Real-world data from the Portuguese Nivolumab Expanded Access Program (EAP) in previously treated Non Small Cell Lung Cancer (NSCLC).

OBJECTIVE: The main aim of the study was to evaluate the efficacy and safety profile of Nivolumab, an immune-checkpoint-inhibitor antibody, in advanced, previously treated, Non-Small Cell Lung Cancer (NSCLC) patients, in a real world setting. METHODS: We performed a retrospective, multicentre data analysis of patients who were included in the Portuguese Nivolumab Expanded Access Program (EAP). Eligibility criteria included histologically or citologically confirmed NSCLC, stage IIIB and IV, evaluable disease, sufficient organ function and at least one prior line of chemotherapy. The endpoints included Overall Response Rate (ORR), Disease Control Rate (DCR), Progression Free Survival (PFS) and Overall Survival (OS). Safety analysis was performed with the National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE), version 4.0, and immune-related Adverse Events (irAEs) were treated according to protocol treatment guidelines. Tumour response was assessed using the Response Evaluation Criteria in Solid Tumours (RECIST) version 1.1. Data was analysed using SPSS, version 21.0 (IBM Statistics). RESULTS: From June 2015 to December 2016, a total of 229 patients with advanced NSCLC were enrolled at 30 Portuguese centres. Clinical data were collected up to the end of July 2018. The baseline median age was 64 years (range 37-83) and the majority of patients were males (70.3%) and former/current smokers (69.4%). Patients with non-squamous histology predominated (88.1%), and 67.6% of the patients had received 2 or more prior lines of chemotherapy. Out of 229 patients, data was available for 219 patients (3 patients did not start treatment, while data was unavailable in 7 patients); of the 219 patients, 15.5% were not evaluated for radiological tumour assessment, 1.4% had complete response (CR), 21% partial response (PR), 31% stable disease (SD) and 31.1% progressive disease (PD). Thus, the ORR was 22.4% and DCR was 53.4% in this population. At the time of survival analysis the median PFS was 4.91 months (95% CI, 3.89-6.11) and median OS was 13.21 months (95% CI, 9.89-16.53). The safety profile was in line with clinical trial data. CONCLUSIONS: Efficacy and safety results observed in this retrospective analysis were consistent with observations reported in clinical trials and from other centres.

Dermatite piogranulomatosa ulcerativa em bovino por Corynebacterium pseudotuberculosis / Ulcerative pyogranulomatous dermatitis in bovine by Corynebacterium pseudotuberculosis

Relata-se um caso de lesão cutânea secundária à infecção por Corynebacterium pseudotuberculosis em bovino. Abscessos e múltiplas lesões cutâneas nodulares, variando de 8x10 para 20x25cm de diâmetro, firmes, sensíveis ao toque e sem mobilidade, algumas com superfície ulcerada, circundada por halo avermelhado e drenando secreção piossanguinolenta, e outras com superfícies alopécicas, foram identificados na região torácica lateral do animal. Exames histopatológicos e o isolamento do agente de fragmentos obtidos após remoção cirúrgica das lesões confirmaram tratar-se da forma cutânea da infecção pelo C. pseudotuberculosis. As lesões microscópicas caracterizaram-se por dermatite nodular piogranulomatosa e ulcerativa. A avaliação da susceptibilidade in vitro do C. pseudotuberculosis a antimicrobianos demonstrou que o agente era resistente à amicacina, kanamicina, neomicina e penicilina G, apresentando sensibilidade à ampicilina adicionada de subactam, amoxicilia com ácido clavulônico, cefalexina, cefalotina, cefotaxima, enrofloxacina, gentamicina e tetraciclina. A retirada cirúrgica das lesões e o tratamento com enrofloxacina resultaram na cura do animal. Lesões de pele observadas em casos de ptiose, carcinoma de células escamosas e na forma atípica da actinobacilose devem ser consideradas no diagnóstico diferencial da forma cutânea da infecção por C. pseudotuberculosis em bovinos. Os dados apresentados demonstram que a infecção pelo C. pseudotuberculosis deve ser considerada no diagnóstico diferencial das lesões de pele em bovinos no Brasil.(AU)

