Anti-inflammatory and antioxidant nanostructured cellulose membranes loaded with phenolic-based ionic liquids for cutaneous application

The utilization of natural compounds, such as phenolic acids and biopolymers, in the healthcare domain is gaining increasing attention. In this study, bacterial nanocellulose (BC) membranes were loaded with ionic liquids (ILs) based on phenolic acids. These ionic compounds, with improved solubility and bioavailability, were prepared by combining the cholinium cation with anions derived from caffeic, ellagic and gallic acids. The obtained BC-ILs membranes were homogeneous, conformable and their swelling ability agreed with the solubility of each IL. These membranes revealed a controlled ILs dissolution rate in the wet state and high antioxidant activity. In vitro assays performed with Raw 264.7 macrophages and HaCaT keratinocytes revealed that these novel BC-ILs membranes are non-cytotoxic and present relevant anti-inflammatory properties. Diffusion studies with Hanson vertical diffusion cells showed a prolonged release profile of the ILs from the BC membranes. Thus, this work, successfully demonstrates the potential of BC-ILs membranes for skin treatment.publishe

Bicistronic DNA vaccines simultaneously encoding HIV, HSV and HPV antigens promote CD8⁺ T cell responses and protective immunity

Millions of people worldwide are currently infected with human papillomavirus (HPV), herpes simplex virus (HSV) or human immunodeficiency virus (HIV). For this enormous contingent of people, the search for preventive and therapeutic immunological approaches represents a hope for the eradication of latent infection and/or virus-associated cancer. To date, attempts to develop vaccines against these viruses have been mainly based on a monovalent concept, in which one or more antigens of a virus are incorporated into a vaccine formulation. In the present report, we designed and tested an immunization strategy based on DNA vaccines that simultaneously encode antigens for HIV, HSV and HPV. With this purpose in mind, we tested two bicistronic DNA vaccines (pIRES I and pIRES II) that encode the HPV-16 oncoprotein E7 and the HIV protein p24 both genetically fused to the HSV-1 gD envelope protein. Mice i.m. immunized with the DNA vaccines mounted antigen-specific CD8⁺ T cell responses, including in vivo cytotoxic responses, against the three antigens. Under experimental conditions, the vaccines conferred protective immunity against challenges with a vaccinia virus expressing the HIV-derived protein Gag, an HSV-1 virus strain and implantation of tumor cells expressing the HPV-16 oncoproteins. Altogether, our results show that the concept of a trivalent HIV, HSV, and HPV vaccine capable to induce CD8⁺ T cell-dependent responses is feasible and may aid in the development of preventive and/or therapeutic approaches for the control of diseases associated with these viruses.CNPqINCTVFAPESPUS

Parenteral adjuvant effects of an enterotoxigenic Escherichia coli natural heat-labile toxin variant

Native type I heat-labile toxins (LTs) produced by enterotoxigenic Escherichia coli (ETEC) strains exert strong adjuvant effects on both antibody and T cell responses to soluble and particulate antigens following co-administration via mucosal routes. However, inherent enterotoxicity and neurotoxicity (following intra-nasal delivery) had reduced the interest in the use of these toxins as mucosal adjuvants. LTs can also behave as powerful and safe adjuvants following delivery via parenteral routes, particularly for activation of cytotoxic lymphocytes. In the present study, we evaluated the adjuvant effects of a new natural LT polymorphic form (LT2), after delivery via intradermal (i.d.) and subcutaneous (s.c.) routes, with regard to both antibody and T cell responses. A recombinant HIV-1 p24 protein was employed as a model antigen for determination of antigen-specific immune responses while the reference LT (LT1), produced by the ETEC H10407 strain, and a non-toxigenic LT form (LTK63) were employed as previously characterized LT types. LT-treated mice submitted to a four dose-base immunization regimen elicited similar p24-specific serum IgG responses and CD4(+) T cell activation. Nonetheless, mice immunized with LT1 or LT2 induced higher numbers of antigen-specific CD8(+) T cells and in vivo cytotoxic responses compared to mice immunized with the non-toxic LT derivative. These effects were correlated with stronger activation of local dendritic cell populations. In addition, mice immunized with LT1 and LT2, but not with LTK63, via s.c. or i.d. routes developed local inflammatory reactions. Altogether, the present results confirmed that the two most prevalent natural polymorphic LT variants (LT1 or LT2) display similar and strong adjuvant effects for subunit vaccines administered via i.d. or s.c. routes.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)National Institute of Science and Technology in Vaccines (INCTV

Reaproveitamento de resíduos alimentares na produção de tanase por Cunninghamella echinulata UCP 1305 através de fermentação submersa / Reuse of food waste in the production of tannase by Cunninghamella echinulata UCP 1305 through submerged fermentation

