Benefícios da condroitinase abc associada a células-tronco mesenquimais na lesão espinhal aguda em ratos / Benefits of chondroitinase abc associated with mesenchymal stem cells in acute spinal cord injury in rats

Com o objetivo de estudar o efeito da condroitinase associada às células-tronco mesenquimais na lesão aguda da medula espinhal, utilizaram-se 50 ratos Lewis, distribuídos igualmente nos grupos: controle negativo (CN), tratamento com placebo (PLA), condroitinase (CDN), células-tronco mesenquimais (CTM) e condroitinase mais células-tronco mesenquimais (CDN+CTM). Todos os animais tiveram a medula espinhal exposta por laminectomia, e os grupos PLA, CDT, CTM e CDT+CTM sofreram também trauma medular compressivo. Após sete dias, procedeu-se à reexposição da medula espinhal, quando os grupos PLA e CTM receberam 4µL de líquido cefalorraquidiano artificial via intralesional, e os grupos CDT e CDT+CTM receberam o mesmo líquido contendo 2,2U de condroitinase. Após 14 dias da cirurgia inicial, todos os animais receberam 0,2mL de PBS via endovenosa, contudo, nos grupos CTM e CDT+CTM, esse líquido continha 1x106 CTM. Avaliou-se a capacidade motora até o 28o dia pós-trauma e, posteriormente, as medulas espinhais foram analisadas por RT-PCR, para quantificação da expressão gênica para BDNF, NT-3, VEGF, KDR e PECAM-1, e por imunoistoquímica, para detecção das células-tronco GFP injetadas (anti-GFP), quantificação dos neurônios (anti-NeuN) e da GFAP e vimentina, para avaliação da cicatriz glial. As análises estatísticas foram realizadas com o auxílio do Prism 5 for Windows, com o nível de significância de 5%. Não houve diferença entre os grupos quanto à capacidade motora. O grupo CDT+CTM apresentou maior imunoexpressão de neurônios viáveis do que o placebo. No CTM, houve maior expressão dos fatores neurotróficos BDNF e VEGF. E no CDT, houve menor imunoexpressão de vimentina. Concluiu-se que a associação CDT+CTM favorece a viabilidade neuronal após o trauma, que o tratamento com CTM promove aumento na expressão dos fatores tróficos BDNF e VEGF e que o tratamento com condroitinase é efetivo na redução da cicatriz glial.(AU)

The aim of this work was to study the effect of chondroitinase associated with mesenchymal stem cells in acute spinal cord injury. Therefore, 50 Lewis rats were distributed in the following groups: negative control (NC), treatment with placebo (PLA), chondroitinase (CDT), mesenchymal stem cells (MSC), and chondroitinase associated with mesenchymal stem cells (CDT + MSC). All animals had their spinal cord exposed by laminectomy, and the groups named PLA, CDT, MSC and CDT + MSC also suffered compressive spinal cord trauma. After seven days, the spinal cord was re-exposed, when the PLA and MSCs groups received 4uL of artificial cerebrospinal fluid through the lesion, and the CDT group and CDT + MSC received the same fluid containing 2,2U of chondroitinase. 14 days after the first surgery, all animals received 0.2ml of PBS intravenously; however, the MSC and CDT + MSC groups received the same liquid also containing 1x106 MSCs. The motor skills were evaluated up to 28 days post-injury and, subsequently, the spinal cords were analyzed by RT-PCR for BDNF, NT-3, VEGF, PECAM-1 and KDR gene expression quantification, immunohistochemistry to detect injected stem cells GFP (anti-GFP), to quantify neurons (anti-NeuN), GFAP and detect vimentin in order to evaluate the glial scar. Statistical analyzes were performed by Prism 5 for Windows using a 5% level of significance. There was no difference between groups with regarding motor capacity. The CDT + MSC group showed increased immunoreactivity of viable neurons than placebo. In MSC, there was a greater expression of neurotrophic factors BDNF and VEGF. Also, there was less vimentin immunostaining in group CDT. It was concluded that CDT + MSC association promotes neuronal viability after trauma, in which treatment with MSC promotes increased expression of BDNF and VEGF trophic factors, and also that treatment with chondroitinase is effective in reducing the glial scar.(AU)