We report a case of secondary skin lesionby infection with Corynebacterium pseudotuberculosis in bovine. Abscesses and multiple nodular lesions, ranging from 8x10 to 20x25 cm in diameter, firm, sensitive to touch, and without mobility, some with ulcerated surface, surrounded by reddish halo and draining piosanguinolenta secretion, and other surfaces with alopecia, were identified in the skin of the animal`s thoracic area. Histopathology and isolation of the agent from fragments obtained after surgical removal of the lesions confirmed the cutaneous infection by C. pseudotuberculosis. Microscopic lesions were characterized by lumpy skin disease and ulcerative pyogranulomatous. Evaluation of in vitro susceptibility to antimicrobial demonstrated that the agent was resistant to amikacin, kanamycin, neomycin and penicillin G, and sensitive to ampicillin + subactam, amoxicilia with clavulonic acid, cephalexin, cephalothin, cefotaxime, enrofloxacin, gentamicin, and tetracycline. The surgical removal of the lesions, and treatment with enrofloxacin resulted in animal cure. Skin lesions observed in case of ptiose, squamous cell carcinoma and atypical form of Actinobacillosis should be considered in the differential diagnosis of cutaneous form of C. pseudotuberculosis infection in cattle. The data presented demonstrate that infection with C. pseudotuberculosis should be considered in the differential diagnosis of skin lesions in cattle in Brazil.(AU)

What Makes Umbilical Cord Tissue-Derived Mesenchymal Stromal Cells Superior Immunomodulators When Compared to Bone Marrow Derived Mesenchymal Stromal Cells?

MSCs derived from the umbilical cord tissue, termed UCX, were investigated for their immunomodulatory properties and compared to bone marrow-derived MSCs (BM-MSCs), the gold-standard in immunotherapy. Immunogenicity and immunosuppression were assessed by mixed lymphocyte reactions, suppression of lymphocyte proliferation and induction of regulatory T cells. Results showed that UCX were less immunogenic and showed higher immunosuppression activity than BM-MSCs. Further, UCX did not need prior activation or priming to exert their immunomodulatory effects. This was further corroborated in vivo in a model of acute inflammation. To elucidate the potency differences observed between UCX and BM-MSCs, gene expression related to immune modulation was analysed in both cell types. Several gene expression profile differences were found between UCX and BM-MSCs, namely decreased expression of HLA-DRA, HO-1, IGFBP1, 4 and 6, ILR1, IL6R and PTGES and increased expression of CD200, CD273, CD274, IL1B, IL-8, LIF and TGFB2. The latter were confirmed at the protein expression level. Overall, these results show that UCX seem to be naturally more potent immunosuppressors and less immunogenic than BM-MSCs. We propose that these differences may be due to increased levels of immunomodulatory surface proteins such as CD200, CD273, CD274 and cytokines such as IL1ß, IL-8, LIF and TGFß2.

Efeitos da suplementação com fósforo em caprinos no semiárido do nordeste brasileiro / Effect of phosphorus supplementation for goats in the Brazilian semiarid region

To evaluate the effect of phosphorus supplementation for goats grazing for the semiarid region, one group of 16 recently weaned Moxotó goats was supplemented with a mineral supplement containing Na, Cl, Zn, Cu, Se, Co, and P during 240 days. Another similar group was supplemented with a similar mineral supplement without P. The mean daily consumption of supplement by animal was of 7.09±2.77g and 7.67±3.14g for the groups with and without P, respectively. The mean weight gain of the P supplemented group (45.20±5.56g) was significantly higher (P<0.05) than the non-supplemented group (40.03±2.80g). The average total P in soil was 30.8mg/kg and in the pasture 0.13 percent in dry matter. These results demonstrate the occurrence of P deficiency in some areas of the Brazilian semiarid region.

Survivin silencing as a promising strategy to enhance the sensitivity of cancer cells to chemotherapeutic agents.

