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Machado-Joseph disease (MJD) is a neurodegenerative disorder caused by an abnormal expansion of citosine-adenine-guanine trinucleotide repeats in the disease-causing gene. This mutation leads to an abnormal polyglutamine tract in the protein ataxin-3 (Atx3), resulting in formation of mutant Atx3 aggregates. Despite several attempts to develop a therapeutic option for MJD, currently there are no available therapies capable of delaying or stopping disease progression. Recently, our group reported that reducing the expression levels of mutant Atx3 lead to a mitigation of several MJD-related behavior and neuropathological abnormalities. Aiming a more rapid translation to the human clinics, in this study we investigate a pharmacological inhibitor of translation-cordycepin-in several preclinical models. We found that cordycepin treatment significantly reduced (i) the levels of mutant Atx3, (ii) the neuropathological abnormalities in a lentiviral mouse model, (iii) the motor and neuropathological deficits in a transgenic mouse model and (iv) the number of ubiquitin aggregates in a human neural model. We hypothesize that the effect of cordycepin is mediated by the increase of phosphorylated adenosine monophosphate-activated protein kinase (AMPK) levels, which is accompanied by a reduction in the global translation levels and by a significant activation of the autophagy pathway. Overall, this study suggests that cordycepin might constitute an effective and safe therapeutic approach for MJD, and probably for the other polyglutamine diseases.

Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is an incurable disorder, widely regarded as the most common form of spinocerebellar ataxia in the world. MJD/SCA3 arises from mutation of the ATXN3 gene, but this simple monogenic cause contrasts with the complexity of the pathogenic mechanisms that are currently admitted to underlie neuronal dysfunction and death. The aberrantly expanded protein product - ataxin-3 - is known to aggregate and generate toxic species that disrupt several cell systems, including autophagy, proteostasis, transcription, mitochondrial function and signalling. Over the years, research into putative therapeutic approaches has often been devoted to the development of strategies that counteract disease at different stages of cellular pathogenesis. Silencing the pathogenic protein, blocking aggregation, inhibiting toxic proteolytic processing and counteracting dysfunctions of the cellular systems affected have yielded promising ameliorating results in studies with cellular and animal models. The current review analyses the available studies dedicated to the investigation of MJD/SCA3 pathogenesis and the exploration of possible therapeutic strategies, focusing primarily on gene therapy and pharmacological approaches rooted on the molecular and cellular mechanisms of disease., (© 2018 International Society for Neurochemistry.)

Chromosome segregation errors during meiosis result in the formation of aneuploid gametes and are the leading cause of pregnancy loss and birth defects in humans. Proper chromosome segregation requires pairwise associations of maternal and paternal homologous chromosomes. Chiasmata, which are the cytological manifestations of crossovers (COs), provide a physical link that holds the homologs together as a pair, facilitating their orientation on the spindle at meiosis I. Although CO-promoting activities ensure a balanced number and position of COs, their identity and mechanism of action in mammals remain understudied. Previous work in yeast and Arabidopsis has shown that Zip2 and Shoc1 are ortholog proteins with an important role in promoting the formation of COs. Our work is the first study in mammals showing the in vivo and in vitro function of mouse and human SHOC1. We show that purified recombinant human SHOC1, an XPF/MUS81 family member, preferentially binds branched DNA molecules but apparently lacks in vitro endonuclease activity, despite its conserved ERCC4-(HhH)2 core structure. Cytological observations suggest that initial steps of recombination are normal in a majority of spermatocytes from SHOC1 hypomorphic mice. However, late stages of recombination appear abnormal, as chromosomal localization of MLH1 is reduced. In agreement, chiasma formation is reduced, and cells arrest at metaphase I with a few lagging chromosomes and subsequent apoptosis. This analysis of SHOC1-deficient mice and the selective localization of SHOC1 to a subset of recombination sites show that SHOC1 acts at key mid-stage steps of the CO formation process. The formation of chromosome axial elements and homologous pairing are apparently normal, but synapsis is altered with SYCP1 frequently failing to extend the full length of the chromosome axes. Finally, we describe that SHOC1 interacts with TEX11, another protein important for the formation of COs, connecting SHOC1 to chromosome axis and structure., Competing Interests: The authors have declared that no competing interest exist.

A DAND5-control human iPSC line was generated from the urinary cells of a phenotypically normal donor. Exfoliated renal epithelial (RE) cells were collected and reprogrammed into iPSCs using Sendai virus reprogramming system. The pluripotency, in vitro differentiation potential, karyotype stability, and the transgene-free status of generated iPSC line were analyzed and confirmed. This cell line can be exploited as a control iPSC line to better understand the mechanisms involved in DAND5-associated cardiac disease., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Machado-Joseph disease (MJD) is a dominantly inherited disorder originally described in people of Portuguese descent, and associated with the expansion of a CAG tract in the coding region of the causative gene MJD1/ATX3. The CAG repeats range from 10 to 51 in the normal population and from 55 to 87 in SCA3/MJD patients. MJD1 encodes ataxin-3, a protein whose physiological function has been linked to ubiquitin-mediated proteolysis. Despite the identification of the causative mutation, the pathogenic process leading to the neurodegeneration observed in the disease is not yet completely understood. In the past years, several studies identified different molecular mechanisms and cellular pathways as being impaired or deregulated in MJD. Autophagy, proteolysis or post-translational modifications, among other processes, were implicated in MJD pathogenesis. From these studies it was possible to identify new targets for therapeutic intervention, which in some cases proved successful in models of disease.

Polyglutamine diseases are hereditary degenerative disorders of the nervous system that have remained, to this date, untreatable. Promisingly, investigation into their molecular etiology and the development of increasingly perfected tools have contributed to the design of novel strategies with therapeutic potential. Encouraging studies have explored gene therapy as a means to counteract cell demise and loss in this context. The current chapter addresses the two main focuses of research in the area: the characteristics of the systems used to deliver nucleic acids to cells and the molecular and cellular actions of the therapeutic agents. Vectors used in gene therapy have to satisfyingly reach the tissues and cell types of interest, while eliciting the lowest toxicity possible. Both viral and non-viral systems have been developed for the delivery of nucleic acids to the central nervous system, each with its respective advantages and shortcomings. Since each polyglutamine disease is caused by mutation of a single gene, many gene therapy strategies have tried to halt degeneration by silencing the corresponding protein products, usually recurring to RNA interference. The potential of small interfering RNAs, short hairpin RNAs and microRNAs has been investigated. Overexpression of protective genes has also been evaluated as a means of decreasing mutant protein toxicity and operate beneficial alterations. Recent gene editing tools promise yet other ways of interfering with the disease-causing genes, at the most upstream points possible. Results obtained in both cell and animal models encourage further delving into this type of therapeutic strategies and support the future use of gene therapy in the treatment of polyglutamine diseases.

Polyglutamine (polyQ) diseases are a family of neurodegenerative disorders with very heterogeneous clinical presentations, although with common features such as progressive neuronal death. Thus, at the time of diagnosis patients might present an extensive and irreversible neuronal death demanding cell replacement or support provided by cell-based therapies. For this purpose stem cells, which include diverse populations ranging from embryonic stem cells (ESCs), to fetal stem cells, mesenchymal stromal cells (MSCs) or induced pluripotent stem cells (iPSCs) have remarkable potential to promote extensive brain regeneration and recovery in neurodegenerative disorders. This regenerative potential has been demonstrated in exciting pre and clinical assays. However, despite these promising results, several drawbacks are hampering their successful clinical implementation. Problems related to ethical issues, quality control of the cells used and the lack of reliable models for the efficacy assessment of human stem cells. In this chapter the main advantages and disadvantages of the available sources of stem cells as well as their efficacy and potential to improve disease outcomes are discussed.

