therapeutic efficiency. However, the ecotoxicological safety of this association has been questioned, given the scarcity of studies in this area. Thus, in this work the toxicity of Poly (ε-Caprolactone) nanocapsules added with ART (ART-NANO) in zebrafish (Danio rerio), embryos and adults was studied. Different endpoints were analyzed in organisms exposed to ART-NANO, including those predictive of embryotoxicity and histopatoxicity. Embryotoxicity was analyzed based on Organization for Economic Co-operation and Development (OECD) test guideline (236) for fish embryo acute toxicity applied to zebrafish (Danio rerio) at 96 hpf under five nominal logarithmic concentrations (0.125 to 2.0 mg/ L). Our results demonstrate, mainly, that fertilized eggs presented increased coagulation, lack of heart rate, vitelline sac displacement and lack of somite formation. On the other hand, adult individuals (exposed to the same concentrations and evaluated after 24 and 96 h of exposure) have shown increased pericarditis. Therefore, the treatment based on ART, poly (ε-caprolactone) nanocapsules and on their combination at different concentrations have shown toxic effects on zebrafish embryos and adult individuals., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Mucoadhesive polymeric nanocapsules have attracted interest of researchers from different fields from natural sciences because of their ability to interact with the mucosa and increase drug permeation. Anesthesia by immersion causes absorption through the skin and gills of fish, so it is important to evaluate the exposure of these organs to drug nanosystems. Benzocaine (BENZ) is one of the most popular anesthetic agents used in fish anesthesia, but it has drawbacks because of its low bioavailability, resulting in weak absorption after immersion. Here we describe method developed for preparing and characterizing chitosan-coated PLGA mucoadhesive nanoparticles containing BENZ (NPMAs) for zebrafish immersion anesthesia. We determined the lowest effective concentration, characterized the interaction of the mucoadhesive system with fish, measured the anesthetic efficacy, and evaluated possible toxic effects in embryos and adults exposed to the nanoformulations. This study opens perspectives for using nanoformulations prepared with BENZ in aquaculture, allowing reduction of dosage as well as promoting more effective anesthesia and improved interaction with the mucoadhesive system of fish., Competing Interests: The authors have read the journal’s policy and have the following potential competing interests: authors LFF and GBdM are members of the PLOS ONE Editorial Board. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.

Much of medical research relies on animal models to deepen knowledge of the causes of animal and human diseases, as well as to enable the development of innovative therapies. Despite rodents being the most widely used research model worldwide, in recent decades, the use of the zebrafish ( Danio rerio ) model has exponentially been adopted among the scientific community. This is because such a small tropical freshwater teleost fish has crucial genetic, anatomical and physiological homology with mammals. Therefore, zebrafish constitutes an excellent experimental model for behavioral, genetic and toxicological studies which unravels the mechanism of various human diseases. Furthermore, it serves well to test new therapeutic agents, such as the safety of new vaccines. The aim of this review was to provide a systematic literature review on the most recent studies carried out on the topic. It presents numerous advantages of this type of animal model in tests of efficacy and safety of both animal and human vaccines, thus highlighting gains in time and cost reduction of research and analyzes., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2020.)

Tahiti lemon juice (Citrus latifolia) (TLJ), as a natural source of flavonoids, has been used as an alternative to anti-inflammatory drugs for the treatment of dysmenorrhea and menstrual excessive bleeding, often associated with an imbalance of the prostaglandins (PG) levels. However, despite the positive effects, the mechanisms that rule menstruation control are still unknown. Therefore, the objectives were to characterize the TLJ and analyze its effect on the production of PGF2α, PGE2 and pro-inflammatory cytokines involved inmenstruation. Flavonoids from TLJ were discriminated by UPLC-DAD-MS/MS (Qq-TOF) and the effects of TLJ were studied in vitro by quantification of the contraction of myoblasts in culture and PGF2α and PGE2 productions. Further, the systemic and menstrual fluid levels of PGF2α, PGE2, IL-1β, TNF-α, IL-6, AK1B1 and AK1C3 enzymes produced by women during the menstrual period were compared after exposition or not to TLJ or meloxicam. The results showed that TLJ induces an increase in the contraction of myoblasts and the PGF2α supernatant level. Regarding in vivo analysis, a higher concentration of PGF2α and an unaltered PGE2 level was also found in the menstrual blood of women treated with TLJ, in contrast with a lower level of PGE2 and PGF2α observed in the meloxicam group. Concerning cytokines, only menstrual TNF-α levels decrease after treatment with TLJ or meloxicam. In conclusion, TLJ may favor the control of menstruation events via a PGF2α mediated muscle contractile response.

Purpose: The aim of this study was to compare the effects of supervised combined physical training and unsupervised physician-prescribed regular exercise on the functional capacity and quality of life of heart failure patients., Methods: This is a longitudinal prospective study composed of 28 consecutive heart failure with reduced ejection fraction patients randomly divided into two age- and gender-matched groups: trained group ( n  = 17) and nontrained group ( n  = 11). All patients were submitted to clinical evaluation, transthoracic echocardiography, the Cooper walk test, and a Quality of Life questionnaire before and after a 12-week study protocol. Categorical variables were expressed as proportions and compared with the chi-square test. Two-way ANOVA was performed to compare the continuous variables considering the cofactor groups and time of intervention, and Pearson correlation tests were conducted for the associations in the same group., Results: No significant differences between groups were found at baseline. At the end of the protocol, there were improvements in the functional capacity and ejection fraction of the trained group in relation to the nontrained group ( p < 0.05). There was time and group interaction for improvement in the quality of life in the trained group., Conclusions: In patients with heart failure with reduced ejection fraction, supervised combined physical training improved exercise tolerance and quality of life compared with the unsupervised regular exercise prescribed in routine medical consultations. Left ventricular systolic function was improved with supervised physical training., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2019 Tainá Fabri et al.)