Since clinical application of conventional cancer therapies is usually limited by drug resistance and toxic side effects, combination of chemotherapeutic agents with gene therapy appears as an attractive therapeutic strategy to overcome these issues. Being selectively expressed in tumor tissues, survivin is a promising target for the development of anticancer strategies aimed at eliminating tumor cells while sparing normal tissues. In this work, we achieved substantial protein knockdown in a number of human cell lines, namely, A549, HeLa and MCF-7 cells which overexpress survivin, after treatment with anti-survivin siRNAs, which was associated with a significant reduction of cell viability, when compared to treatment with control siRNAs. Interestingly, when the survivin-silencing approach was combined with a chemotherapeutic agent, an enhancement of the therapeutic effect was achieved. Treatment with anti-survivin siRNAs resulted in high levels of caspase 3/7 activation, and an enhancement of this effect was observed when survivin silencing was combined with vinblastine. In addition, we showed that for A549 and HeLa cells survivin silencing contributes to the reversion of cell resistance to doxorubicin. Overall, we demonstrate that the combination of a survivin-directed silencing strategy with chemotherapeutic agents constitutes a valuable approach for cancer treatment.

In vitro modulation of Bcl-2 levels in small cell lung cancer cells: effects on cell viability.

Small cell lung cancer (SCLC) is an aggressive disease, representing 15% of all cases of lung cancer, has high metastatic potential and low prognosis that urgently demands the development of novel therapeutic approaches. One of the proposed approaches has been the down-regulation of BCL2, with poorly clarified and controversial therapeutic value regarding SCLC. The use of anti-BCL2 small interfering RNA (siRNA) in SCLC has never been reported. The aim of the present study was to select and test the in vitro efficacy of anti-BCL2 siRNA sequences against the protein and mRNA levels of SCLC cells, and their effects on cytotoxicity and chemosensitization. Two anti-BCL2 siRNAs and the anti-BCL2 G3139 oligodeoxynucleotide (ODN) were evaluated in SCLC cells by the simultaneous determination of Bcl-2 and viability using a flow cytometry method recently developed by us in addition to Western blot, real-time reverse-transcription PCR, and cell growth after single and combined treatment with cisplatin. In contrast to previous reports about the use of ODN, a heterogeneous and up to 80% sequence-specific Bcl-2 protein knockdown was observed in the SW2, H2171 and H69 SCLC cell lines, although without significant sequence-specific reduction of cell viability, cell growth, or sensitization to cisplatin. Our results question previous data generated with antisense ODN and supporting the present concept of the therapeutic interest in BCL2 silencing per se in SCLC, and support the growing notion of the necessity of a multitargeting molecular approach for the treatment of cancer.

In vitro modulation of Bcl-2 levels in small cell lung cancer cells: effects on cell viability

Small cell lung cancer (SCLC) is an aggressive disease, representing 15 percent of all cases of lung cancer, has high metastatic potential and low prognosis that urgently demands the development of novel therapeutic approaches. One of the proposed approaches has been the down-regulation of BCL2, with poorly clarified and controversial therapeutic value regarding SCLC. The use of anti-BCL2 small interfering RNA (siRNA) in SCLC has never been reported. The aim of the present study was to select and test the in vitro efficacy of anti-BCL2 siRNA sequences against the protein and mRNA levels of SCLC cells, and their effects on cytotoxicity and chemosensitization. Two anti-BCL2 siRNAs and the anti-BCL2 G3139 oligodeoxynucleotide (ODN) were evaluated in SCLC cells by the simultaneous determination of Bcl-2 and viability using a flow cytometry method recently developed by us in addition to Western blot, real-time reverse-transcription PCR, and cell growth after single and combined treatment with cisplatin. In contrast to previous reports about the use of ODN, a heterogeneous and up to 80 percent sequence-specific Bcl-2 protein knockdown was observed in the SW2, H2171 and H69 SCLC cell lines, although without significant sequence-specific reduction of cell viability, cell growth, or sensitization to cisplatin. Our results question previous data generated with antisense ODN and supporting the present concept of the therapeutic interest in BCL2 silencing per se in SCLC, and support the growing notion of the necessity of a multitargeting molecular approach for the treatment of cancer.