A human iPSC line was generated from exfoliated renal epithelial (ERE) cells of a patient affected with Congenital Heart Disease (CHD) and Laterality Defects carrying tshe variant p.R152H in the DAND5 gene. The transgene-free iPSCs were generated with the human OSKM transcription factor using the Sendai-virus reprogramming system. The established iPSC line had the specific heterozygous alteration, a stable karyotype, expressed pluripotency markers and generated embryoid bodies that can differentiate towards the three germ layers in vitro. This iPSC line offers a useful resource to study the molecular mechanisms of cardiomyocyte proliferation, as well as for drug testing., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

The consequences of treatment involving the use of a natural product and whole body vibration (WBV) exercise have been investigated. The aim of the present study was to evaluate the effects of the joint treatment with an aqueous extract of Chenopodium ambrosioides and WBV on physiological parameters in rats. Wistar rats ( n =20) were divided equally into four groups: control group (CG), treated with C. ambrosioides (CHE) group, exposed to 50 Hz of mechanial vibration (VBR), and treated with C. ambrosioides and exposed to 50 Hz of mechanical vibration (VBR + CHE) daily for 6 weeks. The body mass of the animals was determined weekly, the feed intake and the stool consistency were measured daily. One day after the 6 weeks of treatment, samples of blood were collected and used for biochemical analysis. Along 6 weeks, there was an increase ( P <0.001) in the feed intake in VBR group and a decrease in the CHE group in comparison with other groups. The levels of the enzyme aspartate aminotransferase (AST) in VBR + CHE group decreased ( P <0.05) in comparison with other groups. No differences were found in body mass and stool consistency. WBV altered the feed intake without directly affecting the body mass. Moreover, WBV in association with C. ambrosioides caused alteration in the enzymatic activity of AST., (© 2017 The Author(s).)

The aim of the present study was to evaluate the effect of the association of whole body vibration (WBV) exercise with an aqueous extract of coriander on the biodistribution of the radiopharmaceutical sodium pertechnetate, on the concentration of some plasma biomarker, on the feed intake, on the body mass, and on the stool consistency in rats. Rats were divided in four groups and submitted to different treatments for 40 days. The control group (CON) received deionized water. The group treated with coriander (COR) received the extract of coriander. The rats that were exposed to WBV exercises (WBV-E) also received deionized water. A group of animals received coriander and was exposed to WBV (COR + WBV-E). We found in testis a decrease (0.13 ± 0.01 to 0.06 ± 0.03) of the percentages of injected radioactivity per gram (%ATI/g) in the WBV-E in comparison with the COR. There is no significant alteration on the concentrations of the plasma biomarkers. The feed intake showed a statistically significant increase in WBV-E. No significant difference on the body mass was found. The stool analysis showed a statistical difference on the consistency between COR (hard and dry, darker) and all the other groups (normal). In conclusion, it was verified that possible modifications in some biochemical/physiological parameters of the rats submitted to WBV exercise would be capable to increase the feed intake without changing the body mass, and normalizing the stool consistency altered by the coriander supplementation. Further studies are needed to try to understand better the biological effects involving the association of WBV exercise and coriander., (© 2017 The Author(s).)

Objective:: To reflect about continuing education from the perspective of Augustine of Hippo and his theories based on the construction of knowledge and the learning process., Method:: This is a theoretical reflection study whose aim is to propose dimensions of analysis, emphasizing the history and challenges of continuing education. Such dimensions analyze the production of knowledge in Augustinian pedagogy, its historical aspects and its close relationship with lifelong education in health professions., Results:: The results show the difficulty of continuing education to reach adequate importance in health services, and that of academia in appropriating the thoughts of renowned philosophers such as Augustine. This is a result of ignorance about the convergence of these principles and their relevance., Final Considerations:: Continuing education and Augustinian pedagogy walk hand in hand in terms of care, meeting the needs that originate from practice and that are reflected in it, challenging isolated knowledge and putting different areas of knowledge to work together., Objetivo:: Refletir sobre a educação permanente na perspectiva de Agostinho de Hipona e suas teorias pautadas na construção do conhecimento e no processo de aprendizagem., Método:: Trata-se de uma reflexão teórica cujo intuito é propor dimensões de análise, enfatizando a história e os desafios da educação permanente. Tais dimensões analisam a produção do conhecimento na pedagogia agostiniana, seus aspectos históricos e sua relação próxima com a educação permanente em saúde., Resultados:: Revela a dificuldade da educação permanente em ter sua dimensão alcançada noserviço de saúde, tal qual a da academia de apropriar-se do pensamento de filósofos renomados como Agostinho, fruto do desconhecimento da convergência desses pressupostos e de sua relevância., Considerações Finais:: Considera-se que a educação permanente e a pedagogia agostiniana caminham juntas no cuidar, atendendo às necessidades advindas da prática e nela refletida, confrontando os saberes isolados e com eles operando em conjunto.

Proteolytic cleavage has been implicated in the pathogenesis of diverse neurodegenerative diseases involving abnormal protein accumulation. Polyglutamine diseases are a group of nine hereditary disorders caused by an abnormal expansion of repeated glutamine tracts contained in otherwise unrelated proteins. When expanded, these proteins display toxic properties and are prone to aggregate, but the mechanisms responsible for the selective neurodegeneration observed in polyglutamine disease patients are still poorly understood. It has been suggested that the neuronal toxicity of polyglutamine-expanded proteins is associated with the production of deleterious protein fragments. This review aims at discussing the involvement of proteolytic cleavage in the six types of spinocerebellar ataxia caused by polyglutamine expansion of proteins. The analysis takes into detailed consideration evidence concerning fragment detection and the mechanisms of fragment toxicity. Current evidence suggests that the proteins involved in spinocerebellar ataxia types 3, 6 and 7 give rise to stable proteolytic fragments. Fragments carrying polyglutamine expansions display increased tendency to aggregate and toxicity, comparing with their non-expanded counterparts or with the correspondent full-length expanded proteins. Data concerning spinocerebellar ataxia types 1, 2 and 17 is still scarce, but available results afford further investigation. Available literature suggests that proteolytic cleavage of expanded polyglutamine-containing proteins enhances toxicity in disease-associated contexts and may constitute an important step in the pathogenic cascade of polyglutamine diseases. Countering protein fragmentation thus presents itself as a promising therapeutic aim., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Tuberculosis (TB) is a major public health concern worldwide; however the factors that account for resistance or susceptibility to disease are not completely understood. Although some studies suggest that the differential expression of miRNAs in peripheral blood of TB patients could be useful as biomarkers of active disease, their involvement during the inflammatory process in lungs of infected individuals is unknown. Here, we evaluated the global expression of miRNAs in the lungs of mice experimentally infected with Mycobacterium tuberculosis on 30 and 60 days post-infection. We observed that several miRNAs were differentially expressed compared to uninfected mice. Furthermore, we verified that the expression of miR-135b, miR-21, miR-155, miR-146a, and miR-146b was significantly altered in distinct leukocyte subsets isolated from lungs of infected mice, while genes potentially targeted by those miRNAs were associated with a diversity of immune related molecular pathways. Importantly, we validated the inhibition of Pellino 1 expression by miR-135b in vitro. Overall, this study contributes to the understanding of the dynamics of miRNA expression in lungs during experimental TB and adds further perspectives into the role of miRNAs on the regulation of immune processes such as leukocyte activation., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