Benefícios da condroitinase abc associada a células-tronco mesenquimais na lesão espinhal aguda em ratos / Benefits of chondroitinase abc associated with mesenchymal stem cells in acute spinal cord injury in rats

Com o objetivo de estudar o efeito da condroitinase associada às células-tronco mesenquimais na lesão aguda da medula espinhal, utilizaram-se 50 ratos Lewis, distribuídos igualmente nos grupos: controle negativo (CN), tratamento com placebo (PLA), condroitinase (CDN), células-tronco mesenquimais (CTM) e condroitinase mais células-tronco mesenquimais (CDN+CTM). Todos os animais tiveram a medula espinhal exposta por laminectomia, e os grupos PLA, CDT, CTM e CDT+CTM sofreram também trauma medular compressivo. Após sete dias, procedeu-se à reexposição da medula espinhal, quando os grupos PLA e CTM receberam 4µL de líquido cefalorraquidiano artificial via intralesional, e os grupos CDT e CDT+CTM receberam o mesmo líquido contendo 2,2U de condroitinase. Após 14 dias da cirurgia inicial, todos os animais receberam 0,2mL de PBS via endovenosa, contudo, nos grupos CTM e CDT+CTM, esse líquido continha 1x106 CTM. Avaliou-se a capacidade motora até o 28o dia pós-trauma e, posteriormente, as medulas espinhais foram analisadas por RT-PCR, para quantificação da expressão gênica para BDNF, NT-3, VEGF, KDR e PECAM-1, e por imunoistoquímica, para detecção das células-tronco GFP injetadas (anti-GFP), quantificação dos neurônios (anti-NeuN) e da GFAP e vimentina, para avaliação da cicatriz glial. As análises estatísticas foram realizadas com o auxílio do Prism 5 for Windows, com o nível de significância de 5%. Não houve diferença entre os grupos quanto à capacidade motora. O grupo CDT+CTM apresentou maior imunoexpressão de neurônios viáveis do que o placebo. No CTM, houve maior expressão dos fatores neurotróficos BDNF e VEGF. E no CDT, houve menor imunoexpressão de vimentina. Concluiu-se que a associação CDT+CTM favorece a viabilidade neuronal após o trauma, que o tratamento com CTM promove aumento na expressão dos fatores tróficos BDNF e VEGF e que o tratamento com condroitinase é efetivo na redução da cicatriz glial.(AU)

The aim of this work was to study the effect of chondroitinase associated with mesenchymal stem cells in acute spinal cord injury. Therefore, 50 Lewis rats were distributed in the following groups: negative control (NC), treatment with placebo (PLA), chondroitinase (CDT), mesenchymal stem cells (MSC), and chondroitinase associated with mesenchymal stem cells (CDT + MSC). All animals had their spinal cord exposed by laminectomy, and the groups named PLA, CDT, MSC and CDT + MSC also suffered compressive spinal cord trauma. After seven days, the spinal cord was re-exposed, when the PLA and MSCs groups received 4uL of artificial cerebrospinal fluid through the lesion, and the CDT group and CDT + MSC received the same fluid containing 2,2U of chondroitinase. 14 days after the first surgery, all animals received 0.2ml of PBS intravenously; however, the MSC and CDT + MSC groups received the same liquid also containing 1x106 MSCs. The motor skills were evaluated up to 28 days post-injury and, subsequently, the spinal cords were analyzed by RT-PCR for BDNF, NT-3, VEGF, PECAM-1 and KDR gene expression quantification, immunohistochemistry to detect injected stem cells GFP (anti-GFP), to quantify neurons (anti-NeuN), GFAP and detect vimentin in order to evaluate the glial scar. Statistical analyzes were performed by Prism 5 for Windows using a 5% level of significance. There was no difference between groups with regarding motor capacity. The CDT + MSC group showed increased immunoreactivity of viable neurons than placebo. In MSC, there was a greater expression of neurotrophic factors BDNF and VEGF. Also, there was less vimentin immunostaining in group CDT. It was concluded that CDT + MSC association promotes neuronal viability after trauma, in which treatment with MSC promotes increased expression of BDNF and VEGF trophic factors, and also that treatment with chondroitinase is effective in reducing the glial scar.(AU)

