A map of neutrophil granules by proteomics / Mapa dos neutrófilos por proteômica (OU) Mapa dos grânulos do neutrófilo por proteômica

Neutrophils are polymorphonuclear leukocytes that play a key role in the organism defense. These cells enroll in a range of actions to ensure pathogen elimination and orchestrate both innate and adaptative immune responses. The main physiological structures of neutrophils are their storage organelles that are essential since the cells activation and participate in all their functions. The storage organelles are divided into 2 types: granules and secretory vesicles. The granules are subdivided into azurophilic, specific and gelatinase. The granules are distinguished by their protein content, and since they play an important role on the neutrophil function, the knowledge of the proteins stored in these organelles can help to better understand these cells. Some proteins are present in high abundance and are used as markers for each storage organelle. These proteins are myeloperoxidase (MPO) for azurophil granules, neutrophil gelatinase associated with lipocalin-2 (NGAL) and lactoferrin (LTF) for specific granules, matrix metalloproteinase-9 (MMP9) for gelatinase granules and alkaline phosphatase (AP) for secretory vesicles. The isolation of neutrophils granules, however, is challenging and the existing procedures rely on large sample volumes, about 400 mL of peripheral blood or 3 x 108 neutrophils, not allowing for multiple biological and technical replicates. Therefore, the aim of this study was to develop a miniaturized neutrophil granules isolation method and to use biochemical assays, mass spectrometry-based proteomics and a machine learning approach to investigate the protein content of the neutrophils storage organelles. With that in mind, 40 mL of the peripheral blood of three apparently healthy volunteers were collected. The neutrophils were isolated, disrupted using nitrogen cavitation and organelles were fractionated with a discontinuous 3-layer Percoll density gradient. The presence of granules markers in each fraction was assessed using western blot , gelatin zymography and enzymatic assays. The isolation was proven successful and allowed for a reasonable separation of all neutrophils storage organelles in a gradient of less than 1 mL, about 37 times smaller than the methodsdescribed in the literature. Moreover, mass spectrometry-based proteomics identified 369 proteins in at least 3 of the 5 samples, and using a machine learning strategy, the localization of 140 proteins was predicted with confidence. Furthermore, this study was the first to investigate the proteome of neutrophil granules using technical and biological replicates, creating a reliable database for further studies. In conclusion, the developed miniaturized method is reproducible, cheaper, and reliable. In addition, it provides a resource for further studies exploring neutrophil granules protein content and mobilization during activation with different stimuli

Neutrófilos são leucócitos polimorfonucleares que possuem papel fundamental na defesa do organismo. Essas células desempenham diversas ações a fim de assegurar a eliminação de um patógeno e, além disso, orquestram a resposta imune inata e adaptativa. O conjunto composto pelos grânulos de armazenamento e as vesículas secretórias compõe a principal estrutura fisiológica dos neutrófilos. Estes componentes são essenciais desde a ativação celular, participando de todas as funcionalidades desta célula. Os grânulos são subdivididos em azurófilos, específicos e gelatinase. Eles podem ser distinguidos por meio de seu conteúdo proteico e, como são importantes na funcionalidade dos neutrófilos, identificar quais proteínas são armazenadas nestas organelas é imprescindível para entender melhor essa célula como um todo. Algumas proteínas, estão presentes de forma abundante e, portanto, são utilizadas como marcadores dos grânulos. Tais proteínas são mieloperoxidase (MPO) para os grânulos azurófilos, gelatinase de neutrófilo associada a lipocalina (NGAL) e lactoferrina (LTF) para os específicos, metaloproteinase de matrix 9 (MMP9) para os grânulos de gelatinase e fosfatase alcalina (AP) para as vesículas secretórias. Isolar estas estruturas, no entanto, é desafiador visto que os protocolos existentes na literatura utilizam grandes volumes de amostra, cerca de 400 mL de sangue ou 3 x 108 neutrófilos, para apenas um isolamento, impedindo a realização de replicatas técnicas e biológicas. Desta forma, o objetivo do presente estudo foi desenvolver um protocolo miniaturizado de isolamento dos grânulos neutrofílicos e utilizar métodos bioquímicos, de proteômica e machine learning para investigar o conteúdo proteico destas estruturas celulares. Para isto, 40 mL de sangue periférico de três voluntários aparentemente saudáveis foi coletado. Os neutrófilos foram então isolados, lisados com cavitação de nitrogênio e o fracionamento subcelular foi realizado baseado em um gradiente descontínuo de 3 camadas de Percoll. O método de isolamento foi avaliado através da investigação dos marcadores utilizando western blotting (WB), zimografia de gelatina e ensaios enzimáticos em cada fração coletada. O isolamento demonstrou-se eficiente e permitiu uma ótima separação dos grânulosem um gradiente menor que 1 mL, cerca de 37 vezes menor que os métodos atualmente descritos na literatura. Além disso, a análise proteômica foi capaz de identificar 369 proteínas presentes em pelo menos 3 das 5 réplicas investigadas e, utilizando ferramentas de machine learning, 140 proteínas foram classificadas como pertencentes a um dos tipos de grânulos ou vesícula secretória com alto nível de confiabilidade. Por fim, o presente estudo foi o primeiro a investigar o proteoma dos grânulos utilizando replicatas técnicas e biológicas, criando e fornecendo uma base de dados robusta que poderá ser utilizada em estudos futuros. Conclui-se, portanto, que a metodologia miniaturizada desenvolvida é eficaz, reprodutível e mais barata, além de permitir estudos mais complexos e profundos sobre o proteoma dos grânulos dos neutrófilos em diferentes momentos celulares, tais como quando ativados via estímulos distintos

Biomarcadores da função renal: do que dispomos atualmente? / Biomarkers of renal function: what is currently available?