The ability to manipulate the structure and function of promising systems via external stimuli is emerging with the development of reconfigurable and programmable multifunctional materials. Increasing antifungal and antitumor activity requires novel, effective treatments to be diligently sought. In this work, the synthesis, characterization, and in vitro biological screening of pure α-Ag 2 WO 4 , irradiated with electrons and with non-focused and focused femtosecond laser beams are reported. We demonstrate, for the first time, that Ag nanoparticles/α-Ag 2 WO 4 composite displays potent antifungal and antitumor activity. This composite had an extreme low inhibition concentration against Candida albicans, cause the modulation of α-Ag 2 WO 4 perform the fungicidal activity more efficient. For tumor activity, it was found that the composite showed a high selectivity against the cancer cells (MB49), thus depleting the populations of cancer cells by necrosis and apoptosis, without the healthy cells (BALB/3T3) being affected.

The live attenuated mycobacterial strain BCG, in use as vaccine against tuberculosis, is considered the gold standard for primary therapy of carcinoma in situ of the bladder. Despite its limitations, to date it has not been surpassed by any other treatment. Our group has developed a recombinant BCG strain expressing the detoxified S1 pertussis toxin (rBCG-S1PT) that proved more effective than wild type BCG (WT-BCG) in increasing survival time in an experimental mouse model of bladder cancer, due to the well-known adjuvant properties of pertussis toxin. Here, we investigated the capacity of rBCG-S1PT to stimulate human immune responses, in comparison to WT-BCG, using an in vitro stimulation assay based on human whole blood cells that allows for a comprehensive evaluation of leukocyte activation. Blood leukocytes stimulated with rBCG-S1PT produced increased levels of IL-6, IL-8, and IL-10 as compared to WT-BCG, but comparable levels of IL-1β, IL-2, IFN-γ, and TNF-α. Stimulation of blood cells with the recombinant BCG strain also enhanced the expression of CD25 and CD69 on human CD4 + T cells. PBMC stimulated with rBCG-S1PT induced higher cytotoxicity to MB49 bladder cancer cells than WT-BCG-stimulated PBMC. These results suggest that the rBCG-S1PT strain is able to activate an immune response in human leukocytes that is higher than that induced by WT-BCG for parameters linked to better prognosis in bladder cancer (regulation of immune and early inflammatory responses), while fully comparable to WT-BCG for classical inflammatory parameters. This establishes rBCG-S1PT as a new highly effective candidate as immunotherapeutic agent against bladder cancer.

Background: There are indications that Th1 polarization of immune response plays an important role in the pathogenesis of recurrent aphthous stomatitis (RAS), and that the use of probiotics can stimulate immune regulatory activity, influencing the course of the disease. The aim of this study was to characterize the initial immune profile of RAS patients and evaluate clinical and serological response following a challenge with symbiotic treatment containing fructooligosaccharide, Lactobacillus, and Bifidobacterium., Methods: The immune responses of the 45 patients with RAS, submitted to symbiotic or placebo for 120 days, in relation to 30 RAS-free controls, were evaluated over a period of 6 months. Peripheral blood was collected from all patients at 0 (T0), 120 (T4), and 180 days (T6) after the start of treatment and Th1 (IL12-p70, IFN-γ), Th2 (IL-4), Treg (IL-10), Th17 (IL-17A), inflammatory (TNF-α, IL-6)-associated cytokines, and clinical parameters were quantified., Results: At T0, significant differences were found in the serological levels of the IFN-γ, IL-4, and IL-6 cytokines of the RAS patients in comparison with the controls. It was observed that the cytokine profile of the RAS group was comprised of 2 distinct clusters: a pure Th2 and a Mixed (Th1/Th2) subtype and that symbiotic treatment induced an improvement in pain and an increase in IFN-γ levels, producing a reduction in Th2 response., Conclusions: In RAS, symbiotic treatment based on a fructooligosaccharide, Lactobacillus, and Bifidobacterium composition produced an alteration in the Th2 serological immune profile in the direction of Th1 and improved pain symptomatology., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

This study evaluated the control of streptococcosis outbreaks in Brazil, isolated from diseased sorubim and identified as Lactococcus garvieae by genetic sequencing. This report determined the potential for lactococcosis control in sorubim Pseudoplatystoma sp. with two vaccines: an aqueous-based, whole-cell inactivated vaccine (bacterin) and an oil-adjuvanted bacterin. Their efficacy was evaluated at 30 days post-vaccination (d.p.v.) by challenge with L. garvieae, and the antibody production response at 15, 30 and 60 d.p.v. and the non-specific immune response were compared amongst treatments. High protection levels (P < 0.05) were achieved with the oil-adjuvanted vaccine with a relative percentage survival value of 81.7% at 30 d.p.v. Additionally, the oil-adjuvanted vaccine increased the immunogenicity of the bacterin as indicated by greater agglutination antibody titres from 15 until 60 d.p.v. This is the first report of a positive effect of vaccine administration on the specific immunity of sorubim, and the study showed that a specific antibody plays an important role in sorubim defence against lactococcosis because the innate immune responses were similar in all of the studied animals. These results demonstrated that oil-adjuvanted vaccine can be an effective alternative for the protection of sorubim from L. garvieae disease., (© 2016 John Wiley & Sons Ltd.)