Clinical and radiographic changes of carpi, tarsi and interphalangeal joints of beef zebu bulls on semen collection regimen / Mudanças clínicas e radiográficas das articulações carpo, tarso e interfalangeana de touros de corte zebuínos em regime de colheita de sêmen

Osteoarthritis and osteochondrosis are highly correlated to reproductive failure in bulls. This study aimed to evaluate the carpal, tarsal and interphalangeal lesions in beef zebu bulls on semen collection regimen. Twenty-one beef cattle bulls, in a total of forty-one animals, were split into three age-based groups: animals from two to four years old (GI), from more than four to eight years old (GII) and above eight years old (GIII). The clinical findings were conformational changes of limbs, synovial effusion, peripheral venous engorgement of joints and prolonged decubitus. The total population showed moderate clinical manifestation and radiographic score. The GIII presented more severe joint lesions. Carpi and tarsi regions had discrete to difuse osteophytosis, subchondral cysts, cartilaginous flaps, bone incongruence and fragmentation, osteitis, and ankylosis. Interphalangeal joints presented osteophytosis, distal phalanx osteitis and enthesophytosis. The digital radiographic examination allowed full identification of articular lesions and their clinical correspondences, besides the positive correlation between age, body weight and radiographic score.(AU)

Osteoartrite e osteocondrose estão altamente correlacionados à falha reprodutiva em touros. Este estudo teve como objetivo avaliar as lesões do carpo, tarso e interfalangianas em touros zebuínos de corte em regime de coleta de sêmen. Vinte e um bovinos de corte, touros, provenientes de um total de 41 animais, foram divididos em três grupos baseados na idade, sendo eles: animais de dois a quatro anos (GI), de mais de quatro a oito anos (GII) e acima de oito anos de idade (GIII). Os sinais clínicos encontrados foram mudanças de conformação dos membros, derrame sinovial, ingurgitamento venoso periférico das articulações e decúbito prolongado. A população total avaliada apresentou manifestação clínica e escore radiográfico de grau moderado. Os touros mais velhos apresentaram maior gravidade nas lesões articulares. O GIII foi o que apresentou as lesões articulares mais severas. Identificou-se nos carpos osteofitose de discreta a difusa, cistos subcondrais, "flaps" cartilaginosos, fragmentação óssea, perda de congruência óssea, osteíte e anquilose; as lesões társicas obedeceram ao mesmo padrão. As articulações interfalangeanas apresentaram somente osteofitose, osteíte nas falanges distais e entesiofitose. O exame radiográfico digital permitiu a identificação completa das lesões articulares e suas correspondências clínicas, além da correlação positiva entre a idade, o peso corporal e o escore radiológico.(AU)

Clinical and radiographic changes of carpi, tarsi and interphalangeal joints of beef zebu bulls on semen collection regimen / Mudanças clínicas e radiográficas das articulações carpo, tarso e interfalangeana de touros de corte zebuínos em regime de colheita de sêmen

Osteoarthritis and osteochondrosis are highly correlated to reproductive failure in bulls. This study aimed to evaluate the carpal, tarsal and interphalangeal lesions in beef zebu bulls on semen collection regimen. Twenty-one beef cattle bulls, in a total of forty-one animals, were split into three age-based groups: animals from two to four years old (GI), from more than four to eight years old (GII) and above eight years old (GIII). The clinical findings were conformational changes of limbs, synovial effusion, peripheral venous engorgement of joints and prolonged decubitus. The total population showed moderate clinical manifestation and radiographic score. The GIII presented more severe joint lesions. Carpi and tarsi regions had discrete to difuse osteophytosis, subchondral cysts, cartilaginous flaps, bone incongruence and fragmentation, osteitis, and ankylosis. Interphalangeal joints presented osteophytosis, distal phalanx osteitis and enthesophytosis. The digital radiographic examination allowed full identification of articular lesions and their clinical correspondences, besides the positive correlation between age, body weight and radiographic score.(AU)