As enzimas microbianas têm aumentado sua produção nas últimas décadas, devido a sua enorme versatilidade em diversos processos industriais. Tanino acil hidrolase (EC 3.1.1.20) ou tanase, é uma enzima que apresenta uma grande versatilidade e está envolvida diretamente na hidrolise de ésteres e ligações laterais de taninos. Esta enzima é produzida principalmente por diversos gêneros de fungos filamentosos, leveduras e bactérias. Foram realizados estudos envolvendo amostras de Cunninghamella echinulata (UCP 1305 e 1308), isoladas de amostras de solo da Caatinga da cidade de Serra Talhada no Estado de Pernambuco, onde inicialmente foram testadas diferentes temperaturas (28 e 37 ºC) e pH (4.5, 5.5, 6.5, 7.0 e 8.0) em meio sólido durante 120 horas, com acompanhamento diário. Após a seleção da melhor amostra produtora, foram realizados estudos de produção enzimática através de fermentação submersa, utilizando quatro meios de composição química diferenciada, durante 120 horas, 37 oC e 150 rpm. Nesses ensaios foram avaliados a curva de crescimento microbiano, variação do pH e a determinação enzimática das amostras a cada 24 hs, Após a seleção do melhor meio de produção, foram realizados ensaios envolvendo planejamento fatorial de 23 com meios contendo resíduos agroindustriais (resíduo de uva, cascas de laranjas e café) nas mesmas condições do meio controle, realizado previamente. Os resultados obtidos revelaram que a amostra Cunninghamella echinulata UCP 1305 demonstrou a produção de tanase em meio sólido através do aparecimento do halo característico da enzima testada (4,5 cm), com valores de pH de 7,0 e temperatura de 37 oC. Nos ensaios de fermentação submersa, os melhores resultados da atividade enzimática foram obtidos no meio denominado 3, pH de 6.58 e atividade de 0,413 U/mL, sendo denominado de meio controle.  Os ensaios utilizando meios alternativos contendo resíduos agroindustriais, demonstraram que o meio contendo resíduo da uva à 3%, apresentou máximo de atividade da tanase (0,357 U/mL) quando comparado aos demais meios testados nas mesmas condições. Verifica-se que a utilização de meios utilizando resíduos agroindustriais ricos em taninos surgem como uma alternativa promissora nos processos fermentativos, pois reduzem a poluição ambiental e geram produtos biotecnológicos de alto valor agregado para as indústrias. 

Epidemiologic Study of Retinoblastoma in Recife, Pernambuco - Brazil: January 1985 - July 1997 Estudo Epidemiológico do Retinoblastoma no Recife - Pernambuco - Brasil: janeiro 1985 - julho 1997

Background: Retinoblastoma is by far the most frequent malignant intraocular tumor of childhood. This study was performed to characterize the clinical, diagnostic, treatment and prognostic aspects in patients with retinoblastoma in three reference centers for this pathology in the city of Recife, Pernambuco - Brazil. Methods: A consecutive series of 85 patients with retinoblastoma was reviewed. The authors selected 66 patients (77.6%) that fulfilled the inclusion criteria for these study. Results: Of a total of 66 patients with retinoblastoma, 4.5% had a previous history of the disease in the family. Males were more affected than females at a ratio (male/female) of 1.12. The mean age of appearance of the first symptoms was 23.8 months, with leukocoria and ocular hyperemia being the most frequent. The mean age at time of diagnosis was 31.7 months and for surgical treatment 32.8 months. The right eye was affected in 42.4% of the cases and the left was involved in 37.9% of cases. The tumor was unilateral in 80.3% and bilateral in 19.7% of the cases. There was extraocular involvement in 62.1%, and it was intraocular in 37.9% of the patients. Treatment was surgery combined with chemotherapy in 47.0% of the patients. 27.3% of the patients died and 19.7% abandoned the treatment. Conclusions: The data on the epidemiology of retinoblastoma found in our city resembles that of other developing countries, concerning the epidemioloy of retinoblastoma.<br>Objetivo: O retinoblastoma é o tumor maligno intraocular mais freqüente da infância. O objetivo deste estudo foi avaliar aspectos clínicos, de diagnóstico, tratamento e prognóstico em pacientes portadores de retinoblastoma atendidos em três centros de referência para esta patologia na cidade do Recife - PE. Métodos: Revisamos 85 prontuários de portadores de retinoblastoma e apresentamos o resultado da análise de 66 destes pacientes, atendidos durante o período de janeiro de 1985 a julho de 1997. Resultados: Apenas 4,5% tinham história pregressa da doença. O sexo masculino foi mais acometido numa razão homem/mulher de 1,12. A média de idade ao surgimento dos primeiros sintomas foi de 23,8 meses, sendo leucocoria e hiperemia ocular os mais freqüentes. A média de idade ao diagnóstico foi de 31,7 meses e a média de idade ao tratamento cirúrgico foi de 32,8 meses. O olho direito, com 42,4% dos casos, foi mais acometido que o esquerdo (37,9%). O tumor era unilateral em 80,3% dos casos e bilateral em 19,7%. Os tumores eram extra-oculares ao diagnóstico em 62,1%, e intra-oculares em 37,9%. O tratamento mais instituído foi a cirurgia associada à quimioterapia, em 47% dos casos. 27,3% dos pacientes tratados foram a óbito e 19,7% abandonaram o tratamento. Conclusões: Os dados encontrados em nosso estado asseme-lham-se àqueles observados em países em desenvolvimento, no que concerne à epidemiologia do retinoblastoma