Purpose: To evaluate genes differentially expressed in ovaries from lean (wild type) and obese (ob/ob) female mice and cyclic AMP production in both groups., Methods: The expression on messenger RNA levels of 84 genes concerning obesity was analyzed through the PCR array, and cyclic AMP was quantified by the enzyme immunoassay method., Results: The most downregulated genes in the Obesity Group included adenylate cyclase-activating polypeptide type 1, somatostatin, apolipoprotein A4, pancreatic colipase, and interleukin-1 beta. The mean decrease in expression levels of these genes was around 96, 40, 9, 4.2 and 3.6-fold, respectively. On the other hand, the most upregulated genes in the Obesity Group were receptor (calcitonin) activity-modifying protein 3, peroxisome proliferator activated receptor alpha, calcitonin receptor, and corticotropin-releasing hormone receptor 1. The increase means in the expression levels of such genes were 2.3, 2.7, 4.8 and 6.3-fold, respectively. The ovarian cyclic AMP production was significantly higher in ob/ob female mice (2,229 ± 52 fMol) compared to the Control Group (1,814 ± 45 fMol)., Conclusions: Obese and anovulatory female mice have reduced reproductive hormone levels and altered ovogenesis. Several genes have their expression levels altered when leptin is absent, especially adenylate cyclase-activating polypeptide type 1.

Background: Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine with pro-inflammatory functions and involved in tumorigenesis. The aim of this study was to evaluate the expression and localization of the macrophage MIF in oral squamous carcinoma (OSC). In addition, the relationship between MIF expression and clinicopathological parameters such as survival data, tobacco use, alcohol habits, TNM stage, tumor graduation, and peritumoral inflammatory infiltrate were evaluated., Methods: Using immunohistochemistry, expression and localization of MIF was detected in 44 specimens of OSC. The absolute number and relative proportions of MIF-positive cells detected were also determined separately for tumor parenchyma vs. stroma. All counts were determined from 10 consecutive high-power fields using an integration graticule. Moreover, some parameters were analyzed separately for lip and intra-oral cancers., Results: Migration inhibitory factor-positive cells were observed in both the tumor parenchyma and in inflammatory cells of all specimens. In contrast, MIF expression was not detected in tumoral nests associated with poorly differentiated tumors. In specimens of lip cancer, a greater number of MIF-positive stromal immune cells were detected than in intra-oral cancer specimens (Mann-Whitney test, P = 0.049)., Conclusions: Oral squamous carcinoma cells consistently express MIF independent of their location. Lip tumors presented more MIF-positive peritumoral inflammatory cells, similar to control, suggesting that immunological differences in leukocyte activation exist between in lip and intra-oral cancers., (© 2012 John Wiley & Sons A/S.)

Objective: The aim of this study was to analyse the visual perceptions of different experts with respect to multilocular radiolucent lesions in circumstances when the diagnosis is either known or unknown., Methods: 6 radiographs of ameloblastomas (AMELs), keratocystic odontogenic tumours (KOTs), central giant cell lesions (CGCLs) and myxomas (MIXs) were analysed by 16 dental experts [stomatologists/oral surgeons (SS) and dental radiologists (R)]. They delimited the lesions prior to having knowledge of the diagnosis (T1) and after 30 days, when they were aware of the histopathological results (T2). For each image, the following morphometric parameters were calculated: area (A), perimeter (P) and shape factor (SF); after image subtraction procedures (T1 - T2), the exclusive area (EA) of the non-overlapping delimited region was also calculated., Results: For both groups, the T2 area was larger than the T1, although the EA of the SS group was higher than that of the R group independently of the type of lesion. The SF from the SS group was greater than that from the R group, and at T2 the SF values were higher for both groups. AMELs and MIXs showed larger SF and A values; the SS group tended to have the greatest changes in the delimitations of the lesions at T2., Conclusions: The methodology allowed us to quantify differences in the spatial perceptions of professionals. The knowledge of the diagnosis and the expertise of examiners influenced the examiner's perception of the limits of the lesions independently of the actual biological behaviour of the lesion.

Objective: The aim of this study was to investigate the local and systemic expression of CC-chemokine ligand 3 (CCL3) and its receptors (CCR1 and CCR5) in tissue samples and peripheral blood mononuclear cells of recurrent aphthous stomatitis (RAS) patients., Study Design: This case-control study enrolled 29 patients presenting severe RAS manifestations and 20 non-RAS patients proportionally matched by sex and age. Total RNA was extracted from biopsy specimens and peripheral blood mononuclear cells for quatitative reverse-transcription polymerase chain reaction. The data obtained by relative quantification were evaluated by the 2(-ΔΔCt) method, normalized by the expression of an endogenous control, and analyzed by Student t test., Results: The results demonstrated overexpression in RAS tissue samples of all of the chemokines evaluated compared with healthy oral mucosa, whereas the blood samples showed only CCR1 overexpression in RAS patients., Conclusions: These findings suggest that the increased expression of CCL3, CCR1, and CCR5 may influence the immune response in RAS by T(H)1 cytokine polarization., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The aim was to analyze the effect of adipose tissue transplantation on growth differentiation factor-9 (GDF-9), insulin growth factor 1 receptor (IGF1R), and leptin receptor (LEPR) protein expression in ovaries of obese anovulatory mice. Leptin-deficient female (ob/ob) and wild-type mice were divided into untreated ob/ob mice and gonadal white adipose tissue transplanted ob/ob mice, with evaluation after 7, 15, and 45 days and compared to control wild-type mice. The corporal weight and glycemia levels increased in the obese group concomitant with polymicrocyst formation and abundant estrone, mimicking anovulatory disease. In the treated group after 45 days, glycemia, weight, ovarian size, and number of follicles were decreased and corpora lutea were decreased. The analysis of GDF-9 revealed that, whereas control ovaries presented follicular localization, the obese ovary lacked this protein. On the other hand, obese ovaries showed elevated expression of IGF1R that was normalized after the transplantation. Finally, LEPR was reduced in obese ovaries, and adipose tissue transplantation was efficient in returning it to normal levels. In conclusion, the adipose tissue transplantation, especially after 45 days, seems to stimulate ovulation, supported by the fact that several proteins involved in ovulation returned to basal levels.