A avaliação da função renal é de extrema importância na prática clínica, tanto para o diagnóstico quanto para e prognóstico e monitoração das doenças renais. Neste contexto, aparticipação do laboratório é de grande importância, uma vez que a maior parte das doenças renais só se manifesta clinicamente quando mais de 50% a 75% da função renal estácomprometida. O desenvolvimento de novos biomarcadores para diagnóstico precoce, estratificação de risco, prognóstico de lesão renal tem sido um dos principais alvos das pesquisas envolvendo o sistema renal. Dessa forma, diversos novos biomarcadores, tais como lipocalina associada à gelatinase de neutrófilos (NGAL), cistatina C, molécula-1 de lesão renal (KIM-1), interleucina-18 (IL-18), enzimas urinárias tubulares e proteínas de baixo peso molecular, dentre outros, têm sido propostos para diagnosticar /monitorar as doenças renais agudas e crônicas. Este estudo visa discutir aspectos associados aos principais biomarcadores utilizados na rotina laboratorial para diagnóstico, prognóstico e acompanhamento do paciente com disfunção renal, bem como apresentar novos marcadores que se destacam na literatura recente e que podem ser promissores na prática clínica

The assessment of renal function is very important in clinical practice, both for diagnosis and for prognosis and monitoring of renal diseases. In this context, the role of the laboratory is of great importance, since most of the kidney disease manifests itself clinically only when more than 50 to 75% of kidney function is compromised. The development of new biomarkers for early diagnosis, risk stratification, prognosis of renal injury has been a major focus of research involving the renal system. Thus, several new biomarkers, such as neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), interleukin-18 (IL18) and low-molecular weight proteins and enzymes, and others, have been proposed to diagnose/monitoring acute and chronic renal diseases. The aim of this study is to discuss aspects related to the main biomarkers used in routine laboratory tests for diagnosis, prognosis and monitoring of patients with renal dysfunction, as well as provide new markers that stand out in the recent literature, and that may be promising in clinical practice

Effects of platelet rich plasma on fascial healing in rats with fecal peritonitis

ABSTRACT PURPOSE : To evaluate the effects of platelet rich plasma (PRP) on the healing of fascia wherein peritonitis has been created. METHODS: Twenty eight Wistar Albino rats were divided into four groups. Only a primary fascial repair following laparotomy was performed on Group 1, a primary fascial repair performed and PRP treatment applied following laparotomy on Group 2, and a fecal peritonitis created following laparotomy and a primary fascial repair carried out on Group 3. A fecal peritonitis was created following laparotomy and primary fascial repair and PRP treatment on the fascia was carried out on Group 4. RESULTS: TNF-α was found to be significantly lower in the control group (Group 1). It was detected at the highest level in the group in which fecal peritonitis was created and PRP applied (Group 4). TGF-β was determined as being significantly higher only in Group 4. Histopathologically, the differences between the groups in terms of cell infiltration and collagen deposition were not found to be significant. CONCLUSION: When platelet rich plasma was given histologically and biochemicaly as wound healing parameters cellular infiltration, collagen accumulation, and tissue hydroxyiproline levels were not increased but neovascularization, fibroblast activation and TNF Alfa levels were increased and PRP accelerated wound healing.

Neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker of kidney damage: a review / Lipocalina associada à gelatinase neutrofílica (NGAL) como um biomarcador de lesão renal: uma revisão

Neutrophil gelatinase- associated lipocalin (NGAL) is a protein molecule predominantly expressed in the distal nephron after the occurrence of renal injury. Unlike the serum creatinine and the glomerular filtration rate, which are the renal function markers, the increased levels of NGAL, both in serum and urine, are closely linked to the structural injury to the nephron. Clinical studies indicate that a few hours after the occurrence of acute renal injury, the serum and urinary levels of NGAL are already significantly elevated, while the serum creatinine levels and renal clearance only undergo significant changes between 24-48h after injury. Thus, the use of renal function markers, usually assessed in the clinical practice, they may have some limitations also hindering the implementation of early measures aimed at protecting the kidneys. This literature review aims at examining the biological aspects and the applications of NGAL measurement in some clinical conditions, including kidney failure, kidney diseases and renal ischemia.

Lipocalina associada à gelatinase neutrofílica (NGAL) é uma molécula proteica predominantemente expressa na parte distal do néfron após a ocorrência de lesão renal. Diferentemente da creatinina sérica e da taxa de filtração glomerular, que são marcadores de função renal, os níveis aumentados de NGAL, tanto no soro quanto na urina, estão intimamente ligados a lesões estruturais do néfron. Os estudos clínicos indicam que poucas horas após a ocorrência da lesão renal aguda os níveis séricos e urinários de NGAL já se apresentam significativamente elevados, enquanto os níveis séricos de creatinina e a sua depuração renal apenas sofrem alterações significativas entre 24-48h após a lesão. Assim, a utilização de marcadores de função renal, usualmente avaliados na prática clínica, pode apresentar algumas limitações além de dificultar a aplicação de medidas precoces que visam a proteção renal. Esta revisão da literatura tem por objetivo analisar os aspectos biológicos e as aplicações da mensuração de NGAL em algumas condições clínicas, incluindo injúria renal, nefropatias e isquemia renal.