GABA (γ-aminobutyric acid) is the major inhibitory neurotransmitter in the central nervous system, and changes in GABAergic neurotransmission modulate the activity of neuronal networks. Gephyrin is a scaffold protein responsible for the traffic and synaptic anchoring of GABAA receptors (GABAAR); therefore, changes in gephyrin expression and oligomerization may affect the activity of GABAergic synapses. In this work, we investigated the changes in gephyrin protein levels during brain ischemia and in excitotoxic conditions, which may affect synaptic clustering of GABAAR. We found that gephyrin is cleaved by calpains following excitotoxic stimulation of hippocampal neurons with glutamate, as well as after intrahippocampal injection of kainate, giving rise to a stable cleavage product. Gephyrin cleavage was also observed in cultured hippocampal neurons subjected to transient oxygen-glucose deprivation (OGD), an in vitro model of brain ischemia, and after transient middle cerebral artery occlusion (MCAO) in mice, a model of focal brain ischemia. Furthermore, a truncated form of gephyrin decreased the synaptic clustering of the protein, reduced the synaptic pool of GABAAR containing γ2 subunits and upregulated OGD-induced cell death in hippocampal cultures. Our results show that excitotoxicity and brain ischemia downregulate full-length gephyrin with a concomitant generation of truncated products, which affect synaptic clustering of GABAAR and cell death.

Machado Joseph Disease (MJD) is the most frequent autosomal dominantly inherited cerebellar ataxia caused by the over-repetition of a CAG trinucleotide in the ATXN3 gene. This expansion translates into a polyglutamine tract within the ataxin-3 protein that confers a toxic gain-of-function to the mutant protein ataxin-3, contributing to protein misfolding and intracellular accumulation of aggregates and neuronal degeneration. Autophagy impairment has been shown to be one of the mechanisms that contribute for the MJD phenotype. Here we investigated whether this phenotype was present in patient-derived fibroblasts, a common somatic cell type used in the derivation of induced pluripotent stem cells and subsequent differentiation into neurons, for in vitro disease modeling. We generated and studied adult dermal fibroblasts from 5 MJD patients and 4 healthy individuals and we found that early passage MJD fibroblasts exhibited autophagy impairment with an underlying mechanism of decreased autophagosome production. The overexpression of beclin-1 on MJD fibroblasts reverted partially autophagy impairment by increasing the autophagic flux but failed to increase the levels of autophagosome production. Overall, our results provide a well-characterized MJD fibroblast resource for neurodegenerative disease research and contribute for the understanding of mutant ataxin-3 biology and its molecular consequences.

In a clinical setting, where multiple administrations of the therapeutic agent are usually required to improve the therapeutic outcome, it is crucial to assess the immunogenicity of the administered nanoparticles. In this data work, we investigated the safety profile of the repeated intravenous administration of brain-targeted stable nucleic acid lipid particles (RVG-9r-targeted SNALPs). To evaluate local activation of the immune system, we performed analysis of mouse tissue homogenates and sections from cerebellum. To investigate peripheral activation of the immune system, we used serum of mice that were intravenously injected with RVG-9r-targeted SNALPs. These data are related and were discussed in the accompanying research article entitled "Intravenous administration of brain-targeted stable nucleic acid lipid particles alleviates Machado-Joseph disease neurological phenotype" (Conceição et al., in press) [1].

Machado-Joseph disease is a progressive neurodegenerative disorder associated with the polyQ-expanded ataxin-3 (encoded by ATXN3), for which no therapy is available. With the aim of clarifying the mechanism of neurodegeneration, we hypothesized that the abnormally long polyQ tract would interact aberrantly with ataxin-2 (encoded by ATXN2), another polyQ protein whose function has recently been linked to translational regulation. Using patient's samples and cellular and animal's models we found that in Machado-Joseph disease: (i) ataxin-2 levels are reduced; and (ii) its subcellular localization is changed towards the nucleus. Restoring ataxin-2 levels by lentiviral-mediated overexpression: (i) reduced mutant ataxin-3 levels; and (ii) rescued behaviour defects and neuropathology in a transgenic mouse model of Machado-Joseph disease. Conversely (i) mutating the ataxin-2 motif that enables binding to its natural interactor and translation activator poly(A)-binding protein; or (ii) overexpressing poly(A)-binding protein, had opposite effects, increasing mutant ataxin-3 translation and aggregation. This work suggests that in Machado-Joseph disease, mutant ataxin-3 drives an abnormal reduction of ataxin-2 levels, which overactivates poly(A)-binding protein, increases translation of mutant ataxin-3 and other proteins and aggravates Machado-Joseph disease. Re-establishment of ataxin-2 levels reduces mutant ataxin-3 and alleviates Machado-Joseph disease pathogenesis opening a new avenue for therapeutic intervention in this and potentially other polyQ disorders., (© The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

In DNA vaccines, the gene of interest is cloned into a bacterial plasmid that is engineered to induce protein production for long periods in eukaryotic cells. Previous research has shown that the intramuscular immunization of BALB/c mice with a naked plasmid DNA fragment encoding the Mycobacterium leprae 65-kDa heat-shock protein (pcDNA3-Hsp65) induces protection against M. tuberculosis challenge. A key stage in the protective immune response after immunization is the generation of memory T cells. Previously, we have shown that B cells capture plasmid DNA-Hsp65 and thereby modulate the formation of CD8+ memory T cells after M. tuberculosis challenge in mice. Therefore, clarifying how B cells act as part of the protective immune response after DNA immunization is important for the development of more-effective vaccines. The aim of this study was to investigate the mechanisms by which B cells modulate memory T cells after DNA-Hsp65 immunization. C57BL/6 and BKO mice were injected three times, at 15-day intervals, with 100 µg naked pcDNA-Hsp65 per mouse. Thirty days after immunization, the percentages of effector memory T (TEM) cells (CD4+ and CD8+/CD44high/CD62Llow) and memory CD8+ T cells (CD8+/CD44high/CD62Llow/CD127+) were measured with flow cytometry. Interferon γ, interleukin 12 (IL-12), and IL-10 mRNAs were also quantified in whole spleen cells and purified B cells (CD43-) with real-time qPCR. Our data suggest that a B-cell subpopulation expressing IL-10 downregulated proinflammatory cytokine expression in the spleen, increasing the survival of CD4+ TEM cells and CD8+ TEM/CD127+ cells.