Imunidade passiva, ingestão de colostro e mortalidade em cabritos Moxotó criados em sistemas extensivo e intensivo / Passive immunity, colostrum ingestion, and mortality of Moxotó kids raised under extensive and intensive breeding systems

Dosou-se a proteína sérica total para avaliar a aquisição de imunidade passiva em cabritos Moxotó. Para tal, formaram-se quatro grupos experimentais, sendo dois sistemas de criação, extensivo e intensivo, e dois manejos de colostro, ingestão natural e artificial. Tanto no sistema intensivo quanto no extensivo, os teores de proteína no soro foram significativamente mais altos nos animais com ingestão natural de colostro, 7,11±0,2g/dL, do que nos com ingestão artificial, 6,35±0,17g/dL. Independentemente da forma de ingestão de colostro, os cabritos do sistema intensivo tiveram teores de proteína sérica total, 7,21±0,19g/dL, mais elevados que os do sistema extensivo, 6,25±0,18g/dL, no entanto a imunidade passiva foi satisfatória nos dois grupos de animais. Ocorreu alta mortalidade de crias no sistema extensivo, 37 por cento, devido ao complexo hipotermia/inanição em decorrência dos baixos níveis de colostro ingeridos. No sistema intensivo de criação não ocorreu mortalidade de cabritos. A produção de colostro das cabras criadas intensivamente, 163,5±14,71mL, foi mais alta que das cabras criadas extensivamente, 53,75±19,12mL. O peso total dos cabritos foi semelhante nos dois sistemas de criação, 2881±252,78g no sistema extensivo, e 2297±194,59g no sistema intensivo. Conclui-se que a ingestão de colostro nos dois sistemas de produção permitiu adequada aquisição de imunidade em cabritos, porém o sistema extensivo determinou severa deficiência nutricional nas mães, com baixa produção de colostro e graves perdas de neonatos.

The acquisition of passive immunity in Moxotó kids was determined by dosages of total serum proteins. Four experimental groups were formed in two breeding systems - extensive and intensive - and two managements of colostrum intake - suckling from the mother or supplying in bottles. In both breeding systems, the serum protein levels were significantly higher in kids with natural ingestion of colostrum, 7.11±0.2g/dL, than in kids with artificial ingestion, 6.35±0.17g/dL. The kids of the intensive system had levels of total serum protein of 7.21±0.19 g/dL which was higher than the one of the extensive breeding system, 6.25±0.18g/dL. However, the passive immunity was satisfactory in all groups. There was high mortality of kids, 37 percent, due to starvation/hypothermia, in the extensive breeding system. This mortality was apparently due to the low levels of colostrum ingestion, 55.83±8.7mL. The production of colostrum by does from intensive breeding sistem, 163.5±14.71mL, was significantly higher than those from extensive breeding system, 53.75±19.12mL. The total weight of the kids born in the extensive breeding system, 2,881±252.78g, was similar to those born in the intensive breeding system, 2,297±194.59g. The colostrum ingestion allowed appropriate immunity acquisition by kid raised under both systems. However, the extensive breeding system determined a severe nutritional deficiency in the does with low colostrum production and high neonatal losses.

Tf-lipoplex-mediated c-Jun silencing improves neuronal survival following excitotoxic damage in vivo.

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.

Transferrin lipoplex-mediated suicide gene therapy of oral squamous cell carcinoma in an immunocompetent murine model and mechanisms involved in the antitumoral response.

Suicide gene therapy has been used for the treatment of a variety of cancers. We reported previously the in vitro efficacy of the Herpes Simplex Virus Thymidine kinase (HSV-tk)/ganciclovir (GCV) system to mediate cytotoxicity in oral squamous cancer cells, using transferrin (Tf)-lipoplexes, prepared from cationic liposomes composed of 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and cholesterol. In the present study, we evaluated the antitumoral efficacy mediated by this lipoplex formulation in two suicide gene therapy strategies, HSV-tk/GCV and cytosine deaminase (CD)/5-fluorocytosine (5-FC), using a syngeneic, orthotopic murine model for head and neck squamous cell carcinoma. The cellular and molecular events associated with the antitumoral response elicited by both the therapeutic approaches were investigated by analyzing tumor cell death, tumor-infiltrating immune cells and tumor cytokine microenvironment. Significant tumor reduction was achieved upon intratumoral delivery of HSV-tk or CD genes mediated by Tf-lipoplexes, followed by intraperitoneal injection of GCV or 5-FC, respectively. Enhanced apoptosis, the recruitment of NK cells, CD4 and CD8 T-lymphocytes and an increase in the levels of several cytokines/chemokines were observed within the tumors. These observations suggest that suicide gene therapy with lipoplexes modifies the tumor microenvironment, and leads to the recruitment of immune effector cells that can act as adjuvants in reducing the tumor size.

