A peptide dataset for target analysis of human complement system proteins.

The targeted LC-MS/MS method has been widely applied for peptide quantification, offering sensibility, specificity, and reproducibility to the analysis. However, it requires the prior selection of targets, including the construction of a spectral library. Here, we present a dataset comprising peptide mass spectra for targeted LC-MS/MS method setup, applied to a set of human complement system proteins. Additionally, we selected a group of peptides and demonstrated their stability and reproducibility in quantification. This dataset is invaluable for studies aiming at the quantification of the complement system proteins by targeted LC-MS/MS, as it provides data for spectral library construction and a list of selected peptides.

Development of monoclonal antibodies against prM of Dengue virus 4 / Desenvolvimento de anticorpos monoclonais para prM de Dengue vírus 4

Introduction: dengue is a most common mosquito-borne viral disease in the Americas and tropical countries. Objective: in this work, mice were hyperimmunized with DENV 4 antigen to produce monoclonal antibodies (mAbs). Methodology: DENV 4 (GenBank KC806069) was inoculated in C6/36 cell monolayers cultivated in Leibovitz's 15 medium supplemented with 5% fetal bovine serum and incubated at 28 oC. The virus stock was submitted to concentration and ultracentrifugation and stored at -80 oC until use (VC DENV 4). Balb/c mice were injected intraperitoneally with 50µg of DENV-4 and successive intraperitoneal injections of 25 µg of VCDENV 4 with Freund's incomplete adjuvant were performed. The spleen cells were fused to SP2/0 myeloma cells with PEG 1540 and distributed in 96-well microplates with Iscove's modified medium with Hipoxantina­Aminopterina­Timidina. Hybridoma screening by indirect ELISA showed positive results for six mAbs, and their characterization was performed by Western blotting and Indirect Immunofluorescence (IFI) techniques. Results: the six mAbs showed strong recognition of prM (24/29 kDa), and minor reaction to E protein (66 kDa), E/E protein dimer (105 kDa), and NS1 (49 kDa) protein in two mAbs. The use of mAbs anti-prM as a diagnostic tool using IFI has been demonstrated to detect DENV-4 antigen in infected cells or tissues. Conclusion: DENV 4 generate mAbs with strong reactivity to prM with potential use to confirm the presence of DENV 4 antigen in tissues or infected cells.

Introdução: a dengue é uma doença viral transmitida por mosquitos comumente das Américas e países tropicais. Objetivo: neste trabalho, camundongos foram hiperimunizados com antígeno DENV 4 para produzir anticorpos monoclonais (mAbs). Metodologia: DENV 4 (GenBank KC806069) foi inoculado em monocamadas de células C6 / 36 cultivadas em meio Leibovitz 15 suplementado com 5% de soro fetal bovino e incubadas a 28oC. O estoque viral foi submetido à concentração, ultracentrifugação e armazenado a -80 oC (VC DENV 4). Camundongos Balb / c foram injetados intraperitonealmente com 50 µg de VC DENV-4 e injeções intraperitoneais sucessivas de 25 µg de antigeno com adjuvante incompleto de Freund. As células do baço foram misturadas a células SP2/0 com PEG 1540 e distribuídas em microplacas de 96 poços com meio Iscove Modificado em presença de Hipoxantina ­ Aminopterina ­ Timidina. A triagem de hibridomas por ELISA indireto apresentou resultados positivos para seis mAbs, e sua caracterização foi realizada por técnicas de Western blotting e Imunofluorescência Indireta (IFI). Resultados: os seis mAbs mostraram forte reconhecimento de prM (24/29 kDa) e reação menor à proteína E (66 kDa), dímero de proteína E / E (105 kDa) e proteína NS1 (49 kDa) em dois mAbs. O uso de mAbs anti-prM como uma ferramenta de diagnóstico utilizando IFI demonstrou eficacia em detectar o antígeno DENV-4 em células ou tecidos infectados. Conclusão: o mAbs produzidos para DENV 4 demonstraram uma forte reatividade contra prM, e poderiam ser uma ferramenta de uso potencial no diagnóstico de DENV 4 .