Isoflavones are phytoestrogens reported to be potent antioxidant agents. In contrast, the antileishmanial meglumine antimoniate has mutagenic activities. This study evaluated the ability of soy isoflavones to reduce DNA damage induced by meglumine antimoniate. Antimutagenic effects (by micronucleus test) were tested using Swiss mice divided into seven groups treated with meglumine antimoniate (425 mg/kg bw pentavalent antimony); cyclophosphamide (50 mg/kg bw); water (negative control); single isoflavones dose (1.6 mg/kg bw), and three groups received one dose of isoflavones via gavage (0.4 mg/kg bw, 0.8 mg/kg bw or 1.6 mg/kg bw) plus meglumine antimoniate via intraperitoneal, simultaneously. To evaluate antigenotoxicity (by Comet assay), each group with 10 animals received the above-mentioned control doses; single dose of isoflavones 0.8 mg/kg bw, and three groups received isoflavones (0.8 mg/kg bw) by gavage along with intraperitoneal meglumine antimoniate, which were treated with isoflavones 24 h before or after receiving meglumine antimoniate (pre-treatment and post-treatment, respectively) or simultaneously. Cells were harvested 24 h after the treatment, and the data were evaluated by ANOVA followed by Tukey's test (p < 0.05). The data from the simultaneous treatment by micronucleus test revealed that isoflavones (0.4 and 0.8 mg/kg) were able to reverse the mutagenic effect of Glucantime. Moreover, all regimes of the treatment with 0.8 mg/kg bw dose were able to reduce the genotoxicity caused by meglumine antimoniate. It is suggested that the protective effect of isoflavones against DNA damage is related to their ability to reduce oxidative stress caused by the trivalent Sb(III) metabolite of meglumine antimoniate.

MicroRNAs (miRNAs) have emerged as a class of small, endogenous, regulatory RNAs that exhibit the ability to epigenetically modulate the translation of mRNAs into proteins. This feature enables them to control cell phenotypes and, consequently, modify cell function in a disease context. The role of inflammatory miRNAs in Alzheimer's disease (AD) and their ability to modulate glia responses are now beginning to be explored. In this study, we propose to disclose the functional role of miR-155, one of the most well studied immune-related miRNAs in AD-associated neuroinflammatory events, employing the 3xTg AD animal model. A strong upregulation of miR-155 levels was observed in the brain of 12-month-old 3xTg AD animals. This event occurred simultaneously with an increase of microglia and astrocyte activation, and before the appearance of extracellular Aβ aggregates, suggesting that less complex Aβ species, such as Aβ oligomers may contribute to early neuroinflammation. In addition, we investigated the contribution of miR-155 and the c-Jun transcription factor to the molecular mechanisms that underlie Aβ-mediated activation of glial cells. Our results suggest early miR-155 and c-Jun upregulation in the 3xTg AD mice, as well as in Aβ-activated microglia and astrocytes, thus contributing to the production of inflammatory mediators such as IL-6 and IFN-β. This effect is associated with a miR-155-dependent decrease of suppressor of cytokine signaling 1. Furthermore, since c-Jun silencing decreases the levels of miR-155 in Aβ-activated microglia and astrocytes, we propose that miR-155 targeting can constitute an interesting and promising approach to control neuroinflammation in AD., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Tephrosia cinerea L. (Pers.) is a tropical species that exhibits antileishmanial activity in Leishmania amazonensis promastigote cultures and is commonly used to treat infections, inflammations, ulcers, nervous conditions, and diarrhea. However, no studies have investigated its effects on genetic material. Therefore, we evaluated the genotoxic potential, antigenotoxic potential, and cytotoxic effects of hydroalcoholic extracts of T. cinerea leaves. In an in vitro genotoxicity study, human peripheral blood leukocytes were treated for 3, 24 (comet assay), or 48 h (cell death assay) with 22, 44, or 88 μg/mL plant extract. In the in vivo assay, Swiss mice were treated with 500, 1000, or 2000 mg extract/kg body weight by intraperitoneal injection and were evaluated 24 h later. Antigenotoxicity was investigated in pre- and post-treatment assays in which the animals received the plant extract (2000 mg/kg) 24 h before or after receiving cyclophosphamide (50 mg/kg), respectively. The extract had no genotoxic effects in the in vitro or in vivo assays. However, the extract reduced apoptotic cell death and induced necrotic cell death at concentrations that presented leishmanicidal activity in vitro. The extract also had an antigenotoxic effect, reducing the levels of genomic damage that were caused by cyclophosphamide in Swiss mice by more than 80%.

Machado-Joseph disease or Spinocerebellar ataxia type 3 is a progressive fatal neurodegenerative disorder caused by the polyglutamine-expanded protein ataxin-3. Recent studies demonstrate that RNA interference is a promising approach for the treatment of Machado-Joseph disease. However, whether gene silencing at an early time-point is able to prevent the appearance of motor behavior deficits typical of the disease when initiated before onset of the disease had not been explored. Here, using a lentiviral-mediated allele-specific silencing of mutant ataxin-3 in an early pre-symptomatic cerebellar mouse model of Machado-Joseph disease we show that this strategy hampers the development of the motor and neuropathological phenotypic characteristics of the disease. At the histological level, the RNA-specific silencing of mutant ataxin-3 decreased formation of mutant ataxin-3 aggregates, preserved Purkinje cell morphology and expression of neuronal markers while reducing cell death. Importantly, gene silencing prevented the development of impairments in balance, motor coordination, gait and hyperactivity observed in control mice. These data support the therapeutic potential of RNA interference for Machado-Joseph disease and constitute a proof of principle of the beneficial effects of early allele-specific silencing for therapy of this disease.

The recently described generation of new neurons in the adult hypothalamus, the center for energy regulation, suggests that hypothalamic neurogenesis is a crucial part of the mechanisms that regulate food intake. Accordingly, neurogenesis in both the adult and embryonic hypothalamus is affected by nutritional cues and metabolic disorders such as obesity, with consequent effects on energy-balance. This review critically discusses recent findings on the contribution of adult hypothalamic neurogenesis to feeding regulation, the impact of energy-balance disorders on adult hypothalamic neurogenesis, and the influence of embryonic hypothalamic neurogenesis upon feeding regulation in the adult. Understanding how hypothalamic neurogenesis contributes to food intake control will change the paradigm on how we perceive energy-balance regulation., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is a fatal, dominant neurodegenerative disorder caused by the polyglutamine-expanded protein ataxin-3. Clinical manifestations include cerebellar ataxia and pyramidal signs culminating in severe neuronal degeneration. Currently, there is no therapy able to modify disease progression. In the present study, we aimed at investigating one of the most severely affected brain regions in the disorder--the cerebellum--and the behavioral defects associated with the neuropathology in this region. For this purpose, we injected lentiviral vectors encoding full-length human mutant ataxin-3 in the mouse cerebellum of 3-week-old C57/BL6 mice. We show that circumscribed expression of human mutant ataxin-3 in the cerebellum mediates within a short time frame--6 weeks, the development of a behavioral phenotype including reduced motor coordination, wide-based ataxic gait, and hyperactivity. Furthermore, the expression of mutant ataxin-3 resulted in the accumulation of intranuclear inclusions, neuropathological abnormalities, and neuronal death. These data show that lentiviral-based expression of mutant ataxin-3 in the mouse cerebellum induces localized neuropathology, which is sufficient to generate a behavioral ataxic phenotype. Moreover, this approach provides a physiologically relevant, cost-effective and time-effective animal model to gain further insights into the pathogenesis of MJD and for the evaluation of experimental therapeutics of MJD.