Simultaneous evaluation of viability and Bcl-2 in small-cell lung cancer.

When designing molecular targeted therapeutic strategies against cancer, it is important to correlate protein expression and cell viability. However, such goal can be difficult if performed in separate assays, especially when only a fraction of cells has been efficiently transfected. Therefore, the aim of the present study was to establish a flow cytometry procedure to assess simultaneously Bcl-2 protein level and viability in small-cell lung cancer (SCLC) cells. Viability assessment was performed by staining cells with Annexin V-fluorescein isothiocyanate (FITC) and 7-aminoactinomycin D (7-AAD). Intracellular detection of Bcl-2 was carried out by immunodetection with monoclonal antibodies. Regarding viability determination, the FSC/7-AAD plot identifies the same percentage of viable cells as the FSC/Annexin V-FITC plot, although with greater sensitivity. The procedures involving cells' fixation with 1% paraformaldehyde and permeabilization with digitonin, required for intracellular Bcl-2 immunostaining did not compromise the association of 7-ADD (nor Annexin V-FITC) previously incubated with SCLC cells. It was therefore possible to simultaneously assess cell viability and Bcl-2 protein in SCLC cells. A simple, sensitive, and versatile procedure was established for the first time for the simultaneous evaluation of cell viability and intracellular detection of Bcl-2 in SCLC.

Non-viral lipid-based nanoparticles for targeted cancer systemic gene silencing.

New molecular biology techniques have uncovered the hidden role of genes in cancer. Identification of activated oncogenes, as fundamental genetic differences relative to normal cells, has made it possible to consider such genes as targets for antitumor therapy, namely by applying gene silencing strategies. In this regard, antisense oligonucleotides or small interfering RNAs, constitute promising therapeutic tools. The widespread clinical application of such molecules as modulators of gene expression, is still dependent on several aspects that limit their bioavailability, including: enhanced biological stability, favourable pharmacokinetics, enhanced tumor cell uptake and, consequently, efficient targeted delivery. One of the most promising strategies to overcome the barriers faced by gene silencing molecules, upon systemic administration, involves the use of lipid-based nanoparticles. The first part of this review aims at providing the reader with the molecular mechanism of action of the most important gene silencing molecules used in anticancer therapy. The primary obstacle for translating gene silencing technology from an effective research tool into a feasible therapeutic strategy remains its efficient delivery to the targeted cell type in vivo. Therefore, an overview of different lipid-based strategies for nucleic acid delivery will be presented on the second part. As we learn more about the pharmacokinetics and pharmacodynamics of the carrier and/or of the gene silencing molecules, it will be possible to further improve the delivery strategy that likely in the future will lead to the ideal non-viral particle for targeted cancer systemic gene silencing.

Relief influence on the spatial distribution of the Atlantic Forest cover on the Ibiúna Plateau, SP

Several studies suggest that, on a large scale, relief conditions influence the Atlantic Forest cover. The aim of this work was to explore these relationships on a local scale, in Caucaia do Alto, on the Ibiúna Plateau. Within an area of about 78 km², the distribution of forest cover, divided into two successional stages, was associated with relief attribute data (slope, slope orientation and altitude). The mapping of the vegetation was based on the interpretation of stereoscopic pairs of aerial photographs, from April 2000, on a scale of 1:10,000, while the relief attributes were obtained by geoprocessing from digitalized topographic maps on a scale of 1:10,000. Statistical analyses, based on qui-square tests, revealed that there was a more extensive forest cover, irrespective of the successional stage, in steeper areas (>10 degrees) located at higher altitudes (>923 m), but no influence of the slope orientation. There was no sign of direct influence of relief on the forest cover through environmental gradients that might have contributed to the forest regeneration. Likewise, there was no evidence that these results could have been influenced by the distance from roads or urban areas or with respect to permanent preservation areas. Relief seems to influence the forest cover indirectly, since agricultural land use is preferably made in flatter and lower areas. These results suggest a general distribution pattern of the forest remnants, independent of the scale of study, on which relief indirectly has a strong influence, since it determines human occupation.