Complement-Mediated Differential Immune Response of Human Macrophages to Sporothrix Species Through Interaction With Their Cell Wall Peptidorhamnomannans.

In this study, the human immune response mechanisms against Sporothrix brasiliensis and Sporothrix schenckii, two causative agents of human and animal sporotrichosis, were investigated. The interaction of S. brasiliensis and S. schenckii with human monocyte-derived macrophages (hMDMs) was shown to be dependent on the thermolabile serum complement protein C3, which facilitated the phagocytosis of Sporothrix yeast cells through opsonization. The peptidorhamnomannan (PRM) component of the cell walls of these two Sporothrix yeasts was found to be one of their surfaces exposed pathogen-associated molecular pattern (PAMP), leading to activation of the complement system and deposition of C3b on the Sporothrix yeast surfaces. PRM also showed direct interaction with CD11b, the specific component of the complement receptor-3 (CR3). Furthermore, the blockade of CR3 specifically impacted the interleukin (IL)-1ß secretion by hMDM in response to both S. brasiliensis and S. schenckii, suggesting that the host complement system plays an essential role in the inflammatory immune response against these Sporothrix species. Nevertheless, the structural differences in the PRMs of the two Sporothrix species, as revealed by NMR, were related to the differences observed in the host complement activation pathways. Together, this work reports a new PAMP of the cell surface of pathogenic fungi playing a role through the activation of complement system and via CR3 receptor mediating an inflammatory response to Sporothrix species.

DENV-3 precursor membrane (prM) glycoprotein enhances E protein immunogenicity and confers protection against DENV-2 infections in a murine model.

To improve a DNA vaccine containing the truncated dengue virus serotype 2 (DENV-2) envelope (E) protein and evaluate the influence of precursor membrane (prM) glycoprotein polymorphism on E protein immunogenicity, two vaccine candidates have been constructed by upstream insertion of the DENV-2 and DENV-3 prM genes into the DENV-2 E gene, named pCID2EtD2prM and pCID2EtD3prM, respectively. Both constructs were able to induce antibody production, which were neutralizing against DENV-2 in a murine model. Splenocytes of immunized groups, when challenged with virus, demonstrated Th1 cytokine pattern and proliferation, in addition to the increase of specific T cells. Vaccine candidates pCID2EtD2prM and pCID2EtD3prM confer 70% and 90% protection against DENV-2, respectively. The pCID2EtD3prM plasmid conferred only 40% protection in the lethal challenge with DENV-2. The results demonstrate that DENV-3 prM has a greater influence on the immunogenicity of the E protein and, probably due to its role as a chaperone, these results may be related to the correct folding and, consequently, an increase in the presentation efficiency of produced transcripts.

Comparing Data-Independent Acquisition and Parallel Reaction Monitoring in Their Abilities To Differentiate High-Density Lipoprotein Subclasses.

High-density lipoprotein (HDL) is a diverse group of particles with multiple cardioprotective functions. HDL proteome follows HDL particle complexity. Many proteins were described in HDL, but consistent quantification of HDL protein cargo is still a challenge. To address this issue, the aim of this work was to compare data-independent acquisition (DIA) and parallel reaction monitoring (PRM) methodologies in their abilities to differentiate HDL subclasses through their proteomes. To this end, we first evaluated the analytical performances of DIA and PRM using labeled peptides in pooled digested HDL as a biological matrix. Next, we compared the quantification capabilities of the two methodologies for 24 proteins found in HDL2 and HDL3 from 19 apparently healthy subjects. DIA and PRM exhibited comparable linearity, accuracy, and precision. Moreover, both methodologies worked equally well, differentiating HDL subclasses' proteomes with high precision. Our findings may help to understand HDL functional diversity.