Lipocalina associada à gelatinase neutrofílica(NGAL) como um biomarcador de lesão renal: uma revisão / Neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker of kidney damage: a review

Lipocalina associada à gelatinase neutrofílica (NGAL) é uma molécula proteica predominantemente expressa na parte distal do néfron após a ocorrência de lesão renal. Diferentemente da creatinina sérica e da taxa de filtração glomerular, que são marcadores de função renal, os níveis aumentados de NGAL, tanto no soro quanto na urina, estão intimamente ligados a lesões estruturais do néfron.Os estudos clínicos indicam que poucas horas após a ocorrência da lesão renal aguda os níveis séricos e urinários de NGAL já se apresentam significativamente elevados, enquanto os níveis séricos de creatinina e a sua depuração renal apenas sofrem alterações significativas entre 24-48hapós a lesão. Assim, a utilização de marcadores de função renal, usualmente avaliados na práticaclínica, pode apresentar algumas limitações além de dificultar a aplicação de medidas precoces quevisam a proteção renal. Esta revisão da literatura tem por objetivo analisar os aspectos biológicos e as aplicações da mensuração de NGAL em algumas condições clínicas, incluindo injúria renal, nefropatias e isquemia renal.

Neutrophil gelatinase-associated lipocalin (NGAL) is a protein molecule predominantly expressed in the distal part of the nephron following the occurrence of renal damage. Differently from serum creatinine and glomerular filtration rate, which are markers of renal function, increased levels of NGAL in both serum and urine are closely linked to structural damage of the nephron. Clinical studies indicate that within a few hours of occurrence Of acute renal injury serum and urinary NGAL levels are already significantly elevated, while serum creatinine levels and renal clearance only change significantly between 24-48 hours after injury. Thus, the use of renal function markers, usually evaluated in practice, may present some limitations, as well as hinder the application of early measures that aim at renal protection. This review of the literature aims to analyze the biological aspects and applications of NGAL measurement in some clinical conditions, including renal injury, nephropathy and renal ischemia.

In ovo administration of human recombinant leptin shows dose dependent angiogenic effect on chicken chorioallantoic membrane

BACKGROUND: Leptin, the cytokine produced by white adipose tissue is known to regulate food energy homeostasis through its hypothalamic receptor. In vitro studies have demonstrated that leptin plays a major role in angiogenesis through binding to the receptor Ob-R present on ECs by stimulating and initiating new capillary like structures from ECs. Various in vivo studies indicate that leptin has diverse effect on angiogenesis. A few reports have showed that leptin exerts pro angiogenic effects while some suggested that it has antiangiogenic potential. It is theoretically highly important to understand the effect of leptin on angiogenesis to use as a therapeutic molecule in various angiogenesis related pathological conditions. Chicken chorio allantoic membrane (CAM) on 9th day of incubation was incubated with 1, 3 and 5 µg concentration of HRL for 72 h using gelatin sponge. Images where taken after every 24 h of incubation and analysed with Angioguant software. The treated area was observed under microscope and histological evaluation was performed for the same. Tissue thickness was calculated morphometrically from haematoxylin and eosin stained cross sections. Reverse transcriptase PCR and immunohistochemistry were also performed to study the gene and protein level expression of angiogenic molecules. RESULTS: HRL has the ability to induce new vessel formation at the treated area and growth of the newly formed vessels and cellular morphological changes occur in a dose dependent manner. Increase in the tissue thickness at the treated area is suggestive of initiation of new capillary like structures. Elevated mRNA and protein level expression of VEGF165 and MMP2 along with the activation of ECs as demonstrated by the presence of CD34 expression supports the neovascularization potential of HRL. CONCLUSION: Angiogenic potential of HRL depends on the concentration and time of incubation and is involved in the activation of ECs along with the major interaction of VEGF 165 and MMP2. It is also observed that 3 µg of HRL exhibits maximum angiogenic potential at 72 h of incubation. Thus our data suggest that dose dependent angiogenic potential HRL could provide a novel role in angiogenic dependent therapeutics such as ischemia and wound healing conditions.

Molecular detection of virulence factors among food and clinical Enterococcus faecalis strains in South Brazil

The present report aimed to perform a molecular epidemiological survey by investigating the presence of virulence factors in E. faecalis isolated from different human clinical (n = 57) and food samples (n = 55) in Porto Alegre, Brazil, collected from 2006 to 2009. In addition, the ability to form biofilm in vitro on polystyrene and the β-haemolytic and gelatinase activities were determined. Clinical strains presented a higher prevalence of aggregation substance (agg), enterococcal surface protein (esp) and cytolysin (cylA) genes when compared with food isolates. The esp gene was found only in clinical strains. On the other hand, the gelatinase (gelE) and adherence factor (ace) genes had similar prevalence among the strains, showing the widespread occurrence of these virulence factors among food and clinical E. faecalis strains in South Brazil. More than three virulence factor genes were detected in 77.2% and 18.2% of clinical and food strains, respectively. Gelatinase and β-haemolysin activities were not associated with the presence of gelE and cylA genes. The ability to produce biofilm was detected in 100% of clinical and 94.6% of food isolates, and clinical strains were more able to form biofilm than the food isolates (Student's t-test, p < 0.01). Results from the statistical analysis showed significant associations between strong biofilm formation and ace (p = 0.015) and gelE (p = 0.007) genes in clinical strains. In conclusion, our data indicate that E. faecalis strains isolated from clinical and food samples possess distinctive patterns of virulence factors, with a larger number of genes that encode virulence factors detected in clinical strains.