Osteoartrite e osteocondrose estão altamente correlacionados à falha reprodutiva em touros. Este estudo teve como objetivo avaliar as lesões do carpo, tarso e interfalangianas em touros zebuínos de corte em regime de coleta de sêmen. Vinte e um bovinos de corte, touros, provenientes de um total de 41 animais, foram divididos em três grupos baseados na idade, sendo eles: animais de dois a quatro anos (GI), de mais de quatro a oito anos (GII) e acima de oito anos de idade (GIII). Os sinais clínicos encontrados foram mudanças de conformação dos membros, derrame sinovial, ingurgitamento venoso periférico das articulações e decúbito prolongado. A população total avaliada apresentou manifestação clínica e escore radiográfico de grau moderado. Os touros mais velhos apresentaram maior gravidade nas lesões articulares. O GIII foi o que apresentou as lesões articulares mais severas. Identificou-se nos carpos osteofitose de discreta a difusa, cistos subcondrais, "flaps" cartilaginosos, fragmentação óssea, perda de congruência óssea, osteíte e anquilose; as lesões társicas obedeceram ao mesmo padrão. As articulações interfalangeanas apresentaram somente osteofitose, osteíte nas falanges distais e entesiofitose. O exame radiográfico digital permitiu a identificação completa das lesões articulares e suas correspondências clínicas, além da correlação positiva entre a idade, o peso corporal e o escore radiológico.(AU)

Nutritional regulation of body condition score at the initiation of the transition period in primiparous and multiparous dairy cows under grazing conditions: milk production, resumption of post-partum ovarian cyclicity and metabolic parameters.

The objective of this study was to investigate the effect of different body condition score (BCS) at 30 days before calving (-30 days) induced by a differential nutritional management from -100 days until -30 days on productive parameters, the interval to first ovulation and blood parameters in primiparous and multiparous Holstein cows under grazing conditions until 60 days post partum. The experimental arrangement was a randomized complete block design, where cows were blocked according to BW and expected calving date and then randomly assigned to different nutritional treatments from -100 to -30 days relative to calving to induce different BCS. As the assignment of cows to treatments was random, cows had to lose, maintain or gain BCS; thus, different planes of nutrition were offered with approximately 7, 14 or 20 kg dry matter per day. The BCS score was assessed every 15 days and animals were reassigned in order to achieve the desired BCS at -30 days. Only animals that responded to nutritional treatment were considered and this was defined as follows: primiparous and multiparous high cows (PH and MH) had to gain 0.5 points of BCS, primiparous low (PL) had to lose 0.5 points of BCS and multiparous low (ML) had to maintain BCS at least in two subsequent observations from -100 to -30 days. From -30 days to calving, primiparous and multiparous cows (P and M cows) were managed separately and cows were offered a diet once a day. From calving to 60 days post partum, cows of different groups grazed in separate plots a second year pasture. Cows were also supplemented individually with whole-plant maize silage and commercial concentrate. Cows had similar BCS at -100 days and differed after the nutritional treatment; however, all groups presented similar BCS at 21 days post partum. The daily milk production and milk yield at 60 days post partum was higher in M than P cows. The percentage of milk fat was higher in PH cows compared with PL cows. Concentrations of non-esterified fatty acids (NEFA) were affected by the BCS at -30 days within parity, and in PH cows the concentration of NEFA was higher than in PL cows. The concentrations of total protein were higher in M cows. A lower probability of cycling was found in PL than in PH cows (P < 0.05) and in ML than in MH cows (P < 0.05). Treatment affected various endocrine/metabolic profiles according to parity, suggesting that the metabolic reserves signal the productive/reproductive axis so as to induce a differential nutrient partitioning in adult v. first-calving cows.