Caracterización de la atención farmacéutica en pacientes poli medicados con enfermedades crónicas no transmisibles del Hospital Nacional Especializado Rosales, atendidos en Farmacia Especializada del MINSAL. Agosto 2017 - Agosto 2018 / Characterization of pharmaceutical care in polymedicated patients with chronic noncommunicable diseases of the Rosales Specialized National Hospital, treated in the specialized pharmacy of MINSAL. August 2017 - August 2018

OBJETIVO: Caracterizar la atención farmacéutica en pacientes poli medicados con enfermedades crónicas no transmisibles, del Hospital Nacional Especializado Rosales, atendidos en la farmacia especializada del MINSAL. Agosto 2017 - Agosto 2018. METODOLOGÍA: Se realizó un estudio descriptivo de corte transversal; La muestra fue 378 expedientes de pacientes diagnosticados con Enfermedades Crónicas no Transmisibles, con seguimiento en el área de atención farmacéutica; Se diseñó una matriz para recolectar la información y facilitar su análisis haciendo uso de frecuencias de variables múltiples de SPSS. RESULTADOS: el 40% de las edades estaban en el rango de 66 años o más, el sexo femenino predominó con el 69%, más del 50% de los participantes estaban solteros, la ocupación más frecuente del estudio, fue ama de casa con un 43%, el 56% de las personas sabían leer, pero no habían realizado estudios de nivel básico, Las características personales fueron el PRM que predominó en el estudio con un 39% de los casos, la educación en medidas no farmacológicas fue la intervención que más se realizó de parte de los profesionales farmacéuticos con un 50% del total de casos, y el 71% de los casos reportados logró una mejoría en el control de su presión arterial con el seguimiento farmacoterapéutico. CONCLUSIONES: Se encontró que las características personales son el problema relacionado con la medicación más frecuente en pacientes con Enfermedades Crónicas no Transmisibles, la educación en medidas no farmacológicas es la intervención que más se realiza en atención farmacéutica y los logros en pacientes se evidenciaron en un mejor control de la presión arterial

The Roles of prM-E Proteins in Historical and Epidemic Zika Virus-mediated Infection and Neurocytotoxicity.

The Zika virus (ZIKV) was first isolated in Africa in 1947. It was shown to be a mild virus that had limited threat to humans. However, the resurgence of the ZIKV in the most recent Brazil outbreak surprised us because it causes severe human congenital and neurologic disorders including microcephaly in newborns and Guillain-Barré syndrome in adults. Studies showed that the epidemic ZIKV strains are phenotypically different from the historic strains, suggesting that the epidemic ZIKV has acquired mutations associated with the altered viral pathogenicity. However, what genetic changes are responsible for the changed viral pathogenicity remains largely unknown. One of our early studies suggested that the ZIKV structural proteins contribute in part to the observed virologic differences. The objectives of this study were to compare the historic African MR766 ZIKV strain with two epidemic Brazilian strains (BR15 and ICD) for their abilities to initiate viral infection and to confer neurocytopathic effects in the human brain's SNB-19 glial cells, and further to determine which part of the ZIKV structural proteins are responsible for the observed differences. Our results show that the historic African (MR766) and epidemic Brazilian (BR15 and ICD) ZIKV strains are different in viral attachment to host neuronal cells, viral permissiveness and replication, as well as in the induction of cytopathic effects. The analysis of chimeric viruses, generated between the MR766 and BR15 molecular clones, suggests that the ZIKV E protein correlates with the viral attachment, and the C-prM region contributes to the permissiveness and ZIKV-induced cytopathic effects. The expression of adenoviruses, expressing prM and its processed protein products, shows that the prM protein and its cleaved Pr product, but not the mature M protein, induces apoptotic cell death in the SNB-19 cells. We found that the Pr region, which resides on the N-terminal side of prM protein, is responsible for prM-induced apoptotic cell death. Mutational analysis further identified four amino-acid residues that have an impact on the ability of prM to induce apoptosis. Together, the results of this study show that the difference of ZIKV-mediated viral pathogenicity, between the historic and epidemic strains, contributed in part the functions of the structural prM-E proteins.

New approaches for the standardization and validation of a real-time qPCR assay using TaqMan probes for quantification of yellow fever virus on clinical samples with high quality parameters.