Prevalence and phenotypic characterization of Enterococcus spp. isolated from food in Brazil

We evaluated the frequency of enterococci from food and found 95.2% of positivity, being E. faecium and E. faecalis the most frequent species. High-level streptomycin resistance was observed, as well as gelatinase and hemolysis activity, showing the potential role of environmental strains as reservoir of virulence and resistance traits.

Presence of virulence factors in Enterococcus faecalis and Enterococcus faecium susceptible and resistant to vancomycin

Despite the increasing importance of Enterococcus as opportunistic pathogens, their virulence factors are still poorly understood. This study determines the frequency of virulence factors in clinical and commensal Enterococcus isolates from inpatients in Porto Alegre, Brazil. Fifty Enterococcus isolates were analysed and the presence of the gelE, asa1 and esp genes was determined. Gelatinase activity and biofilm formation were also tested. The clonal relationships among the isolates were evaluated using pulsed-field gel electrophoresis. The asa1, gelE and esp genes were identified in 38%, 60% and 76% of all isolates, respectively. The first two genes were more prevalent in Enterococcus faecalis than in Enterococcus faecium, as was biofilm formation, which was associated with gelE and asa1 genes, but not with the esp gene. The presence of gelE and the activity of gelatinase were not fully concordant. No relationship was observed among any virulence factors and specific subclones of E. faecalis or E. faecium resistant to vancomycin. In conclusion, E. faecalis and E. faecium isolates showed significantly different patterns of virulence determinants. Neither the source of isolation nor the clonal relationship or vancomycin resistance influenced their distribution.

Zimography is an effective method for detection of matrix metalloproteinase 2 (MMP-2) activity in cultured human fibroblasts

PURPOSE: To describe a method to characterize the gelatinase activity of cultured human periodontal fibroblasts stimulated with Pam3Cys and E. coli LPS, ligands of TLR2 and TLR4 respectively, and by centrifugation of the cultures, simulating an orthodontic force. METHODS: To study MMP-2 activity, primary cultures of human periodontal fibroblasts were stimulated with the addition of TLRs 2 and 4 ligands and the application of mechanical force by centrifugation at 141 x g for 30 min. Supernatant media was collected 24 hours later to perform protein quantification and zymography. RESULTS: MMP-2 activity suffered an increase in cultures co-stimulated with TLRs 2 and 4 ligands alone or with the presence of mechanical force application compared to basal levels. CONCLUSION: Zymography, one of the several methods to study MMPs activities, is a simple, qualitative and efficient method based on electrophoresis of bis-acrylamide gels copolymerized with a protein substrate.

Heavy metal tolerance (Cr, Ag and Hg) in bacteria isolated from sewage

Samples of sewage from a university hospital and a chemistry technical school were analysed for the percentage of bacterial tolerance to chromium (Cr), silver (Ag) and mercury (Hg). Additionally, we investigated the effect of these metals on pigmentation and on some enzymatic activities of the metal tolerant strains isolated, as well as antimicrobial resistance in some metal tolerant Enterobacteriaceae strains. Tolerance to Cr was observed mainly in Gram positive bacteria while in the case of Ag and Hg the tolerant bacteria were predominately Gram negative. Hg was the metal for which the percentage of tolerance was significantly higher, especially in samples from the hospital sewage (4.1%). Mercury also had the most discernible effect on color of the colonies. Considering the effect of metals on the respiratory enzymes, one strain of Ag-tolerantBacillus sp. and one of Hg-tolerant P. aeruginosa were unable to produce oxidase in the presence of Ag and Hg, respectively, while the expression of gelatinase was largely inhibited in various Gram negative strains (66% by Cr). Drug resistance in Hg-tolerant Enterobacteriaceae strains isolated from the university hospital sewage was greater than 80%, with prevalence of multiple resistance, while the Ag-tolerant strains from the same source showed about 34% of resistance, with the predominance of mono-resistance. Our results showed that, despite the ability of metal tolerant strains to survive and grow in the presence of these elements, the interactions with these metals may result in metabolic or phisiological changes in this group of bacteria.

Genes de virulencia y bacteriocinas en cepas de Enterococcus faecalis aisladas desde diferentes muestras clínicas en la Región del Maule, Chile / Virulence genes and bacteriocins in Enterococcus faecalis strains isolated from different clinical samples in Maule Region, Chile

The presence of virulence genes (VG) and bacteriocins from different clinical samples was studied in Enterococcus faecalis isolated from urinary tract infections (UTI), bacteremia and endodontitis and was correlated with haemolysin and gelatinase activity. We evaluated the presence of VG by PCR in 150 strains of E. faecalis including cylA, aggA, efaA, eep, gelE, esp, as-48, bac31, entL50A/B, entA, entP, entB, enlA andentl071. Haemolysin and gelatinase activity was studied. gelE and cylA genes expressed hemolysin and gelatinase, respectively. This activity was observed in some strains of bacteremia, UTI and endodontitis. The highest number of VG was detected in bacteremic strains, being aggA and entA genes the most frequent. efaA, esp, entA, entL50A/B were associated with their clinical origin (p < 0.05). The most common genetic profile was aggA-eep-enlA-entL50A/B. E. faecalis from UTI, bacteremia and endodontitis presented different gene combinations. Some of the genes studied were related to their clinical origin. The results obtained in this study are similar to those reported in other countries.