Effect of plant neutrophil elastase inhibitor on leucocyte migration, adhesion and cytokine release in inflammatory conditions.

BACKGROUND AND PURPOSE: The serine and cysteine peptidase inhibitor, BbCI, isolated from Bauhinia bauhinioides seeds, is similar to the classical plant Kunitz inhibitor, STI, but lacks disulphide bridges and methionine residues. BbCI blocks activity of the serine peptidases, elastase (K(iapp) 5.3 nM) and cathepsin G (K(iapp) 160.0 nM), and the cysteine peptidase cathepsin L (K(iapp) 0.2 nM). These three peptidases play important roles in the inflammatory process. EXPERIMENTAL APPROACH: We measured the effects of BbCI on paw oedema and on leucocyte accumulation in pleurisy, both induced by carrageenan. Leucocyte-endothelial cell interactions in scrotal microvasculature in Wistar rats were investigated using intravital microscopy. Cytokine levels in pleural exudate and serum were measured by elisa. KEY RESULTS: Pretreatment of the animals with BbCI (2.5 mg·kg(-1)), 30 min before carrageenan-induced inflammation, effectively reduced paw oedema and bradykinin release, neutrophil migration into the pleural cavity. The number of rolling, adhered and migrated leucocytes at the spermatic fascia microcirculation following carrageenan injection into the scrotum were reduced by BbCI pretreatment. Furthermore, levels of the rat chemokine cytokine-induced neutrophil chemo-attractant-1 were significantly reduced in both pleural exudates and serum from animals pretreated with BbCI. Levels of interleukin-1ß or tumour necrosis factor-α, however, did not change. CONCLUSIONS AND IMPLICATIONS: Taken together, our data suggest that the anti-inflammatory properties of BbCI may be useful in investigations of other pathological processes in which human neutrophil elastase, cathepsin G and cathepsin L play important roles.

Effect of plant neutrophil elastase inhibitor on leucocyte migration, adhesion and cytokine release in inflammatory conditions

The serine and cysteine peptidase inhibitor, BbCI, isolated from Bauhinia bauhinioides seeds, is similar to the classical plant Kunitz inhibitor, STI, but lacks disulphide bridges and methionine residues. BbCI blocks activity of the serine peptidases, elastase (Kiapp 5.3 nM) and cathepsin G (Kiapp 160.0 nM), and the cysteine peptidase cathepsin L (Kiapp 0.2 nM). These three peptidases play important roles in the inflammatory process. We measured the effects of BbCI on paw oedema and on leucocyte accumulation in pleurisy, both induced by carrageenan. Leucocyte–endothelial cell interactions in scrotal microvasculature in Wistar rats were investigated using intravital microscopy. Cytokine levels in pleural exudate and serum were measured by elisa.Pretreatment of the animals with BbCI (2.5 mg·kg−1), 30 min before carrageenan-induced inflammation, effectively reduced paw oedema and bradykinin release, neutrophil migration into the pleural cavity. The number of rolling, adhered and migrated leucocytes at the spermatic fascia microcirculation following carrageenan injection into the scrotum were reduced by BbCI pretreatment. Furthermore, levels of the rat chemokine cytokine-induced neutrophil chemo-attractant-1 were significantly reduced in both pleural exudates and serum from animals pretreated with BbCI. Levels of interleukin-1â or tumour necrosis factor-á, however, did not change.Taken together, our data suggest that the anti-inflammatory properties of BbCI may be useful in investigations of other pathological processes in which human neutrophil elastase, cathepsin G and cathepsin L play important roles.