The development and production of viral vaccines, in general, involve several steps that need the monitoring of viral load throughout the entire process. Applying a 2-step quantitative reverse transcription real time PCR assay (RT-qPCR), viral load can be measured and monitored in a few hours. In this context, the development, standardization and validation of a RT-qPCR test to quickly and efficiently quantify yellow fever virus (YFV) in all stages of vaccine production are extremely important. To serve this purpose we used a plasmid construction containing the NS5 region from 17DD YFV to generate the standard curve and to evaluate parameters such as linearity, precision and specificity against other flavivirus. Furthermore, we defined the limits of detection as 25 copies/reaction, and quantification as 100 copies/reaction for the test. To ensure the quality of the method, reference controls were established in order to avoid false negative results. The qRT-PCR technique based on the use of TaqMan probes herein standardized proved to be effective for determining yellow fever viral load both in vivo and in vitro, thus becoming a very important tool to assure the quality control for vaccine production and evaluation of viremia after vaccination or YF disease.

Indicadores do Serviço de Atenção Farmacêutica (SAF) da Universidade do Sul de Santa Catarina / Indicators of the pharmaceutical care service at the University of Southern Santa Catarina

A Atenção Farmacêutica tem se constituído numa nova prática clínica para o farmacêutico. O estudo tem por objetivo apresentar os indicadores do Serviço de Atenção Farmacêutica da Universidade do Sul de Santa Catarina. Foi realizada a análise documental dos prontuários farmacêuticos de 58 pacientes entre setembro de 2007 a março de 2008. A maioria dos pacientes eram mulheres (77,6 por cento) com idade média de 54 anos. Identificou-se no primeiro encontro a média de 4,6 problemas de saúde por paciente e no último 4,3. As classes farmacológicas mais prevalentes foram a cardiovascular (30,2 por cento) e no sistema nervoso (27,5 por cento). Detectou-se média de 2,7 problemas relacionados com medicamentos por paciente, sendo os mais comuns aqueles relacionados à efetividade e à segurança. Dos problemas identificados 82,2 por cento foram classificados como evitáveis e 63,7 por cento como manifestados. Das intervenções farmacêuticas registradas soube-se da aceitação de 79,0 por cento com 78,9 por cento de resultados positivos. Encontrou-se média de 2,6 necessidades relacionadas ao paciente sendo, principalmente, dúvidas quanto à terapia farmacológica (30,4 por cento) sendo em 84,8 por cento dos casos supridas.

Pharmaceutical Care has represented a new clinical practice for pharmacists. This study aims to show the indicators of the Pharmaceutical Care Service at the University of Southern Santa Catarina. Documentary analysis of pharmaceutical records of 58 patients was conducted between September 2007 and March 2008 to identify the indicators. Most patients were women (77.6 percent), with a mean age of 54 years. Average number of health problems per patient was 4.6 in the first visit and 4.3 in the last one. The most widely used drug classes were cardiovascular (30.2 percent) and nervous system (27.5 percent). On average, 2.7 drug-related problems per patient were detected, the most common being those related to effectiveness and safety. Of the problems identified, 82.2 percent were classified as preventable and 63.7 percent as manifested. Pharmaceutical interventions registered 79.0 percent of acceptance, of which 78.9 percent had positive results. On average, there were 2.6 patient-related needs, especially regarding questions about drug therapy (30.4 percent), which were positively answered in 84.8 percent of cases. These indicators show that the pharmaceutical care service is very active in promoting health education.

Drug-related problems observed in a pharmaceutical care service, Belo Horizonte, Brazil / Problemas relacionados com a droga observados em um serviço de atenção farmacêutica, Belo Horizonte, Brasil