Desde diferentes muestras clínicas se determinó la presencia de genes codificantes de factores de virulencia (FV) y bacteriocinas en Enterococcus faecalis aislados desde infecciones del tracto urinario (ITU), bacteriemias y endodontitis, correlacionándose con la actividad hemolisina y gelatinasa. En 150 cepas de E. faecalis fue evaluada mediante RPC la presencia de cylA, aggA, efaA, eep, gelE, esp, as-48, bac31, entL50A/B, entA, entP, entB, enlA, y ent1071 determinándose actividad hemolisina y gelatinasa. Los genes cylA y gelE expresaron hemolisina y gelatinasa, respectivamente. Esta actividad fue observada en algunas de las cepas causantes de bacteriemia, ITU y endodontitis. El mayor número de genes estudiados se detectó en cepas bacteriémicas. Los genes aggA y entA, fueron los más frecuentes. Los genes efaA, esp, entL50/AB y entA se asociaron a su origen clínico (p < 0,05). El perfil genético más recurrente fue aggA-eep-enlA-entL50A/B. Enterococcusfaecalis de ITU, bacteriemias y endodontitis presentaron distintas combinaciones génicas. AAlgunos de los genes estudiados se relacionaron con su origen clínico. Los resultados obtenidos son similares a los reportados en otros países.

Transplante de células da medula óssea na insuficiência cardíaca chagásica: relato da primeira experiência humana / Bone marrow cell transplantation in chagas' disease heart failure: report of the first human experience / Transplante de células de la médula ósea en la insuficiencia cardíaca de la enfermedad de chagas: relato de la Primera Experiencia Humana

FUNDAMENTO: Insuficiência cardíaca (IC) causada por Doença de Chagas (DC) é uma cardiomiopatia inflamatória progressiva que afeta milhões de pessoas na América Latina. Estudos com modelos de camundongo de IC devido à DC indicam que o transplante de células mononucleares derivadas da medula óssea (TCDMO) pode reduzir a inflamação, fibrose e melhorar a função miocárdica. OBJETIVO: O propósito desse estudo foi avaliar, pela primeira vez em seres humanos, a segurança e a eficácia de TCDMO no miocárdio de pacientes com IC devido à DC. MÉTODOS: Um total de 28 pacientes com IC devido à DC (média de idade de 52,2 ± 9,9 anos) com classe funcional NYHA III e IV foram submetidos à TCDMO através de injeção coronariana. Os efeitos na fração de ejeção do ventrículo esquerdo (FEVE), capacidade funcional, qualidade de vida, arritmias e parâmetros bioquímicos, imunológicos e neuro-humorais foram avaliados. RESULTADOS: Não houve complicações diretamente relacionadas ao procedimento. A FEVE foi 20,1 ± 6,8 por cento e 28,3 ± 7,9 por cento, p < 0,03 a nível basal e 180 dias após o procedimento, respectivamente. No mesmo período, melhoras significantes foram observadas na classe funcional NYHA (3,1 ± 0,3 para 1,8 ± 0,5; p < 0,001), qualidade de vida (50,9 ± 11,7 para 25,1 ± 15,9; p < 0,001), e no teste de caminhada de seis minutos (355 ± 136 m para 437 ± 94 m; p < 0,01). Não houve alterações nos marcadores de ativação imune ou neurohormonais. Nenhuma complicação foi registrada. CONCLUSÃO: Nossos dados sugerem que a injeção intracoronariana de células derivadas da medula óssea é segura e potencialmente efetiva em pacientes com IC devido à DC. A extensão do benefício, entretanto, parece ser discreta e precisa ser confirmada em estudos clínicos maiores, randomizados, duplo-cegos, controlados com placebo.

BACKGROUND: Heart failure due to Chagas' disease (HFCD) is a progressive inflammatory cardiomyopathy that affects millions of individuals in Latin America. Studies using mice models of HFCD indicate that bone marrow mononuclear cell transplantation (BMCT) may reduce inflammation, fibrosis, and improve myocardial function. OBJECTIVE: The purpose of this study was to evaluate, for the first time in humans, the safety and efficacy of BMCT to the myocardium of patients with HFCD. METHODS: A total of 28 HFCD patients (mean age 52.2 ± 9.9 years) with NYHA class III and IV were submitted to BMCT through intracoronary injection. Effects on the left ventricle ejection fraction (LVEF), functional capacity, quality-of-life, arrhythmias, biochemical, immunological, and neuro-humoral parameters, were evaluated. RESULTS: There were no complications directly related to the procedure. LVEF was 20.1 ± 6.8 percent and 28.3 ± 7.9 percent, p < 0.03 at baseline and 180 days after the procedure, respectively. In the same period, significant improvements were observed in the NYHA class (3.1 ± 0.3 to 1.8 ± 0.5; p < 0.001), quality-of-life (50.9 ± 11.7 to 25.1 ± 15.9; p < 0.001), and in the six-minute walking test (355 ± 136 m to 437 ± 94 m; p < 0,01). There were no changes in markers of immune or neurohormonal activation. No complications were registered. CONCLUSION: Our data suggest that the intracoronary injection of BMCT is safe and potentially effective in patients with HFCD. The extent of the benefit, however, appears to be small and needs to be confirmed in a larger randomized, double blind, placebo controlled clinical trial.