Objective: Machado-Joseph disease (MJD) is a neurodegenerative disorder associated with an abnormal CAG expansion, which translates into an expanded polyglutamine tract within ataxin-3. There is no therapy to prevent or modify disease progression. Because caffeine (a nonselective adenosine receptor antagonist) and selective adenosine A2A receptor (A2A R) blockade alleviate neurodegeneration in different brain diseases, namely at early stages of another polyglutamine-related disorder such as Huntington's disease, we now tested their ability to control MJD-associated neurodegeneration., Methods: MJD was modeled by transducing the striatum of male adult C57Bl/6 mice with lentiviral vectors encoding mutant ataxin-3 in one hemisphere and wild-type ataxin-3 in the other hemisphere (as internal control). Caffeine (1g/L) was applied through the drinking water. Mice were killed at different time points (from 2 to 12 weeks) to probe for the appearance of different morphological changes using immunohistochemical analysis., Results: Mutant ataxin-3 caused an evolving neuronal dysfunction (loss of DARPP-32 staining) leading to neurodegeneration (cresyl violet and neuronal nuclei staining) associated with increased number of mutant ataxin-3 inclusions in the basal ganglia. Notably, mutant ataxin-3 triggered early synaptotoxicity (decreased synaptophysin and microtubule-associated protein-2 staining) and reactive gliosis (glial fibrillary acidic protein and CD11b staining), which predated neuronal dysfunction and damage. Caffeine reduced the appearance of all these morphological modifications, which were also abrogated in mice with a global A2A R inactivation (knockout)., Interpretation: Our findings provide a demonstration that synaptotoxicity and gliosis are precocious events in MJD and that caffeine and A2A R inactivation decrease MJD-associated striatal pathology, which paves the way to consider A2A Rs as novel therapeutic targets to manage MJD., (Copyright © 2013 American Neurological Association.)

Malignant small round cell tumors represent a diagnostic challenge for cytologists and pathologists. This case series describes four cases of unusual metastasis of small round cell tumors subtypes into body cavities generating effusions in which fluid cytological examination suggested the neuroendocrine origin of the tumors. Tumor diagnosis (Ewing sarcoma/primitive neuroectodermal tumor and desmoplastic small round cell tumors) were unknown at the cytological evaluation. We can highlight the importance of the accurate analyses of body fluids, both for early diagnosis of metastatic disease, and for the diagnosis of primary tumor when serous effusion is the first manifestation of the neoplasia., (Copyright © 2011 Wiley Periodicals, Inc.)

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is an autosomal dominantly-inherited neurodegenerative disorder caused by the over-repetition of a CAG codon in the MJD1 gene. This expansion translates into a polyglutamine tract that confers a toxic gain-of-function to the mutant protein--ataxin-3, leading to neurodegeneration in specific brain regions, with particular severity in the cerebellum. No treatment able to modify the disease progression is available. However, gene silencing by RNA interference has shown promising results. Therefore, in this study we investigated whether lentiviral-mediated allele-specific silencing of the mutant ataxin-3 gene, after disease onset, would rescue the motor behavior deficits and neuropathological features in a severely impaired transgenic mouse model of MJD. For this purpose, we injected lentiviral vectors encoding allele-specific silencing-sequences (shAtx3) into the cerebellum of diseased transgenic mice expressing the targeted C-variant of mutant ataxin-3 present in 70% of MJD patients. This variation permits to discriminate between the wild-type and mutant forms, maintaining the normal function of the wild-type allele and silencing only the mutant form. Quantitative analysis of rotarod performance, footprint and activity patterns revealed significant and robust alleviation of gait, balance (average 3-fold increase of rotarod test time), locomotor and exploratory activity impairments in shAtx3-injected mice, as compared to control ones injected with shGFP. An important improvement of neuropathology was also observed, regarding the number of intranuclear inclusions, calbindin and DARPP-32 immunoreactivity, fluorojade B and Golgi staining and molecular and granular layers thickness. These data demonstrate for the first time the efficacy of gene silencing in blocking the MJD-associated motor-behavior and neuropathological abnormalities after the onset of the disease, supporting the use of this strategy for therapy of MJD.

In the last several years, the use of dendritic cells has been studied as a therapeutic strategy against tumors. Dendritic cells can be pulsed with peptides or full-length protein, or they can be transfected with DNA or RNA. However, comparative studies suggest that transfecting dendritic cells with messenger RNA (mRNA) is superior to other antigen-loading techniques in generating immunocompetent dendritic cells. In the present study, we evaluated a new therapeutic strategy to fight tuberculosis using dendritic cells and macrophages transfected with Hsp65 mRNA. First, we demonstrated that antigen-presenting cells transfected with Hsp65 mRNA exhibit a higher level of expression of co-stimulatory molecules, suggesting that Hsp65 mRNA has immunostimulatory properties. We also demonstrated that spleen cells obtained from animals immunized with mock and Hsp65 mRNA-transfected dendritic cells were able to generate a mixed Th1/Th2 response with production not only of IFN-γ but also of IL-5 and IL-10. In contrast, cells recovered from mice immunized with Hsp65 mRNA-transfected macrophages were able to produce only IL-5. When mice were infected with Mycobacterium tuberculosis and treated with antigen-presenting cells transfected with Hsp65 mRNA (therapeutic immunization), we did not detect any decrease in the lung bacterial load or any preservation of the lung parenchyma, indicating the inability of transfected cells to confer curative effects against tuberculosis. In spite of the lack of therapeutic efficacy, this study reports for the first time the use of antigen-presenting cells transfected with mRNA in experimental tuberculosis.

Background: Although plasmid DNA encoding an antigen from pathogens or tumor cells has been widely studied as vaccine, the use of plasmid vector (without insert) as therapeutic agent requires further investigation., Results: Here, we showed that plasmid DNA (pcDNA3) at low doses inhibits the production of IL-6 and TNF-α by lipopolysaccharide (LPS)-stimulated macrophage cell line J774. These findings led us to evaluate whether plasmid DNA could act as an anti-inflammatory agent in a Wistar rat endotoxemia model. Rats injected simultaneously with 1.5 mg/kg of LPS and 10 or 20 μg of plasmid DNA had a remarkable attenuation of mean arterial blood pressure (MAP) drop at 2 hours after treatment when compared with rats injected with LPS only. The beneficial effect of the plasmid DNA on MAP was associated with decreased expression of IL-6 in liver and increased concentration of plasma vasopressin (AVP), a known vasoconstrictor that has been investigated in hemorrhagic shock management. No difference was observed in relation to nitric oxide (NO) production., Conclusion: Our results demonstrate for the first time that plasmid DNA vector at low doses presents anti-inflammatory property and constitutes a novel approach with therapeutic potential in inflammatory diseases.