Vários estudos sugerem que as condições do relevo influenciam, em larga escala, a cobertura da Mata Atlântica. Este trabalho teve por objetivo explorar estas relações em escala local, na região do Planalto de Ibiúna, denominada de Caucaia do Alto. Numa área de cerca de 78 km², procurou-se associar a cobertura florestal, dividida em dois estádios sucessionais, com atributos do relevo (declividade, orientação de vertente e altitude). O mapeamento da vegetação foi feito a partir da interpretação de pares estereoscópicos de fotografias aéreas de abril de 2000, na escala 1:10.000, enquanto os atributos do relevo foram gerados por geoprocessamento a partir de cartas topográficas digitalizadas, em escala 1:10.000. As análises estatísticas, baseadas em testes de qui-quadrado, revelam que há maior cobertura florestal, independentemente do estádio sucessional, em áreas mais íngremes (>10 graus) e situadas em altitudes mais elevadas (>923 m), porém não há influência da orientação de vertente. Não há indícios de influência direta do relevo sobre a cobertura florestal, através de gradientes ambientais que poderiam agir na regeneração florestal. Também não foram obtidas evidências de que estes resultados possam ser influenciados pelo distanciamento a estradas ou centros urbanos, ou ainda pelo respeito às áreas de preservação permanente. O relevo parece determinar o recobrimento florestal, principalmente por condicionar o uso agrícola dos solos, que se dá preferencialmente em áreas mais planas e baixas. Estes resultados sugerem um padrão geral de influência do relevo sobre a distribuição dos remanescentes florestais, independentemente da escala de estudo, onde o relevo atua indiretamente ao condicionar a ocupação humana.

Cellular and molecular events associated with the antitumor response induced by the cytosine deaminase/5-fluorocytosine suicide gene therapy system in a rat liver metastasis model.

The bacterial cytosine deaminase (CD) gene converts the non-toxic prodrug 5-fluorocytosine (5-FC) into 5-fluorouracil. We have previously shown, in a rat liver metastasis model from colon carcinoma, that intratumoral injection of a CD-expressing plasmid into the animals followed by 5-FC treatment results in the regression of the treated tumor as well as distant uninjected tumors. The aim of this study was to further analyze the mechanisms associated with tumor regression induced upon application of suicide CD/5-FC strategy. Tumor regression was associated with an increased apoptosis, the recruitment of natural killer cells, CD4- and CD8 T lymphocytes within the tumors and an increased expression of several cytokines/chemokines mRNAs. These data indicate that the CD/5-FC suicide strategy is associated with the triggering of cellular and molecular events leading to an efficient antitumor immune response involving both innate and acquired immunity.

siRNA delivery by a transferrin-associated lipid-based vector: a non-viral strategy to mediate gene silencing.

BACKGROUND: RNA interference provides a powerful technology for specific gene silencing. Therapeutic applications of small interfering RNA (siRNA) however require efficient vehicles for stable complexation, protection, and extra- and intracellular delivery of these nucleic acids. Here, we evaluated the potential of transferrin (Tf)-associated liposomes for siRNA complexation and gene silencing. METHODS: Cationic liposomes composed of DOTAP : Cholesterol associated with or without transferrin (Tf) were complexed with siRNA at different lipid/siRNA charge ratios. Complexation and protection of siRNA from enzymatic degradation was assessed with the PicoGreen intercalation assay and gel electrophoresis. Cellular internalization of these siRNA Tf-lipoplexes was detected by confocal microscopy. Luciferase assay, immunoblot and fluorescence-activated cell sorting (FACS) analysis were used to evaluate reporter gene silencing in Huh-7 hepatocarcinoma and U-373 glioma cells. c-Jun knockdown in HT-22 cells was evaluated by quantitative real-time polymerase chain reaction (RT-PCR). Cytotoxicity of the siRNA complexes was assessed by Alamar blue, lactate dehydrogenase and MTT assays. RESULTS: Complexation of siRNA with the cationic liposomes in the presence of Tf results in the formation of stable particles and prevents serum-mediated degradation. Confocal microscopy showed fast cellular internalization of the Tf-lipoplexes via endocytosis. In the GFP glioma cells Tf-lipoplexes showed enhanced gene silencing at minimum toxicity in comparison to Tf-free lipoplexes. Targeting luciferase in the hepatocarcinoma cell line resulted in more than 70% reduction of luciferase activity, while in HT-22 cells 50% knockdown of endogenous c-Jun resulted in a significant protection from glutamate-mediated toxicity. CONCLUSIONS: Cationic liposomes associated with Tf form stable siRNA lipoplexes with reduced toxicity and enhanced specific gene knockdown activity compared to conventional lipoplexes. Thus, such formulations may constitute efficient delivery systems for therapeutic siRNA applications.