FUNDAMENTO: La insuficiencia cardíaca (IC), causada por la enfermedad de Chagas (EC), es una cardiomiopatía inflamatoria progresiva que afecta a millones de personas en Latinoamérica. Estudios con modelos experimentales de IC en razón de la EC, nos indican que el transplante de células mononucleares derivadas de la médula ósea (TCMO), puede reducir la inflamación y la fibrosis, mejorando así la función miocárdica. OBJETIVO:El objetivo de este estudio fue evaluar, por primera vez en seres humanos, la seguridad y la eficacia del TCMO en el miocardio de pacientes con IC debido a la EC. MÉTODOS:Fueron estudiados un total de 28 pacientes con IC debido a la EC (con edad promedio 52,2 ± 9,9 años), en clases funcionales III y IV (NYHA), al TCMO por medio de una inyección coronaria. Se evaluaron los efectos en la fracción de eyección del ventrículo izquierdo (FEVI), capacidad funcional, calidad de vida, arritmias y parámetros bioquímicos, inmunológicos y neurohumorales. RESULTADOS:No se registraron complicaciones relacionadas directamente con el procedimiento. La FEVI pasó de 20,1 ± 6,8 por ciento para 28,3 ± 7,9 por ciento, p < 0,03, cuando se comparó con el período basal y 180 días después del procedimiento, respectivamente. En el mismo período, también se observaron mejorías en la clase funcional NYHA promedio (3,1 ± 0,3 para 1,8 ± 0,5; p < 0,001), puntuación de calidad de vida de Minnesota (50,9 ± 11,7 para 25,1 ± 15,9; p < 0,001), y en el test de esfuerzo de seis minutos (355 ± 136 m para 437 ± 94 m; p < 0,01). No hubo alteraciones en los marcadores de activación inflamatoria o neurohormonales. Ninguna complicación fue registrada. CONCLUSIÓN:Nuestros datos sugieren que la inyección intracoronaria de las células derivadas de la médula ósea es segura y potencialmente efectiva en pacientes con IC debido a la EC. La extensión del beneficio, sin embargo, parece ser discreta, y necesita ser confirmada en los ensayos clínicos randomizados, doble ciegos, controlados con placebo.

Fatores envolvidos na cronicidade das lesões cutâneas e na progressão da forma cutânea para a forma mucosa tardia da leishmaniose tegumentar americana / Factors involved in the chronicity of cutaneous lesions and in the progression of cutaneous form to late mucosal form of american tegumentary leishmaniasis

A forma de apresentação mais comum da leishmaniose tegumentar americana (LTA) é o aparecimento de uma úlcera auto-limitada no local da picada do vetor. Embora estas lesões requeiram até 6 meses para atingirem a cura completa, o tratamento com antimoniais pentavalentes pode reduzir significativamente este tempo de cicatrização. Entretanto, a quimioterapia nem sempre apresenta resultados satisfatórios, seja por não resolver a injúria tecidual, seja por permitir a persistência parasitária e desenvolvimento de lesões na mucosa oro-nasal. Desta forma, o presente estudo teve como objetivos principais avaliar fatores envolvidos na cronicidade das lesões cutâneas e na progressão da forma cutânea da leishmaniose para a forma mucosa tardia. A cronicidade de lesões cutâneas de diversas etiologias tem sido relacionada ao desequilíbrio da atividade de alguma metaloproteínases de matriz (MMPs), em especial, das gelatinases MMP-2 e MMP-9. Portanto, decidimos avaliar a participação destas enzimas e de seus moduladores nos danos teciduais causados por LTA. Verificamos que a atividade destas gelatinases atinge extensa área das lesões de má resposta, a qual estava associada a um elevado número de células produzindo IFN-gama, TGF-beta e IL-10, com preponderância da citocina pró-proteolítica IFN-gama. O oposto foi observado nas lesões de boa resposta, onde, paralelamente aos baixos níveis de atividade gelatinolítica e prevalência da citocina anti-inflamatória IL-10, pode-se observar altos níveis de mRNA para MMP-2 e elevados valores da razão MMP-2:TIMP-2. Sugere-se então que o perfil imunilógico in situ controla o balanço entre MMPs e inibidores, determinando assim, o sucesso ou fracasso da cicatrização...

Mechanical ventilation and volutrauma: study in vivo of a healthy pig model

Mechanical ventilation is essential in intensive care units. However, it may itself induce lung injury. Current studies are based on rodents, using exceptionally large tidal volumes for very short periods, often after a "priming" pulmonary insult. Our study deepens a clinically relevant large animal model, closely resembling human physiology and the ventilator setting used in clinic settings. Our aim was to evaluate the pathophysiological mechanisms involved in alveolo/capillary barrier damage due to mechanical stress in healthy subjects. We randomly divided 18 pigs (sedated with medetomidine/tiletamine-zolazepam and anesthetised with thiopental sodium) into three groups (n=6): two were mechanically ventilated (tidal volume of 8 or 20 ml/kg), the third breathed spontaneously for 4 hours, then animals were sacrificed (thiopental overdose). We analyzed every 30' hemogasanalysis and the main circulatory and respiratory parameters. Matrix gelatinase expression was evaluated on bronchoalveolar lavage fluid after surgery and before euthanasia. On autoptic samples we performed zymographic analysis of lung, kidney and liver tissues and histological examination of lung. Results evidenced that high Vt evoked profound alterations of lung mechanics and structure, although low Vt strategy was not devoid of side effects, too. Unexpectedly, also animals that were spontaneously breathing showed a worsening of the respiratory functions.

Efecto de la concentración de hidróxido de calcio y tiempo de cocción del grano de maíz (Zea mays L) nixtamalizado, sobre las características fisicoquímicas y reológicas del nixtamal / Effect of calcium hydroxide concentration and cooking time of corn grain (Zea mays L) nixtamalized, over physicochemical and rheological nixtamal characteristics

Se evaluó el efecto de la concentración de hidróxido de calcio (Ca(OH)2) y tiempo de cocción del grano de maíz sobre las propiedades fisicoquímicas y reológicas durante la nixtamalización tradicional. Se observó que a mayor concentración de Ca(OH)2 y mayor tiempo de cocción del grano, la temperatura de gelatinización aumentó significativamente. El maíz nixtamalizado con 2 g/100g de Ca(OH)2 y 60 min de tiempo de cocción, presentó mayor absorción de calcio, 0.152 g/100g, mostrando un aumento significativo respecto a la concentración inicial (500 g/100g). Todas las muestras presentaron un patrón de difracción de rayos X de almidón tipo A, con diferencias en la intensidad de la difracción. La viscosidad aparente del nixtamal disminuyó considerablemente al utilizar condiciones de procesamiento más altas. Se encontró que la capacidad de retención de agua aumentó con la temperatura.