This present observational, longitudinal, and non-concurrent study was developed with the purposes of evaluate the profile of patients attended by a pharmacotherapeutic follow-up service and describe the Drug-Related Problems (DRPs) found over there; determine the proportion of DRPs between the health problems presented by the studied population, classifying them and identifying the situations related with their appearance. The study was developed at the School Pharmacy of Newton Paiva University Center, Belo Horizonte, MG, during the period from 2001 November up to 2003 November. Ninety seven patients have been evaluated, the majority of female sex (66.0 percent), with up to 8 years of scholarship (45.4 percent), mean age of 56.7 ± 13.0 years; mean of 4 ± 2 diagnosed diseases; 7 ± 6 complaints and 4 ± 2 medications per patient. Nine hundred and twelve health problems have been identified: 56.5 percent uncontrolled. From the uncontrolled problems, 380 (73.6 percent) were DRPs and between these, 81 (21.3 percent) were risks for DRP. From the 97 followed-up patients, 89 (91.7 percent) have presented at least one DRP during the follow-up. The more frequent DRPs were related to effectiveness (53.2 percent), to necessity (25.2 percent) and to safety (21.6 percent). A great number of uncontrolled problems was observed, as well as the possibility to resolve them by means of pharmaceutical care, indicating so the resolutive potential of this practice.

O presente estudo observacional, longitudinal, não concorrente teve por objetivos avaliar o perfil dos pacientes atendidos por um serviço de acompanhamento farmacoterapêutico e descrever os 'problemas relacionados com medicamento' (PRM) encontrados; determinar a proporção de PRM dentre os problemas de saúde apresentados pela população estudada, classificá-los e identificar situações relacionadas com seu surgimento. O estudo foi realizado na Farmácia Escola do Centro Universitário Newton Paiva, em Belo Horizonte, MG, durante o período de novembro de 2001 a novembro de 2003. Foram avaliados 97 pacientes, maioria mulheres (66,0 por cento), com até 8 anos de escolaridade (45,4 por cento), média de idade de 56,7 ± 13,0 anos; média de 4 ± 2 doenças com diagnóstico; 7 ± 6 queixas e 4 ± 2 medicamentos por paciente. Foram identificados 912 problemas de saúde: 56,5 por cento não controlados. Dos problemas não controlados, 380 (73,6 por cento) eram PRM e desses 81 (21,3 por cento) eram riscos de PRM. Dos 97 pacientes acompanhados 89 (91,7 por cento) apresentaram pelo menos um PRM durante o acompanhamento. Os PRMs mais frequentes foram relacionados à efetividade (53,2 por cento), à necessidade (25,2 por cento) e à segurança (21,6 por cento). Observou-se grande número de problemas não controlados e a possibilidade de resolvê-los por meio da atenção farmacêutica, demonstrando o potencial resolutivo dessa prática.

Eficácia da atenção farmacêutica em dislipidemia: revisão sistemática / Efficacy of pharmaceutical care in dyslipidemia: systematic review

Grande parte dos pacientes em tratamento com hipolipemiantes não atinge a meta terapêutica por diversas razões: falta de adesão, polifarmácia, reações adversas. A atenção farmacêutica tem como metas aumentar a adesão terapêutica, diminuir a toxicidade e otimizar resultados, prevenir e solucionar problemas relacionados a medicamentos (PRMs). O objetivo desse trabalho foi de revisar a literatura de forma sistemática para avaliar a eficácia da atenção farmacêutica no manejo da dislipidemia. Foram pesquisados artigos na base de dados MEDLINE (1966 – maio de 2006) e no banco de ensaios clínicos da biblioteca Cochrane. Foram incluídos estudos com delineamento de ensaios clínicos randomizados (ECR) que avaliassem a eficácia da atenção farmacêutica em dislipidemia com limitação de idiomas. A busca resultou em seis artigos selecionados no MEDLINE. A pesquisa na base de ensaios clínicos da Cochrane não acrescentou artigos diferentes. Todos os artigos analisados demonstraram resultados positivos da intervenção farmacêutica em dislipidemia. As doenças cardiovasculares, em especial a dislipidemia, possuem características que necessitam de orientação e monitorização do seu tratamento. De acordo com os artigos revisados, a atenção farmacêutica contribui para melhor tratamento da dislipidemia.