Evaluation of the antitumoral effect mediated by IL-12 and HSV-tk genes when delivered by a novel lipid-based system.

In the present work, we used a novel albumin-associated lipoplex formulation, containing the cationic lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPOPC) and cholesterol (Chol), to evaluate the antitumoral efficacy of two gene therapy strategies: immuno-gene therapy, mediated by IL-12 gene expression, and "suicide" gene therapy, mediated by HSV-tk gene expression followed by ganciclovir (GCV) treatment. Our data show that, in an animal model bearing a subcutaneous TSA (mouse mammary adenocarcinoma) tumor, intratumoral administration of the albumin-associated complexes containing the plasmid encoding IL-12 results in a strong antitumoral effect, as demonstrated by the smaller tumor size, the higher T-lymphocyte tumor infiltration and the more extensive tumor necrotic and hemorrhagic areas, as compared to that observed in animals treated with control complexes. On the other hand, the application of the "suicide" gene therapy strategy results in a significant antitumoral activity, which is similar to that achieved with the immuno-gene therapy strategy, although involving different antineoplastic mechanisms. For the tested model, albumin-associated complexes were shown to efficiently mediate intratumoral delivery of therapeutic genes, thus leading to a significant antitumoral effect. This finding is particularly relevant since TSA tumors are characterized for being poorly immunogenic, aggressive and exhibiting high proliferation capacity.

Inflamação do parênquima pulmonar na asma e sua relação com as vias aéreas / Pulmonary parenchyma inflammation in asthma

A inflamação da asma não está limitada às vias aéreas e pode comprometer também o parênquima pulmonar periférico, no entanto, não há estudos na literatura que enfoquem a participação do parênquima pulmonar na asma. Objetivo: Caracterizar o infiltrado inflamatótrio do parênquima peribronquiolar e distal em vítimas de asma fatal, comparando-o ao de outras regiões de vias aéreas e a de tecido pulmonar de não asmáticos. Fragementos de tecido pulmonar obtidos de 20 pacientes com asma fatal e 10 controles, necropsiados no Serviço de verificacão de óbito da capital - São Paulo, foram submetidos a estudo de imuno-histoquímica e marcados com anticorpos anti-proteína básica principal (eosinófilos), anti-triptase (mastócitos), anti-elastase neutrofílica (neutrófilos) e anti-marcadores de superfície de linfócitos (CD3, CD$, CD* e CD20). Foram determinadas as densidades celulares no parênquima pulmonar periférico peribronquiolar e distal e nas áreas interna e externa das vias aéreas de grande e de pequeno calibre. Resultados: A densidade de eosinófilos foi significativamente maior nas duas regiões do parênquima pulmonar de asmáticos comparados aos controles, bem como as vias aéreas (p<0,02). Nos pacientes asmáticos a densidade eosinófilica se mostrou menos no parênquima distal em relação à da área interna da via aérea grande apenas (p<0,01). A densidade dos mastócitos foi maior no parênquima peribronquiolar bem como na área externa das vias aéreas de grande e de pequeno calibre de asmáticos comparados aos controles (p<0.04). Diferenças significativas quanto à densidade de linfócitos se restringiram às vias aéreas de asmáticos comparadas as de controles. Maior densidade de neutrófilos foi observada apenas no parênquima pulmonar de asmáticos (p=0.029). Conclusões: O parênquima pulmonar participa do processo inflamatório na asma fatal com aumento de células efetoras (mastócitos, neutrófilos e eosinófilos). A área externa da via aérea foi a região que melhor diferenciou o asmáticodo controle e com o maior número de diferenças significativas. A inflamação do parênquima peribronquiolar, associado ao da área externa da via áerea pequena, reforça o papel do pulmão distal na patofisiologia da asma.

Two African swine fever virus proteins derived from a common precursor exhibit different nucleocytoplasmic transport activities.