The effect of calcium hydroxide (Ca(OH)2) concentration and cooking time on physicochemical and pasting properties of maize, during traditional nixtamalization was evaluated. It was observed that the higher the Ca(OH)2 concentration and cooking time the higher the gelatinization temperature. Grain samples treated with 2 g/100g of Ca(OH)2 and 60 min of cooking time presented higher calcium absorption (0.152 g/100g) showing a significant increment (500 g/100g). Results of X-ray diffraction showed that all the samples had X-ray diffraction pattern of starch type A, showing differences in the intensity of the diffraction as indicated by the percentage of cristallinity. The apparent viscosity of the nixtamal considerably decreased when the processes conditions were higher. Water retention capacity increased with temperature.

Gene expression and characterization of 2-keto-3-deoxy-gluconate kinase, a key enzyme in the modified Entner-Doudoroff pathway of Serratia marcescens KCTC 2172

We cloned 2-keto-3-deoxy-gluconate kinase (KDGK), which catalyzes the phosphorylation of 2-keto-3-deoxygluconate (KDG) to 2-keto-3-deoxy-6-phophogluconate (KDPG) from Serratia marcescens KCTC 2172. The nucleotide sequence revealed a single open reading frame containing 1,208 bp and encoding for 309 amino acids, with a molecular weight of 33,993 Da. The enzyme was purified via GST affinity chromatography. The putative KdgT binding site was detected upstream of the initial codon. The KDG kinase utilized 2-ketogluconate (KG) and KDG as substrates. The optimal temperature and pH for KDGK activity were 50ºC and 8.0, respectively.

Análise do padrão de expressão de MMP-2, -9 e -8 em tecido humano pulpar normal e inflamado / Analysis of the expression perfile of MMP-2, -9 and -8 in normal and inflamed human pulp tissue

As metaloproteinases da matriz (MMPs) foram relacionadas a diversas doenças inflamatórias como artrite e também ao câncer. O presente trabalho tem por objetivo estabelecer o papel da MMP-2, MMP-9 e MMP-8 no processo de inflamação pulpar. Foram adotadas as seguintes hipóteses nulas: (1) o padrão de expressão das MMP-2, MMP-9 e MMP-8 não sofre alteração nos diferentes estágios da polpa humana: normal, reversível, transição, irreversível ou necrose; (2) não há diferença de expressão das MMP-2, -9 e MMP-8, considerando-se um mesmo estágio de inflamação tecidual pulpar. Os métodos utilizados foram: (I) Obtenção dos espécimes, que foram divididos em grupos de acordo com critérios adotados de semiologia subjetiva e objetiva. Obtiveram-se os seguintes grupos: GI (Controle) dentes hígidos (n=7); GII (Pulpite Reversível n=4); GIII (Pulpite Transição n=4); GIV (Pulpite Irreversível/Necrose n=8). Logo após exodontia, os dentes obtidos foram cortados ligeiramente abaixo da junção amelodentinária e fixados em formol a 10% por 48h. Foram lavados em água corrente (24h) para então serem processados histologicamente. Foram obtidas secções de 4m, aderidas em lâminas silanizadas e submetidas à imunomarcação (Técnica da Peroxidase), utilizando os anticorpos anti MMP-2, MMP-9 e MMP-8 humanos. A presença de imunomarcação foi realizada através da análise semi-quantitativa por escores, sendo que a quantificação de marcação por corte seguiu o seguinte escore: 0= ausente; 1= leve; 2= moderada; 3= intensa. Realizou-se teste estatístico não paramétrico Kruskal-Wallis, p<0,05. As comparações intergrupos revelaram, para CO: (1)MMP-2 - GI=GII=GIII, GIII=GIV, GI>GIV (p<0,01) e GII>GIV (p<0,05); (2)MMP-9 GI=GII=GIV, GII=GIII e GIII>GI (p<0,01); (3)MMP-8 GI=GII=GIII=GIV. Na região central da polpa, obteve-se: (1)MMP-2 GI=GII=GIII, GIII=GIV, GI>GIV (p<0,001) e GII>GIV (p<0,01); (2)MMP-9 GI=GII=GIII, GIII=GIV, GIV>GI (p<0,001) e GIV>GII (p<0,01); (3)MMP-8 GI=GII, GIII=GIV, GIII>GI (p<0,05),...