Many patients undergoing treatment with lipid-lowering drugs do not reach their therapeutic goal for a number of reasons: nonadherence, polypharmacy, adverse reactions. Pharmaceutical care aims at increasing therapeutic adherence, decreasing toxicity and improving outcomes, preventing and solving drug-related problems. We performed a systematic review of the literature to evaluate pharmaceutical care efficacy in the management of dyslipidemia. The MEDLINE database (1966 to May 2006) was searched for articles, and the Cochrane Library for clinical trials. Inclusion criteria were studies consisting of randomized clinical trials evaluating pharmaceutical care efficacy in dyslipidemia, with language restriction. MEDLINE search yielded six articles. Search in the Cochrane Library did not add any new clinical trial. All selected articles had positive results of pharmaceutical intervention in dyslipidemia. Cardiovascular diseases, especially dyslipidemia, have characteristics suggesting need of treatment guidance and monitoring. According to reviewed articles, pharmaceutical care contributes to better treatment of dyslipidemia.

Estudo do conhecimento dos profissionais farmacêuticos sobre a metodologia dáder de atenção farmacêutica / Study of the knowledge of pharmaceutical professionals on the methodology of pharmaceutical care

O estudo teve por objetivo verifi car o nível de conhecimento de profi ssionais farmacêuticos sobre a metodologia Dáder de Atenção Farmacêutica de um município da região Oeste do Paraná. Foi aplicado um questionário padrão aprovado pelo comitê de ética em pesquisa envolvendo seres humanos da UNIPAR (CEPEH/UNIPAR) protocolo no. 0043/05. A pesquisa foi realizada nos meses de junho e julho de 2005 e nesta ocasião foram entrevistados 45 farmacêuticos responsáveis técnicos pelas farmácias e drogarias do município. Foram coletados dados referentes a atividades de Atenção Farmacêutica desenvolvidas nas farmácias e do entendimento dos profi ssionais farmacêuticos em relação a metodologia Dáder de Atenção Farmacêutica. A maior parte dos entrevistados entende como atenção farmacêutica uma série de atividades desenvolvidas na farmácia como: informar os pacientes sobre posologia, indicação e interações medicamentosas. No entanto, essas atividades são inerentes à assistência farmacêutica. Verifi cou-se que 84,4% desenvolvem Atenção Farmacêutica em sua farmácia, e, apenas 13,33% destes, conhecem o Consenso Brasileiro de Atenção Farmacêutica e 11,11% conhecem e utilizam o método Dáder para o desenvolvimento das atividades farmacêuticas. Outro dado importante foi que 55,55% dos entrevistados sabem o que é um Problema Relacionado a Medicamento (PRM) e apenas 11,11% conhece o Consenso de Granada que defi ne os PRMs. Os resultados demonstram que ocorre uma confusão entre Atenção e Assistência Farmacêutica por parte dos entrevistados, o que demonstra a necessidade de melhor preparo e informação para a Prática da Atenção Farmacêutica. PALAVRAS CHAVE: Atenção farmacêutica; Método dáder; Problema relacionado a medicamento (PRM).

The main objective of this study was to verify the pharmacists’ knowledge level concerning Dáder’s methodology for pharmaceutical care in a municipal district of Paraná State, West area. A standard questionnaire approved by the UNIPAR Ethics Committee in Human Research (CEPEH/UNIPAR), Registry # 0043/05 was applied. The research was done from June to July, 2005, and 45 pharmacists in charge were interviewed in municipal pharmacies and drugstores. Data regarding Pharmaceutical Care activities in drugstores, and others related to the comprehension of these procedures by pharmacists were collected. Most of the interviewees defi ned as “Pharmaceutical Care” the general orientations given about the drug use (dosage, indication, drug interactions). It was verifi ed that 84.4% of the pharmacists apply the Pharmaceutical Care procedures in the drugstore. However, only 13.33% of them know the Brazilian Consensus of Pharmaceutical Care. In addition, only 11.11% know and use Dáder’s methodology. On the other hand, 55.55% of the interviewees know what PRD (Problem Related to Drug) is, but only 11.11% know the Grenade Consensus that defi nes PRD. The results demonstrate a distortion between the concept of Attention and Pharmaceutical Care, in the case of pharmacists submitted to interview and the need of better information to these professionals.