Objective: To evaluate ertapenem disk performance to predict Klebsiella pneumonie carbapenemase production by Gram-negative bacilli., Methods: All Gram-negative bacilli isolated between January 2010 and June 2011 were tested by disk diffusion (Oxoid™) for sensitivity to ertapenem, meropenem and imipenem. Resistant or intermediate sensitivity strains (diameter < 22 mm for ertapenem) were also tested for the blaKPC gene by polymerase chain reaction. Disk predictive positive value for Klebsiella pneumoniae carbapenemase and specificity were calculated., Results: Out of the 21839 cultures performed, 3010 (13.78%) were positive, and Gram-negative bacilli were isolated in 708 (23.52%) of them. Zone of inhibition diameter for ertapenem disk was < 22 mm for 111 isolates, representing 15.7% of all Gram-negative isolates. The PCR assay for blaKPC detected 40 Klebsiella pneumoniae carbapenemase-producing strains. No strains intermediate or resistant to meropenem and imipenem were sensitive to ertapenem. The ertapenem disk presented a positive predictive value of 36% to predict blaKPC and 89% specificity., Conclusion: The resistance of Gram-negative bacilli detected by disk diffusion against ertapenem does not predict Klebsiella pneumoniae carbapenemase production. Other mechanisms, such as production of other betalactamases and porin loss, may be implicated. The need to confirm the presence of the blaKPC is suggested. Therefore, ertapenem was a weak predictor for discriminating strains that produce Klebsiella pneumoniae carbapenemase.

Ankylosing spondylitis (AS) is a chronic inflammatory disease that affects mainly the axial skeletal system, causing pain and functional incapacity. The peripheral joint involvement occurs in 30 to 40% of cases. Osteomyelitis of the mandible was relatively common before the advent of preventive antibiotic therapy and restorative dentistry. Currently, the infection of the facial bones is a rare condition, being the odontogenic infection the most responsible for cases with mandibular involvement. The authors report the case of a EA patient, with severe peripheral involvement, which progressed to osteomyelitis of the jaw, secondary to the odontogenic infection due to delay in diagnosis and treatment.

Background: Various endonucleases can be engineered to induce double-strand breaks (DSBs) in chosen DNA sequences. These DSBs are spontaneously repaired by nonhomologous-end-joining, resulting in micro-insertions or micro-deletions (INDELs). We detected, characterized and quantified the frequency of INDELs produced by one meganuclease (MGN) targeting the RAG1 gene, six MGNs targeting three introns of the human dystrophin gene and one pair of zinc finger nucleases (ZFNs) targeting exon 50 of the human dystrophin gene. The experiments were performed in human cells (i.e. 293 T cells, myoblasts and myotubes)., Methods: To analyse the INDELs produced by the endonucleases the targeted region was polymerase chain reaction amplified and the amplicons were digested with the Surveyor enzyme, cloned in bacteria or deep sequenced., Results: Endonucleases targeting the dystrophin gene produced INDELs of different sizes but there were clear peaks in the size distributions. The positions of these peaks were similar for MGNs but not for ZFNs in 293 T cells and in myoblasts. The size of the INDELs produced by these endonucleases in the dystrophin gene would have permitted a change in the reading frame. In a subsequent experiment, we observed that the frequency of INDELs was increased by re-exposition of the cells to the same endonuclease., Conclusions: Endonucleases are able to: (i) restore the normal reading of a gene with a frame shift mutation; (ii) delete a nonsense codon; and (iii) knockout a gene. Endonucleases could thus be used to treat Duchenne muscular dystrophy and other hereditary diseases that are the result of a nonsense codon or a frame shift mutation., (Copyright © 2011 John Wiley & Sons, Ltd.)

Paget's disease of bone is an osteometabolic focal disease characterized by defects in bone remodeling. It may be asymptomatic, but often is associated with bone pain, deformity, pathological fracture, secondary osteoarthrosis and deafness. The diagnosis is usually made by radiological and laboratory findings. This report describes a male patient, 74 years old, native of Amazon, without European ancestry, with polyostotic Paget's disease, with clinical, radiological and laboratory diagnosis after 30 years of disease. The authors emphasize several complications of Paget's disease due to delayed diagnosis and the rarity of the disease in this population group.

We evaluated all Candida spp. isolates obtained from patients admitted to two tertiary care hospitals between 1999 and 2003 in the city of São Paulo, Brazil. The in vitro activities of fluconazole (FCZ) and voriconazole were determined by the agar disk diffusion test using the Clinical and Laboratory Standards Institute M44-A guidelines. The inhibition zone diameters were read and interpreted automatically by the BIOMIC(®) image-analysis plate reader system. We tested a total of 4,625 strains, including 2,393 strains of C. albicans (51.7%), 658 of C. tropicalis (14.2%), 503 of C. glabrata (10.9%), 495 of C. parapsilosis (10.7%), 292 of C. rugosa (6.3%), 195 of C. guilliermondii (4.2%) and 89 of other Candida species (2.0%). Only 2.0% of the strains tested were classified as dose-dependent susceptible (DDS), and 5.8% of them were resistant to FCZ. The resistance or DDS to fluconazole was verified mainly among C. glabrata (7.8%), C. krusei (67.9%) and C. rugosa (65.1%). Voriconazole exhibited better activity in vitro than fluconazole, even in isolates fluconazole resistant. The resistance of fluconazole and voriconazole did not increase in the isolates of Candida spp. during the evaluated period., (© Springer Science+Business Media B.V. 2010)

Some pathological conditions with feeding pattern alterations, including obesity and Huntington disease (HD) are associated with hypothalamic dysfunction and neuronal cell death. Additionally, the hypothalamus is a neurogenic region with the constitutive capacity to generate new cells of neuronal lineage, in adult rodents. The aim of the present work was to evaluate the expression of feeding-related neuropeptides in hypothalamic progenitor cells and their capacity to differentiate to functional neurons which have been described to be affected by hypothalamic dysfunction. Our study shows that hypothalamic progenitor cells from rat embryos grow as floating neurospheres and express the feeding-related neuropeptides Neuropeptide Y (NPY), Agouti-related Protein (AGRP), Pro-OpioMelanocortin (POMC), Cocaine-and-Amphetamine Responsive Transcript (CART) and Orexin-A/Hypocretin-1. Moreover the relative mRNA expression of NPY and POMC increases during the expansion of hypothalamic neurospheres in proliferative conditions.Mature neurons were obtained from the differentiation of hypothalamic progenitor cells including NPY, AGRP, POMC, CART and Orexin-A positive neurons. Furthermore the relative mRNA expression of NPY, CART and Orexin-A increases after the differentiation of hypothalamic neurospheres. Similarly to the adult hypothalamic neurons the neurospheres-derived neurons express the glutamate transporter EAAT3. The orexigenic and anorexigenic phenotype of these neurons was identified by functional response to ghrelin and leptin hormones, respectively. This work demonstrates the presence of appetite-related neuropeptides in hypothalamic progenitor cells and neurons obtained from the differentiation of hypothalamic neurospheres, including the neuronal phenotypes that have been described by others as being affected by hypothalamic neurodegeneration. These in vitro models can be used to study hypothalamic progenitor cells aiming a therapeutic intervention to mitigate feeding dysfunction that are associated with hypothalamic neurodegeneration.

Disease-modifying antirheumatic biological and non-biological therapies are associated with reduced disease progression and joint destruction. Suggestions have been made that total knee and hip joint arthroplasty indications are decreasing as a beneficial effect of the new forms of therapy for rheumatoid arthritis. We present findings of our institution on the incidence of joint arthroplasty in the past few years in patients with rheumatoid arthritis and the increase in the numbers of procedures not associated with inflammatory arthritis.