The current study investigated the capacity for tumor factors secreted by head and neck squamous cell carcinoma (HNSCC) cell lines, KB, KB16, and HEP, to induce the secretion of various cytokines from peripheral blood mononuclear cells (PBMCs). PBMCs were isolated from blood samples collected from six healthy volunteers and these cells were incubated for 6, 24, 48, or 72 hours in the presence of 50% conditioned medium collected from cultured cell lines pretreated with, or without, stimulants such as phytohemagglutinin (PHA) or lipopolysaccharides (LPS). Aliquots of each supernatant were then assayed for levels of IFN-Γ, vascular endothelial growth factor (VEGF), TNF-α, and IL-4 using enzyme linked immunosorbent assays (ELISAs). Data collected were analyzed using Student's t-test, an ANOVA test followed by Tukey's test, and tests of Pearson's Correlation. PBMCs cultured with KB16-conditioned medium produced the highest levels of IFN-Γ. VEGF was also detected in conditioned media collected from all of the squamous cell carcinoma (SCC) cell lines used, and a significant difference in VEGF levels between control and KB- or KB16-conditioned media was observed. TNF-α was secreted by all PBMC groups within 6 hours of receiving conditioned media, and these levels increased up to the 24 hour timepoint, after which levels of TNF-α stabilized. In contrast, none of the supernatant samples contained detectable levels of IL-4. In combination, these data suggest that direct contact between fresh human PBMCs and conditioned media from tumor cells induces the secretion of TNF-α and VEGF by PBMCs, and this represents an initial angiogenic response.

Purpose: To study the proinflammatory and anti-inflammatory cytokines present in the acute phase of trinitrobenzene sulfonic acid (TNBS)-induced experimental colitis treated with Saccharomyces boulardii., Methods: Thirty male Wistar rats were divided into three groups: (1) treated group--received Saccharomyces boulardii for 14 days; (2) non-treated group--received sodium chloride solution for 14 days; (3) control group. Colitis was induced on the seventh day of the study in the treated and the non-treated groups using TNBS (10 mg) dissolved in 50% ethanol. Quantification of cytokines, including interleukin (IL)-1beta (IL-1beta), IL-6, transforming growth factor-beta (TGF-beta), IL-10 and tumor necrosis factor-alpha (TNF-alpha), in the serum and colonic tissue collected on day 14 were carried out using an enzyme-linked immunosorbent assay (ELISA)., Results: The mean concentrations of TGF-beta in both the serum and the colonic tissue of the treated group were statistically higher than that of the control group. The mean concentration of TGF-beta in the colonic tissue of the non-treated group was also statistically higher than the control group., Conclusion: The group treated with Saccharomyces boulardii showed increased amounts of TGF-beta, an anti-inflammatory cytokine, during the acute phase of colitis. There were no differences in the amount of TNF-alpha, IL-1beta, IL-6, and IL-10 between the treated and the non-treated or the control groups during the acute phase of experimental colitis induced by TNBS.

Purpose: Bacillus Calmette-Guerin (BCG) continues to be employed as the most effective immunotherapy against superficial bladder cancer. We have developed an rBCG-S1PT strain that induces a stronger cellular immune response than BCG. This preclinical study was designed to test the potential of rBCG-S1PT as an immunotherapeutic agent for intravesical bladder cancer therapy., Materials and Methods: A tumor was induced in C57BL/6 mice after chemical cauterization of the bladder and inoculation of the tumor cell line MB49. Next, mice were treated by intravesical instillation with BCG, rBCG-S1PT, or PBS once a week for 4 weeks. After 35 days, the bladders were removed and weighed, Th1 (IL-2, IL-12, INOS, INF-gamma, TNF-alpha), and Th2 (IL-5, IL-6, IL-10, TGF-beta) cytokine mRNA responses in individual mice bladders were measured by quantitative real time PCR, and the viability of MB49 cells in 18-hour coculture with splenocytes from treated mice was assessed. In an equivalent experiment, animals were observed for 60 days to quantify their survival., Results: Both BCG and rBCG-S1PT immunotherapy resulted in bladder weight reduction, and rBCG-S1PT increased survival time compared with the control group. There were increases in TNF-alpha in the BCG treated group, as well as increases in TNF-alpha and IL-10 mRNA in the rBCG-S1PT group. The viability of MB49 cells cocultured with splenocytes from rBCG-S1PT-treated mice was lower than in both the BCG and control groups., Conclusions: rBCG-S1PT therapy improved outcomes and lengthened survival times. These results indicate that rBCG could serve as a useful substitute for wild-type BCG., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