Effect of pressure on refolding of recombinant pentameric cholera toxin B

The production of recombinant proteins is an essential tool for the expansion of modern biological research and biotechnology. The expression of heterologous proteins in Escherichia coli often results in an incomplete folding process that leads to the accumulation of inclusion bodies (IB), aggregates that hold a certain degree of native-like secondary structure. High hydrostatic pressure (HHP) impairs intermolecular hydrophobic and electrostatic interactions, leading to dissociation of aggregates under non-denaturing conditions and is therefore a useful tool to solubilize proteins for posterior refolding. Cholera toxin (CT) is composed of a non-toxic pentamer of B subunits (CTB), a useful adjuvant in vaccines, and a toxic subunit A (CTA). We studied the process of refolding of CTB using HHP. HHP was shown to be effective for dissociation of CTB monomers from IB. Posterior incubation at atmospheric pressure of concentrated CTB (1mg/ml) is necessary for the association of the monomers. Pentameric CTB was obtained when suspensions of CTB IB were compressed at 2.4kbar for 16h in the presence of Tween 20 and incubated at 1bar for 120h. Soluble and biologically active pentameric CTB was obtained, with a yield of 213mg CTB/liter of culture. The experience gained in this study can be important to improve the refolding of proteins with quaternary structureState of São Paulo Research Foundation – FAPESP (process 10/13353-0)134211/2010-3 National Council for Scientific and Technological Development – CNP

Human respiratory syncytial virus N, P and M protein interactions in HEK-293T cells

Characterization of Human Respiratory Syncytial Virus (HRSV) protein interactions with host cell components is crucial to devise antiviral strategies. Viral nucleoprotein, phosphoprotein and matrix protein genes were optimized for human codon usage and cloned into expression vectors. HEK-293T cells were transfected with these vectors, viral proteins were immunoprecipitated, and co-immunoprecipitated cellular proteins were identified through mass spectrometry. Cell proteins identified with higher confidence scores were probed in the immunoprecipitation using specific antibodies. The results indicate that nucleoprotein interacts with arginine methyl-transferase, methylosome protein and Hsp70. Phosphoprotein interacts with Hsp70 and tropomysin, and matrix with tropomysin and nucleophosmin. Additionally, we performed immunoprecipitation of these cellular proteins in cells infected with HRSV, followed by\ud detection of co-immunoprecipitated viral proteins. The results indicate that these interactions also occur in the context of viral infection, and their potential contribution for a HRSV replication model is discussed.Fundação de Amparo à Pesquisa do Estado de São Paulo, FAPESP (proc. 2009/17587-9)Conselho Nacional de Desenvolvimento Científico e Tecnológico, CNPq (proc. 477734/2009-0

Human respiratory syncytial virus detection in children admitted at a community hospital in Botucatu, SP, Brazil

O Vírus Respiratório Sincicial Humano (VRSH) é descrito como o mais importante patógeno viral causador de doenças respiratórias agudas das vias respiratórias inferiores em crianças. Neste estudo 84 amostras de crianças com idade abaixo dos dois anos apresentando sintomas de doença respiratória aguda, foram obtidas no período de setembro de 2000 a novembro de 2001. Analise por imunofluorescência indireta e transcrição reversa seguida de PCR, revelou que 18% (15/84) das amostras foram positivas, sendo que em 80% (12/15) dos casos a detecção de VRSH foi observada em crianças abaixo dos seis meses, e também que os subgrupos A e B co-circularam. Estes são os primeiros dados obtidos para a cidade de Botucatu, sendo que a sazonalidade mostrou-se evidente pela maior circulação desse vírus entre os meses de maio e julhoHuman Respiratory Syncytial Virus (HRSV) is recognized as the single most important pathogen responsible for acute viral infection of the lower respiratory tract in young children. In this study, 84 samples from children under two years of age presenting acute respiratory symptoms were obtained from September 2000 to November 2001. Analysis by immunofluorescence and reverse-transcription followed by PCR, revealed that 18% (15/84) of all the samples were positive for HRSV, in which 80% (12/15) of the cases was observed in children under six months of age and the subgroups A and B co-circulated in the studied period. These are the first data obtained for Botucatu city and seasonality is evident by the higher circulation of the virus between May and July