Introduction: Nephrolithiasis is com>mon and has a high rate of recurrence., Objectives: To assess the prevalence of the main metabolic and anatomical changes and the chemical analysis of stone found in patients with nephrolithiasis in the West region of Paraná., Methods: Retrospective study with 681 adult patients with nephrolithiasis. The laboratory investigation included at least two samples of 24-hour urine test with doses of calcium, uric acid, citrate, oxalate, sodium and creatinine; qualitative cystinuria, urinary pH following 12-hour fast and water restriction, urine culture and chemical analysis, when the stones were available. Renal imaging techniques included at least renal ultrasound and excretory urogram., Results: The metabolic changes most frequently found were: hypercalciuria (51.8%), hyperuricosuria (27.6%), and hypocitraturia (23.5%). Chemical analysis of stones showed calcium oxa late in 85.7% of the cases. The most frequently anatomical changes were renal cyst, duplicated ureter, and ureteropelvic junction obstruction., Conclusions: This paper served as a base for knowing the characteristics of patients with nephrolithiasis in the West area of Paraná.

GABA is the major inhibitory neurotransmitter in the CNS and changes in GABAergic neurotransmission affect the overall activity of neuronal networks. The uptake of GABA into synaptic vesicles is mediated by the vesicular GABA transporter (VGAT), and changes in the expression of the transporter directly regulate neurotransmitter release. In this work we investigated the changes in VGAT protein levels during ischemia and in excitotoxic conditions, which may affect the demise process. We found that VGAT is cleaved by calpains following excitotoxic stimulation of hippocampal neurons with glutamate, giving rise to a stable truncated cleavage product (tVGAT). VGAT cleavage was also observed after transient middle cerebral artery occlusion in mice, a cerebral ischemia model, and following intrahippocampal injection of kainate, but no effect was observed in transgenic mice overexpressing calpastatin, a calpain inhibitor. Incubation of isolated cerebrocortical synaptic vesicles with recombinant calpain also induced the cleavage of VGAT and formation of stable tVGAT. Immunoblot experiments using antibodies targeting different regions of VGAT and N-terminal sequencing analysis showed that calpain cleaves the transporter in the N-terminal region, at amino acids 52 and 60. Immunocytochemistry of GABAergic striatal neurons expressing GFP fusion proteins with the full-length VGAT or tVGAT showed that cleavage of the transporter induces a loss of synaptic delivery, leading to a homogeneous distribution of the protein along neurites. Our results show that excitotoxicity downregulates full-length VGAT, with a concomitant generation of tVGAT, which is likely to affect GABAergic neurotransmission and may influence cell death during ischemia.

Background: Although B cells are important as antigen presenting cells (APC) during the immune response, their role in DNA vaccination models is unknown., Methods: In this study in vitro and in vivo experiments were performed to evaluate the ability of B cells to protect mice against Mycobacterium tuberculosis challenge., Results: In vitro and in vivo studies showed that B cells efficiently present antigens after naked plasmid pcDNA3 encoding M. leprae 65-kDa heat shock protein (pcDNA3-Hsp65) internalization and protect B knock-out (BKO) mice against Mycobacterium tuberculosis infection. pcDNA3-Hsp65-transfected B cells adoptively transferred into BKO mice rescued the memory phenotypes and reduced the number of CFU compared to wild-type mice., Conclusions: These data not only suggest that B cells play an important role in the induction of CD8 T cells but also that they improve bacterial clearance in DNA vaccine model.

Alzheimer's and Parkinson's diseases represent the most prevalent neurodegenerative disorders worldwide. Current pharmacological or surgical treatments provide symptomatic benefits, particularly in the early stages, but none can delay or stop the progression of these diseases. There is an urgent need for new therapies able to modify disease progression. Gene therapy, mainly based on viral vectors, is presently being assumed as an important alternative to conventional treatments. After decades of preclinical developments, we are now facing an important period, in which several viral vector-mediated gene therapies are being tested in Phase 1 and Phase 2 clinical trials, with some of them showing promising results. This review intends to present an overview of the current efforts in the field for the treatment of Alzheimer's and Parkinson's diseases.

Neuropeptide Y (NPY) produced by arcuate nucleus (ARC) neurons has a strong orexigenic effect on target neurons. Hypothalamic NPY levels undergo wide-ranging oscillations during the circadian cycle and in response to fasting and peripheral hormones (from 0.25 to 10-fold change). The aim of the present study was to evaluate the impact of a moderate long-term modulation of NPY within the ARC neurons on food consumption, body weight gain and hypothalamic neuropeptides. We achieved a physiological overexpression (3.6-fold increase) and down-regulation (0.5-fold decrease) of NPY in the rat ARC by injection of AAV vectors expressing NPY and synthetic microRNA that target the NPY, respectively. Our work shows that a moderate overexpression of NPY was sufficient to induce diurnal over-feeding, sustained body weight gain and severe obesity in adult rats. Additionally, the circulating levels of leptin were elevated but the immunoreactivity (ir) of ARC neuropeptides was not in accordance (POMC-ir was unchanged and AGRP-ir increased), suggesting a disruption in the ability of ARC neurons to response to peripheral metabolic alterations. Furthermore, a dysfunction in adipocytes phenotype was observed in these obese rats. In addition, moderate down-regulation of NPY did not affect basal feeding or normal body weight gain but the response to food deprivation was compromised since fasting-induced hyperphagia was inhibited and fasting-induced decrease in locomotor activity was absent.These results highlight the importance of the physiological ARC NPY levels oscillations on feeding regulation, fasting response and body weight preservation, and are important for the design of therapeutic interventions for obesity that include the NPY.

Background: mRNAs are highly versatile, non-toxic molecules that are easy to produce and store, which can allow transient protein expression in all cell types. The safety aspects of mRNA-based treatments in gene therapy make this molecule one of the most promising active components of therapeutic or prophylactic methods. The use of mRNA as strategy for the stimulation of the immune system has been used mainly in current strategies for the cancer treatment but until now no one tested this molecule as vaccine for infectious disease., Results: We produce messenger RNA of Hsp65 protein from Mycobacterium leprae and show that vaccination of mice with a single dose of 10 μg of naked mRNA-Hsp65 through intranasal route was able to induce protection against subsequent challenge with virulent strain of Mycobacterium tuberculosis. Moreover it was shown that this immunization was associated with specific production of IL-10 and TNF-alpha in spleen. In order to determine if antigen presenting cells (APCs) present in the lung are capable of capture the mRNA, labeled mRNA-Hsp65 was administered by intranasal route and lung APCs were analyzed by flow cytometry. These experiments showed that after 30 minutes until 8 hours the populations of CD11c+, CD11b+ and CD19+ cells were able to capture the mRNA. We also demonstrated in vitro that mRNA-Hsp65 leads nitric oxide (NO) production through Toll-like receptor 7 (TLR7)., Conclusions: Taken together, our results showed a novel and efficient strategy to control experimental tuberculosis, besides opening novel perspectives for the use of mRNA in vaccines against infectious diseases and clarifying the mechanisms involved in the disease protection we noticed as well.

Renal tubular acidosis is a rare disease that can present in a primary, resulting from genetic defects in transport mechanisms of the renal tubules, or secondary, consequent to systemic diseases or drugs. The authors report a case of a patient with renal tubular acidosis type II who developed bilateral femoral fracture secondary to severe osteomalacia, with the intention of highlighting the importance of understanding this disease since the late diagnosis and treatment may generate serious repercussions for the patient.