The peptides Tx2-5 and Tx2-6, isolated from the whole venom of "armed-spider"Phoneutria nigriventer venom, are directly linked with the induction of persistent and painful erection in the penis of mammals. The erection induced by Tx2-6 has been associated with the activation of nitric oxide synthases. There is a scarcity of studies focusing on the outcome of Tx2-6 at the molecular level, by this reason we evaluated the gene profile activity of this toxin at the nitric oxide (NO) pathway. After microarray analyses on cavernous tissue of mice inoculated with Tx2-6 we found that only 10.4% (10/96) of these genes were differentially expressed, showing a limited effect of the toxin on the NO pathway. We found the genes sparc, ednrb, junb, cdkn1a, bcl2, ccl5, abcc1 over-expressed and the genes sod1, s100a10 and fth1 under-expressed after inoculation of Tx2-6. The differential expressions of sparc and ednrb genes were further confirmed using real-time PCR. Interestingly, ednrb activates the L-arginine/NO/cGMP pathway that is involved in the relaxation of the cavernous body. Therefore the priapism induced by Tx2-6 is a consequence of a highly specific interference of this neurotoxin with the NO pathway.

Resazurin dye has been broadly used as indicator of cell viability in several types of assays for evaluation of the biocompatibility of medical and dental materials. Mitochondrial enzymes, as carriers of diaphorase activities, are probably responsible for the transference of electrons from NADPH + H+ to resazurin, which is reduced to resorufin. The level of reduction can be quantified by spectrophotometers since resazurin exhibits an absorption peak at 600 etam and resorufin at 570 etam wavelengths. However, the requirement of a spectrophotometer and specific filters for the quantification could be a barrier to many laboratories. Digital cameras containing red, green and blue filters, which allow the capture of red (600 to 700 etam) and green (500 to 600 etam) light wavelengths in ranges bordering on resazurin and resorufin absorption bands, could be used as an alternative method for the assessment of resazurin and resorufin concentrations. Thus, our aim was to develop a simple, cheap and precise method based on a digital CCD camera to measure the reduction of resazurin. We compared the capability of the CCD-based method to distinguish different concentrations of L929 and normal Human buccal fibroblast cell lines with that of a conventional microplate reader. The correlation was analyzed through the Pearson coefficient. The results showed a strong association between the measurements of the method developed here and those made with the microplate reader (r(2) = 0.996; p < 0.01) and with the cellular concentrations (r(2) = 0.965; p < 0.01). We concluded that the developed Colorimetric Quantification System based on CCD Images allowed rapid assessment of the cultured cell concentrations with simple equipment at a reduced cost.

Background: Toll-like receptors (TLR) are membrane proteins that recognize conserved molecules derived from bacterial, virus, fungal or host tissues. Activation of TLRs causes the production of cytokines that mediate inflammatory responses and drive T helper (Th) 1 and 2 cell development. As an exaggerated Th1 immune response is supposed to be involved in pathogenesis of Recurrent Aphthous Ulceration (RAU), we suggest that RAU patients may have an imbalance in TLR pathways., Methods: To study the function of TLR activation ex vivo, peripheral blood mononuclear cells (PBMCs) from RAU patients (n = 17) and controls (n = 17) were exposed to TLR2 [lipoteichoic acid (LTA), heat-killed Listeria monocytogenes (HKLM) and PamC3CSK4], TLR3 [Poly(I:C)], TLR4 [lipopolysaccharide (LPS)], TLR5 (flagellin) and TLR7 (imiquimod) ligands, and the time course of supernatant tumor necrosis factor-alpha (TNF-alpha) levels was quantified by enzyme-linked immunosorbent assay. In addition, serological and salivary TNF-alpha and soluble CD14 levels were quantified. The TNF-alpha produced by PBMCs in contact with each TLR ligand and autologous serum or saliva at the same time was also investigated. The data were analyzed by statistical multivariate tests., Results: The control group had a higher response to LTA, whereas RAU had a higher response to HKLM. LTA and LPS interfered with the salivary stimulation of the RAU PBMC and HKLM with the stimulation of the control. Autologous serum was capable of inhibiting TLR2 responsiveness to LTA and enhancing LPS stimulation. Salivary and serological levels of sCD14 and TNF-alpha were not significantly different., Conclusion: Recurrent Aphthous Ulceration patients have an anomalous activity of the TLR2 pathway that probably influences the stimulation of an abnormal Th1 immune response.

Background: Since successful treatment of superficial bladder cancer with BCG requires proper induction of Th1 immunity, we have developed a rBCG-S1PT strain that induced a stronger cellular immune response than BCG. This preclinical study was designed to compare the modulatory effects of BCG and rBCG-S1PT on bladder TNF-alpha and IL-10 expression and to evaluate antitumour activity., Methods: For Experiment I, the MB49 bladder cancer cell line was used in C57BL/6 mice. Chemical cauterization of the bladder was performed to promote intravesical tumor implantation. Mice were treated by intravesical instillation with BCG, rBCG-S1PT or PBS once a week for four weeks. After 35 days the bladders were removed and weighed. TNF- and IL-10 cytokine responses were measured by qPCR. Experiment II was performed in the same manner as Experiment I, except the animals were not challenged with MB49 tumor cells., Results: rBCG-S1PT immunotherapy resulted in bladder weight reduction, compared to the BCG and control group. There were increases in TNF-alpha in the BCG-treated group, as well as increases in TNF-alpha and IL-10 mRNA in the rBCG-S1PT group., Conclusion: These data indicate a significant reduction of bladder tumor volume for the rBCG group, compared to the BCG and PBS groups. This suggests that rBCG could be a useful substitute for wild-type BCG and that the potential modulation between TNF-alpha and IL-10 cytokine productions may have therapeutic value.