African swine fever virus (ASFV), a large icosahedral deoxyvirus, is the causative agent of an economically relevant hemorrhagic disease that affects domestic pigs. The major purpose of the present study was to investigate the nuclear transport activities of the ASFV p37 and p14 proteins, which result from the proteolytic processing of a common precursor. Experiments were performed by using yeast-based nucleocytoplasmic transport assays and by analysis of the subcellular localization of different green fluorescent and Myc fusion proteins in mammalian cells. The results obtained both in yeast and mammalian cells clearly demonstrated that ASFV p14 protein is imported into the nucleus but not exported to the cytoplasm. The ability of p37 protein to be exported from the nucleus to the cytoplasm of both yeast and mammalian cells was also demonstrated, and the results clearly indicate that p37 nuclear export is dependent on the interaction of the protein with the CRM-1 receptor. In addition, p37 was shown to exhibit nuclear import activity in mammalian cells. The p37 protein nuclear import and export abilities described here constitute the first report of a nucleocytoplasmic shuttling protein encoded by the ASFV genome. Overall, the overlapping results obtained for green fluorescent protein fusions and Myc-tagged proteins undoubtedly demonstrate that ASFV p37 and p14 proteins exhibit nucleocytoplasmic transport activities. These findings are significant for understanding the role these proteins play in the replication cycle of ASFV.

Role of nitric oxide of the median preoptic nucleus (MnPO) in the alterations of salivary flow, arterial pressure and heart rate induced by injection of pilocarpine into the MnPO and intraperitoneally

We investigated the effect of L-NAME, a nitric oxide (NO) inhibitor and sodium nitroprusside (SNP), an NO-donating agent, on pilocarpine-induced alterations in salivary flow, mean arterial blood pressure (MAP) and heart rate (HR) in rats. Male Holtzman rats (250-300 g) were implanted with a stainless steel cannula directly into the median preoptic nucleus (MnPO). Pilocarpine (10, 20, 40, 80, 160 æg) injected into the MnPO induced an increase in salivary secretion (P<0.01). Pilocarpine (1, 2, 4, 8, 16 mg/kg) ip also increased salivary secretion (P<0.01). Injection of L-NAME (40 æg) into the MnPO prior to pilocarpine (10, 20, 40, 80, 160 æg) injected into the MnPO or ip (1, 2, 4, 8, 16 mg/kg) increased salivary secretion (P<0.01). SNP (30 æg) injected into the MnPO or ip prior to pilocarpine attenuated salivary secretion (P<0.01). Pilocarpine (40 æg) injection into the MnPO increased MAP and decreased HR (P<0.01). Pilocarpine (4 mg/kg body weight) ip produced a decrease in MAP and an increase in HR (P<0.01). Injection of L-NAME (40 æg) into the MnPO prior to pilocarpine potentiated the increase in MAP and reduced HR (P<0.01). SNP (30 æg) injected into the MnPO prior to pilocarpine attenuated (100 percent) the effect of pilocarpine on MAP, with no effect on HR. Administration of L-NAME (40 æg) into the MnPO potentiated the effect of pilocarpine injected ip. SNP (30 æg) injected into the MnPO attenuated the effect of ip pilocarpine on MAP and HR. The present study suggests that in the rat MnPO 1) NO is important for the effects of pilocarpine on salivary flow, and 2) pilocarpine interferes with blood pressure and HR (side effects of pilocarpine), that is attenuated by NO

The possibility of B-cell-dependent T-cell development.

The development of T cells is thought to be independent of B cells. However, defects in cell-mediated immunity in individuals with B-cell deficiency suggest the contrary. To test whether B cells affect T-lymphocyte development, we constructed mice with a monoclonal T-cell compartment (MT) and monoclonal B- and T-cell compartments (MBTs). In these mice, the T cells expressed a DO 11.10 transgenic (DO-T) cell receptor restricted to major histocompatibility complex (MHC) class IId. While CD4+ DO-T lymphocytes are rare in transgenic H-2b MT mice, we found that in H-2b MBT mice under the influence of B cells, DO-T lymphocytes mature into large numbers of CD4+ peripheral T cells. H-2b MBT mice have more CD4+ thymocytes than H-2b MT mice. These data are consistent with the view that B cells play some role in thymocyte development.