Background: Osteosarcoma is a malignant tumor of connective tissue whose tumor cells produce bone tissue. It can be classified as osteoblastic, chondroblastic, or fibroblastic, according to the predominant histologic type of cells. Its dissemination is hematogenous, and the lungs are the most frequent site of clinically evident metastasis. Extrapulmonary metastases are rare and more frequently diagnosed at necropsy. We present a case of osteosarcoma with peritoneal dissemination that developed neoplastic ascites., Case: A 46-year-old patient came to the hospital with a 4-month history of lumbar pain and weakness in the lower limbs. Computed tomography showed blastic lesions in the L3 vertebral body. Surgical resection and histologic analysis revealed a mixed osteoblastic and chondroblastic osteosarcoma. After only one session of chemotherapy, the patient presented a marked clinical worsening with extensive metastatic dissemination and occurrence of voluminous ascites. The cytologic examination of the ascitic fluid demonstrated frequent poorly differentiated tumor cells. The patient died a little more than 2 months after the diagnosis., Conclusion: This case is the only report of osteosarcoma primarily focused on the vertebral column affected by peritoneal metastasis shown by cytologic examination of ascitic fluid.

Machado-Joseph disease or spinocerebellar ataxia type 3 (MJD/SCA3) is a fatal, autosomal dominant disorder caused by a cytosine-adenine-guanine expansion in the coding region of the MJD1 gene. RNA interference has potential as a therapeutic approach but raises the issue of the role of wild-type ataxin-3 (WT ATX3) in MJD and of whether the expression of the wild-type protein must be maintained. To address this issue, we both overexpressed and silenced WT ATX3 in a rat model of MJD. We showed that (i) overexpression of WT ATX3 did not protect against MJD pathology, (ii) knockdown of WT ATX3 did not aggravate MJD pathology and that (iii) non-allele-specific silencing of ataxin-3 strongly reduced neuropathology in a rat model of MJD. Our findings indicate that therapeutic strategies involving non-allele-specific silencing to treat MJD patients may be safe and effective.

The present study aimed to provide additional information on the prevalence of mucosal human papillomavirus (HPV) types in Portuguese women by using polymerase chain reaction, restriction fragment length polymorphism and sequencing. HPV was detected in 15.5% (15/97) of the control samples, 23.5% (12/51) of atypical squamous cells of undetermined significance, 52.8% (28/53) of low-grade lesions, 82.4% (28/34) of high-grade lesions, and 100% (44/44) of carcinomas. Overall, 28 HPV types were detected: 11 high-risk (HPV 16, 18, 31, 33, 35, 39, 45, 52, 56, 58, and 59), 3 probable high-risk (HPV 53, 66, and 73), 6 low-risk (HPV 6, 11, 44, 61, 70, and 81), and 8 unknown-risk types (HPV 34, 62, 67, 71, 83, 84, 102, and 108). The most prevalent type was HPV 16, detected in 33.8% of women infected with HPV, followed by HPV 58 (9.2%), HPV 33 (7.0%), HPV 18 (6.3%), HPV 53 (5.6%), HPV 31 and 56 (4.9% each), HPV 6 (3.5%), and HPV 66 and 81 (2.8% each). Of 44 cervical carcinoma samples, 71% were associated with HPV 16 (60%) and HPV 18 (11.1%), followed by the high-risk types 33 (11.1%), 35 (4.4%), 45 (4.4%), and 56 (2.2%), the probable high-risk type 53 (4.4%) and the unknown-risk type 67 (2.2%). This study provides information on the most common HPV types in Portuguese women and suggests that the current prophylactic HPV 16/18 vaccine may be useful for the prevention of cervical cancer in this population., ((c) 2010 Wiley-Liss, Inc.)

Systemic Lupus Erythematosus (SLE) is an autoimmune disease, with multisystemic involvement. Gastrointestinal symptoms are common, like nausea, vomiting and dyspepsia. Acute pancreatitis is an unusual manifestation of SLE, being an important differential diagnosis in evaluation of abdominal pain. The patients usually presents with pain of variable intensity, some occasions simulating acute abdomen. Several factors have been implicated in the pathogenesis of this condition, such as vasculitis, drugs and antiphospholipid antibodies. The role of corticosteroids as etiologic factor remains controversial. Due to the rarity of SLE associated to pancreatitis, we report two cases of patients with severe inflammatory process. In one case, it was used corticosteroids in high doses during treatment, with good outcome. In another, the patient died because of pancreatic pseudocyst rupture and its postoperative hemodynamic complications. In the reported cases, predisposing factors for acute pancreatitis were not verified, so it was considered a primary manifestation of SLE activity.

Excitotoxicity is one of the main features responsible for neuronal cell death after acute brain injury and in several neurodegenerative disorders, for which only few therapeutic options are currently available. In this work, RNA interference was employed to identify and validate a potential target for successful treatment of excitotoxic brain injury, the transcription factor c-Jun. The nuclear translocation of c-Jun and its upregulation are early events following glutamate-induced excitotoxic damage in primary neuronal cultures. We present evidence for the efficient knockdown of this transcription factor using a non-viral vector consisting of cationic liposomes associated to transferrin (Tf-lipoplexes). Tf-lipoplexes were able to deliver anti-c-Jun siRNAs to neuronal cells in culture, resulting in efficient silencing of c-Jun mRNA and protein and in a significant decrease of cell death following glutamate-induced damage or oxygen-glucose deprivation. This formulation also leads to a significant c-Jun knockdown in the mouse hippocampus in vivo, resulting in the attenuation of both neuronal death and inflammation following kainic acid-mediated lesion of this region. Furthermore, a strong reduction of seizure activity and cytokine production was observed in animals treated with anti-c-Jun siRNAs. These findings demonstrate the efficient delivery of therapeutic siRNAs to the brain by Tf-lipoplexes and validate c-Jun as a promising therapeutic target in neurodegenerative disorders involving excitotoxic lesions., ((c) 2009 Elsevier B.V. All rights reserved.)

Although RNAi-based gene silencing holds a great potential for treatment of neurological disorders, its application to the CNS has been restricted by low levels of tissue distribution and cellular uptake. In this work we report that cationic lipid-based vectors can enhance siRNA delivery to neurons both in vitro and in vivo. DOTAP:Chol liposomes associated with transferrin (Tf) and complexed with siRNAs (Tf-lipoplexes) were delivered to primary cultures of luciferase-expressing cortical neurons. Confocal microscopy studies revealed efficient cellular uptake of Cy3-labelled siRNAs after Tf-lipoplex delivery, which was reduced but not completely inhibited by blocking the Tf-receptor with excess Tf. Gene silencing was also evaluated after delivery of anti-luciferase or anti-c-Jun siRNAs. Our results demonstrate that Tf-lipoplexes achieve up to 50% luciferase and c-Jun knockdown, 48 h after transfection, without significant cytotoxicity. Similar results were observed in vivo, where a 40% reduction of luciferase activity was found in the striatum of luciferase mice. In addition, fluorescence microscopy studies showed extensive local distribution and internalization of Tf-lipoplex-associated Cy3-siRNAs without tissue toxicity. Overall, our results demonstrate that Tf-lipoplexes can mediate efficient gene silencing in neuronal cells, both in vitro an in vivo, which may prove useful in therapeutic approaches to neuronal protection and repair.