Background: Recently, abnormal cellular immune response has been considered responsible for the oral lesion in the recurrent aphthous ulceration (RAU). For reasons not yet defined, antigens of the oral microbiota would trigger abnormal Th1 immune response against epithelial cells. On the other hand, studies have demonstrated that heat shock proteins (HSP) can block the production of proinflammatory cytokine through inhibition of NF-kappaB and mitogen-activated protein kinase pathways or activate anti-inflammatory cytokines and therefore control the magnitude of the immune response. HSP27 has been considered a powerful inductor of IL-10, a major inhibitor of Th1 response., Methods: Using immunohistochemistry, we studied the expression and location of HSP27 and IL-10 in ulcerated lesions clinically diagnosed as RAU (n = 27) and to compare it with that of oral clinically normal mucosa (CT; n = 6) and of other inflammatory chronic diseases such as oral fibrous inflammatory hyperplasia (FIH; n = 18), Crohn's disease (CD; n = 10) and ulcerative colitis (UC; n = 9)., Results: A lower proportion of HSP27-positive epithelial cells in RAU and CD were observed when compared with CT and FIH (P < 0.001**; P = 0.013**). A lower proportion of IL-10-positive interstitial cells in RAU was observed when compared with FIH, UC, CT and CD (P < 0.001**; P < 0.001**; P < 0.001**; P = 0.034*)., Conclusion: Altogether the data suggest that a reduced cellular expression of HSP27 and IL-10 in RAU might be related with the aetiopathogenesis of the ulcerated oral lesions.

Purpose: We developed and characterized by histopathology and immunohistochemistry a syngeneic murine bladder tumor model derived from the MB49 tumor cell line., Materials and Methods: Bladder tumor implantation was achieved by intravesical instillation of 5 x 105 MB49 tumor cells in C57BL/6 mice. A chemical lesion of the bladder was performed in order to promote intravesical tumor implantation. The bladder wall lesion was accomplished by transurethral instillation of silver nitrate (AgNO3). After 15 days, the animals were sacrificed, examined macroscopically for intravesical tumor and bladder weight. Histology and immunohistochemistry were performed using cytokeratin 7 (CK7), carcinoembrionic antigen (Dako-CEA), p53 and c-erbB2 oncoprotein (Her2/neu)., Results: Twenty-nine out of 30 animals (96.7%) developed intravesical tumors in a 15-day period. Macroscopically, the mean bladder weight was 0.196g (0.069-0.538g), 10 to 15 times the normal bladder weight. The immunohistochemical analysis showed significant membrane expression of CEA and CK7: a similar finding for human urothelial cancer. We also characterized absence of expression of p53 and anti-Her2/neu in the murine model., Conclusions: High tumor take rates were achieved by using the chemical induction of the bladder tumor. Although electric cauterization is widely described in the literature for syngeneic orthotopic animal models, the technique described in this study represents an alternative for intravesical bladder tumor implantation. Moreover, the histopathology and immunohistochemical analysis of the murine bladder tumor model derived from the MB49 cell line showed a resemblance to human infiltrating urothelial carcinoma, allowing clinical inference from experimental immunotherapy testing.

Objective: To evaluate differential gene expression in penile tissue after treatment with the phosphodiesterase 5 (PDE5) inhibitor tadalafil, as of the three clinically available PDE5 inhibitors (sildenafil, tadalafil, and vardenafil) used for the treatment of erectile dysfunction (ED), tadalafil has a long half-life and low incidence of side-effects., Materials and Methods: In all, 32 adult rats were divided into two groups. The control group received 0.5 mL of drinking water alone, while the tadalafil group was treated with tadalafil at a dose of 0.27 mg/kg. At 4 h after treatment with water or tadalafil the rats were killed and the penile tissue was removed. The total RNA was isolated from the penile tissue from both groups and differentially expressed genes were identified by cDNA microarray analysis. To validate the expression data from the microarray analysis, quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry were used., Results: In all, 153 genes were differentially expressed between the control group and the tadalafil group. We validated the microarray results by quantitative PCR for the insulin-like growth factor binding protein 6 (IGFBP-6) gene and the neuronal calcium sensor 1 (NCS-1) gene, both of which were up-regulated in the tadalafil group, and for the natriuretic peptide receptor 1 (NPR-1) gene that was down-regulated in this group. Immunohistochemistry showed localization of the NCS-1 protein in sinusoid trabeculae of the corpus cavernosum in control and tadalafil-treated rats., Conclusions: There was differential expression in 153 genes after tadalafil treatment. Some of these genes such as IGFBP-6, NPR-1 and NCS-1, might result in new targets in the treatment of ED.

Our purpose, in the present work, was to further comprehend the genetic events underlying the response to steroids of human endometrium from the mRNA as well as protein expression point of view. In order to achieve this goal we undertook 10,000-oligonucleotide, three-dimensional microarray analysis, followed by immunohistochemistry, on human normal endometrium in the proliferative and secretory phases of the menstrual cycle. The results revealed that a myriad of genes involved in immune response, calcium metabolism and thyroid hormone response were frequently overexpressed in the second or luteal phase of the menstrual cycle. During the follicular phase, in contrast, overexpression of genes was mainly restricted to those encoding proteins involved in cell proliferation.