The matrix metalloproteinases (MMPs) have been related to various inflammatory diseases, such as arthritis, as well as to cancer. The aim of the present study was to establish the role of MMP-2, MMP-9 and MMP-8 in the process of dental pulp inflammation. The following null hypotheses were adopted: (1) the pattern of MMP-2, MMP-9 and MMP-8 expression does not undergo alteration in the following different stages of human pulp: normal, reversible, transition, irreversible or necrosis; (2) there is no difference in the expression of MMP-2, -9 and MMP-8, when considering the same stage of pulp tissue inflammation. The methods used were: (I) Obtainment of specimens, which were divided into groups according to the subjective and objective criteria of semiology adopted. The following groups were obtained: GI (Control) healthy teeth (n=7); GII (Reversible Pulpitis n=4); GIII (Transition Pulpitis n=4); GIV (Irreversible Pulpitis/Necrosis n=8). Soon after extraction the teeth obtained were cut slightly below the amelodentinal junction and fixed in 10% formol for 48h. They were washed under running water (24h) and were histologically processed afterwards. Sections of 4m were obtained, adhered to silanized slides, and submitted to immunomarking (Peroxidase Technique), using human anti MMP-2, MMP-9 and MMP- 8 antibodies. The presence of immunomarking was determined through semi-quantitative analysis by scores, and marking by cut was quantified using the following score: 0= absent; 1= slight; 2= moderate; 3= intense. The Kruskal-Wallis non-parametric statistical test was performed, p<0.05. Intergroup comparisons revealed the following: for CO: (1)MMP-2 - GI=GII=GIII, GIII=GIV, GI>GIV (p<0.01) and GII>GIV (p<0.05); (2)MMP-9 GI=GII=GIV, GII=GIII and GIII>GI (p<0,01); (3)MMP-8 GI=GII=GIII=GIV. In the central region of the pulp, the following results were obtained: (1)MMP-2 GI=GII=GIII, GIII=GIV, GI>GIV (p<0.001) and GII>GIV (p<0.01); (2)MMP-9 GI=GII=GIII, GIII=GIV, GIV>GI...

Virulence factors in vancomycin-resistant and vancomycin - susceptible Enterococcus faecalis from Brazil / Fatores de virulência em Enterococcus faecalis resistentes e suscetível à vancomicina isolados no Brasil

Enterococci are members of commensal flora of animals and insects, but are also important opportunistic pathogens. Our objective was to observe if there was any difference of virulence in several groups of E. faecalis, mainly between vancomycin-resistant E. faecalis (VREFS) of colonization and infection. VREFS and vancomycin-sensitive E. faecalis from Brazil were screened for the presence of virulence factor genes. Phenotypic assays were used to assess in vitro expression, to understand the pathogenic potential of these isolates and to determine whether a correlation exists between virulence and antibiotic resistance. Different virulence profiles were found suggesting that the disseminating clone may have generated several variations. However, our study showed that one constellation of traits appeared most commonly: gelatinase, aggregation substance and esp (GEA). These factors are important because they have been implicated in cell aggregation and biofilm formation. Biofilm formation may promote the conjugation of plasmids harboring resistance and virulence genes, enhancing the probability of entry of new resistance genes into species. Curiously, the profile GEA was not exclusive to VREFS, it was the second most observed in VSEFS isolates from colonization and infection in hospitalized patients and also from rectal swabs of healthy volunteers. Such strains appear to represent the entry gateway to new resistance genes into E. faecalis and may contribute to the spreading of E. faecalis mainly in hospitals.

Enterococci são membros da microbiota comensal de animais e insetos, mas também são importantes patógenos oportunistas. Nosso objetivo foi observar se há qualquer diferença na virulência nos diversos grupos de Enterococcus faecalis, principalmente nos E. faecalis resistente à vancomicina (VREFS) isolados de colonização e infecção. VREFS e E. faecalis sensíveis à vancomicina (VSEFS) do Brasil foram pesquisadas quanto a presença de fatores de virulência. Ensaios fenotípicos foram usados para obter a expressão in vivo, entender o potencial patogênico destas amostras e determinar se existe correlação entre virulência e resistência a antibióticos. Diferentes perfis de virulência foram encontrados sugerindo que o clone que está se disseminado pode ter gerado diversas variações. No entanto, nosso estudo mostrou que um conjunto de fatores parece ser mais comum entre as amostras: gelatinase, substância de agregação e esp (GEA). Estes fatores tem sido correlacionados com a agregação de células e formação de biofilmes. A formação de biofilme pode promover a conjugação de plasmídeos contendo genes de resistência entre as espécies. Curiosamente, o perfil GAE não foi exclusivo para VREFS, foi o segundo mais observado em amostras VSEFS provenientes de colonização e infecção em pacientes hospitalizados e também de swabs retais de voluntários saudáveis. Tais linhagens pacerem representar a "porta de entrada" para novos genes de resistência em E. faecalis e podem contribuir para a disseminação de E. faecalis principalmente nos hospitais.

Study of biological characteristics of Pseudomonas aeruginosa strains isolated from patients with cystic fibrosis and from patients with extra-pulmonary infections

A total of 120 strains of Pseudomonas aeruginosa, isolated from cystic fibrosis (CF) patients (n = 80) and from patients having extra-pulmonary infections (n = 40) were studied regarding the presence of some virulence factors (hemolysin, gelatinase and elastase production) and presence of the algD and algU genes as detected by polymerase chain reaction-PCR. There was not a significant difference for the production of gelatinase and hemolysin between non-mucoid strains from CF patients and other isolates from extra-pulmonary infections and mucoid strains. The production of elastase was found to be significant among these strains. The algD gene was detected by PCR in all studied strains but the algU gene was detected only in 25 percent of the mucoid strains. Conclusion withdrawn from the results were: (i) hemolysin and gelatinase production although present in many strains of P aeruginosa should not be considered as general virulence factors for the mucoid phenotype but could help in the pathogenic process; (ii) elastase production could be a necessary virulence factor for the initial pathogenesis process; (iii) mucoid and non-mucoid phenotypes could also be expressed according to the host's tissues or environment, and finally, (iv) more than one regulator system for alginate production is probably present in each strain.