Background: Making diagnoses in oral pathology are often difficult and confusing in dental practice, especially for the less-experienced dental student. One of the most promising areas in bioinformatics is computer-aided diagnosis, where a computer system is capable of imitating human reasoning ability and provides diagnoses with an accuracy approaching that of expert professionals. This type of system could be an alternative tool for assisting dental students to overcome the difficulties of the oral pathology learning process. This could allow students to define variables and information, important to improving the decision-making performance. However, no current open data management system has been integrated with an artificial intelligence system in a user-friendly environment. Such a system could also be used as an education tool to help students perform diagnoses. The aim of the present study was to develop and test an open case-based decision-support system., Methods: An open decision-support system based on Bayes' theorem connected to a relational database was developed using the C++ programming language. The software was tested in the computerisation of a surgical pathology service and in simulating the diagnosis of 43 known cases of oral bone disease. The simulation was performed after the system was initially filled with data from 401 cases of oral bone disease., Results: The system allowed the authors to construct and to manage a pathology database, and to simulate diagnoses using the variables from the database., Conclusion: Combining a relational database and an open decision-support system in the same user-friendly environment proved effective in simulating diagnoses based on information from an updated database.

Paracoccidioidomycosis, a systemic mycosis, is rarely diagnosed in its initial phase and can remain latent for up to 40 years. Although PCR is sensitive for the identification of Paracoccidioides brasiliensis (Pb) in different samples, no study using paraffin-embedded human tissue has been published. The size of the amplicon, the fixation method and the time of the storage may affect the reaction. Recently the more sensitive Primer-Extension-Preamplification (PEP)-Nested-PCR has been used for amplification of small samples. Our aims were to detect Pb in paraffin embedded biopsies using (PEP)-Nested-PCR and to correlate the data with histopathological parameters. Analyses were carried out in 107 biopsies from tegument, lymph node, lung and tongue. The fungal DNA was detected in 29.9% of the biopsies by (PEP)-nested-PCR against 5% of Nested-PCR. The positivity correlated with numbers of fungi and fungal viable cells, and there was no correlation with the granuloma pattern.

Background: Chronic actinic cheilitis (AC) is a precursor of squamous cell carcinoma (SCC) of the lip., Objectives: To evaluate the histopathological characteristics that may help to identify AC more susceptible to carcinomatous transformation, to assess the p53 protein expression in AC, and to determine the value of the p53 expression as a marker of transformation into SCC of the lip., Methods: Seventy cases of chronic AC were reviewed, 31 of which were associated with SCCs. The samples were obtained from pathology reports of AC and SCC of the lip. Histopathology and immunohistochemical expression of the p53 protein were evaluated in isolated AC and in AC adjacent to SCC., Results: The intensity of the inflammatory infiltrate in the corium was the only histopathological finding significantly associated both with the presence of an invasive tumor and with the degree of epithelial atypia. Most AC (85%) were immunoreactive to the p53 protein. The p53 protein expression in cheilitis was not statistically associated with any other histopathological criteria., Conclusions: An intense inflammatory infiltrate in AC was predictive of an adjacent invasive SCC. In this study, the p53 protein immunoreactivity was not a marker of malignant transformation.

To investigate the molecular events involved in the pathogenesis and/or progression of thyroid tumors, we compared the gene expression profiles of three thyroid carcinoma cell lines, which represent major tumor subtypes of thyroid cancer and normal thyroid tissue. Using cDNA array methodology, we investigated the expression of 1807 open reading frame expressed sequence tags (ORESTES), selected from head and neck tumor libraries generated through the Brazilian Human Cancer Project-LICR/FAPESP. We found that 505 transcripts were differentially expressed in the thyroid carcinoma cell lines. Using a more stringent criterion, transcripts underexpressed or overexpressed more than fivefold in 1 of 3 or 3 of 3 carcinoma cell lines, a list of 55 ESTs were detected. Five candidate genes were further validated by quantitative polymerase chain reaction (qPCR) in an independent set of 52 thyroid tumors and 22 matched normal thyroid tissues. DCN was found underexpressed in a high percentage of the follicular thyroid adenomas, follicular thyroid carcinomas, and follicular variant of papillary thyroid carcinomas. DIO1 and DIO2 were underexpressed in nearly all papillary thyroid carcinomas. These genes not only could help to better define a tumor signature for thyroid tumors, but may, in part, also become useful as potential targets for thyroid tumor treatment.

Our purpose was to identify tamoxifen (TAM) responsive genes after 30 days of TAM treatment in tumor tissues obtained from women with breast cancer using microarray expression analysis. In our study, we identified 12 candidates to be considered as tamoxifen-modulated genes. Among them, we selected two candidates the TEGT BI-1 (testis enhanced gene transcript Bax Inhibitor-1) and the CD63 gene in order to further confirm their differential expression under tamoxifen effects. We observed that both were down-regulated in tumor tissues of patients during TAM treatment. TEGT is able to inhibit the expression of Bax, which is known to promote apoptosis. On the other hand, CD63 encodes a cell membrane protein and it seems to be involved in mechanisms of platelet activation, cell adhesion and cell motility. We therefore hypothesize that TAM would be able to modulate tumor growth by down-regulating genes involved in mechanisms such as cell cycle control, tumor invasion and metastasis.

Paracoccidioidomycosis (PCM) is a severe disease caused by the dimorphic fungus Paracoccidioides brasiliensis, which is characterized by granulomatous pulmonary and systemic lesions, affecting mainly men between 20 and 60 years of age. Reports of PCM disease in animals are rare, but the disease has been described in armadillos. On the other hand, PCM infection of domestic and wild animals detected by serological or cutaneous tests in the absence of apparent disease has been frequently reported. We present here the case of a female adult Doberman that developed cervical lymphadenomegaly. Histopathological examination of a cervical biopsy specimen revealed active PCM, with an epithelioid, granulomatous inflammation containing numerous yeast-like, multiple budding fungal forms. The diagnosis of PCM was confirmed by immunohistochemistry using a specific antibody anti-gp43 and by nested PCR using primers for the amplification of the gp43 gene region. This is the first report of PCM disease occurring in a dog, an animal that has been shown to play an important role in the natural history of North American blastomycosis.

Ischemia-reperfusion (IR) injury is a common early feature that contributes to graft damage by impairing resident cell function. Our previous results showed that IR injury impaired renal function, by causing extensive tubular necrosis and increasing MHC class II and ICAM-1 molecule expression by mesangial cells (MC). MCs are likely candidates to come into close contact with immune cells such as monocytes or lymphocytes. It has been suggested that under inflammatory circumstances, there is increased MC expression of MHC class II, of adhesion molecules (such as ICAM-1), of cytokines receptors, and of molecules associated with cellular death (apoptosis). The immunosuppressive properties of FTY720 have been shown in clinical and experimental situations. It has also been shown to be protective against IR injury in rats. We sought to evaluate the role of FTY720 in a murine IR model by measuring renal function, tubular necrosis, and surface molecule expression by cultured mesangial cells. Intravenous administration of FTY720 (1 mg/kg) immediately before IR induction did not improve the short-term (24 hours) outcome of renal function or reduced MHC class II and ICAM-1 surface molecule expression. However, there was a decreased percentage of tubular necrosis in mice treated with FTY720 (51.3% +/- 1.6%) compared with vehicle-treated mice (66% +/- 5.5%). These results suggest a protective role of FTY720 in an IR injury model. More studies are required to identify the mechanisms involved in the protective activity of FTY720 in the IR injury model.

Background: The reduced ability to activate oral tolerance plays a role in the pathogenesis of some gastrointestinal inflammatory diseases. This activation may reflect a preferential reduction of a T-helper (Th)2- or Th3-type response. In recurrent aphthous ulceration (RAU), genetic and environmental factors may contribute to low tolerance, permitting a cytotoxic reaction against the oral epithelium. The cytokine profile has not permitted the definition of RAU as resulting from enhanced Th1 or Th2 responses. A cDNA microarray study would allow the identification of differentially expressed genes and provide a basis for classification of the immune response., Methods: The cDNA from 29 samples of aphthae and from 11 samples of normal mucosa from aphthae-free volunteers were hybridized on microarray membranes with 1176 genes., Results: Forty-one differentially expressed genes were identified, and a higher expression level of the Th1 gene cluster in RAU was found., Conclusions: Microarrays permitted us definition of the gene expression profile of the lesion and identify an increased Th1 activity in RAU lesions.

Insulin-like growth factor (IGF) binding protein 5 (IGFPB-5) is abundant in the uterus and is implicated in the sex steroid-induced growth of this tissue. The purpose of this study was to investigate the potential for modulation of the action of IGFPB-5 at the uterus level in the rat by estrogen and selective estrogen receptor modulators (SERMs). One hundred and twenty adult rats, 2-3 months of age, were included. Among them 100 animals were ovariectomized 4 days prior to the use of drugs for 48 days. Rats were divided into six groups: non-ovariectomized (group 1); ovariectomized (group 2); and those receiving conjugated equine estrogens, 50 microg/day (group 3), tamoxifen 250 microg/day (group 4), raloxifene 3 mg/kg (group 5) and toremifene 2.5 mg/kg (group 6). Total RNA was isolated from the uterus and IGFBP-5 mRNA levels were assessed by the semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Our results demonstrate that conjugated equine estrogens were able to up-regulate mRNA levels of the IGFBP-5 gene, while oophorectomy alone as well as associated with hormone therapy such as tamoxifen, raloxifene and toremifene resulted in down-regulation of uterine IGFBP-5 gene expression. The up-regulation of IGFBP-5 expression induced by estrogens suggests that, in vivo, the uterotrophic effects of estrogen replacement therapy are mediated, at least inpart, by the IGF pathways. On the other hand, the use of SERMs (tamoxifen, raloxifene and toremifene) was associated with severe down-regulation of this gene at the transcription level.

Introduction: In the present study, our aim was to identify differentially expressed genes involved in estrogen actions at the endometrium level in rats., Methods: Thirty adult rats were ovariectomized four days prior to drug administration for 48 days. Rats were divided in 2 groups: I, control and II, conjugated equine estrogens (CCE). Total RNA was isolated from uterus, and differential expression was analyzed by array technology and RT-PCR., Results: A total of 32 candidate genes were shown to be upregulated or downregulated in groups I or II. Among them, differential expression was already confirmed by RT-PCR for IGFBP5, S12, c-kit, and VEGF, genes whose expression was up regulated during CCE therapy, and casein kinase II and serine kinase expression was the same level in both groups., Conclusion: We have demonstrated that cDNA array represents a powerful approach to identify key molecules in the estrogens therapy. A number of the candidates reported here should provide new markers that may contribute to the detection of target estrogen receptor. This information may also aid the development of new approaches to therapeutic intervention.