Evaluación de ingesta en pacientes con hepatopatías crónicas / Evaluation of ingestion in patients with chronic liver diseases

La malnutrición calórico-protéica (MCP) es frecuente entre los pacientes con hepatopatías crónicas (HC) estableciéndose hasta en un 80 por ciento. El problema es multifactorial, siendo la disminución de la ingesta una de las causas más relevantes. Objetivo: evaluar la ingesta calórico-protéica en pacientes con HC. Se evaluaron prospectivamente 60 pacientes (26 mujeres, 43,3 por ciento) con una edad media de 54,1 años, con los siguientes diagnósticos: cirrosis biliar primaria 11 (18,3 por ciento), cirrosis alcohólica 26 (46,3 por ciento), hepatopatías autoinmunes 12 (20 por ciento), cirrosis criptogenética 6 (10 por ciento), Budd-Chiari 1 (1,7 por ciento), Hemocromatosis 1 (1,7 por ciento), cirrosis viral 3 (5 por ciento). Se utilizó el recordatorio de 3 días para la recolección de datos de ingesta, la evaluación global subjetiva EGS) para evaluación nutricional y la clasificación de Child-Pugh para evaluar severidad de la hepatopatía...

Evaluación de ingesta en pacientes con hepatopatías crónicas / Evaluation of ingestion in patients with chronic liver diseases

La malnutrición calórico-protéica (MCP) es frecuente entre los pacientes con hepatopatías crónicas (HC) estableciéndose hasta en un 80 por ciento. El problema es multifactorial, siendo la disminución de la ingesta una de las causas más relevantes. Objetivo: evaluar la ingesta calórico-protéica en pacientes con HC. Se evaluaron prospectivamente 60 pacientes (26 mujeres, 43,3 por ciento) con una edad media de 54,1 años, con los siguientes diagnósticos: cirrosis biliar primaria 11 (18,3 por ciento), cirrosis alcohólica 26 (46,3 por ciento), hepatopatías autoinmunes 12 (20 por ciento), cirrosis criptogenética 6 (10 por ciento), Budd-Chiari 1 (1,7 por ciento), Hemocromatosis 1 (1,7 por ciento), cirrosis viral 3 (5 por ciento). Se utilizó el recordatorio de 3 días para la recolección de datos de ingesta, la evaluación global subjetiva EGS) para evaluación nutricional y la clasificación de Child-Pugh para evaluar severidad de la hepatopatía...(AU)

Prontuário eletrônico de pacientes: integrando informações clínicas e imagens médicas / Eletronic patient record: integrating clinical information and image data

O Instituto do Coração tem envidado esforços para integrar todas as informações clínicas dentro da Instituição. Nos últimos anos o InCor implementou com sucesso um sistema para transmissão, arquivamento, recuperação, processamento e visualização de Imagens Médicas e um Sistema de Informações Hospitalares (HIS) que armazena as informações administrativas e clínicas. A integração desses subsistemas forma o Prontuário Eletrônico do Paciente (PEP). O InCor é um dos seis Institutos que compõem o Hospital das Clínicas da Universidade de São Paulo. Como cada um dos Institutos possui o seu próprio sistema de informações, a troca de informações entre os Institutos é também uma questão muito relevante. Este trabalho apresenta a experiência no desenvolvimento de um Prontuário Eletrônico funcional e completo, que inclui controle de acesso, exames laboratoriais, imagens (estáticas, dinâmicas e 3D), laudos, documentos e mesmo sinais vitais de tempo real. Este artigo também discute a modelagem e implantação de um protótipo de um PEP distribuído e homogêneo. Atualmente, um volume superior a 2,5 TB de imagens DICOM já foi armazenado utilizando a arquitetura proposta. Diariamente, o PEP armazena mais de 5GB de dados e tem uma quantidade de acessos superior a 300 usuários. O sistema de armazenamento permite uma visibilidade de seis meses para acesso imediato e mais de dois anos para acesso automático utilizando uma jukebox

The Heart Institute (InCor) of São Paulo has been committed to the goal of integrating all clinical information within the institution. In the last few years, InCor has successfully created a system for transmission, archiving, retrieval, processing and visualization of Medical Images and a Hospital Information System (HIS) that stores the institution administrative and clinical information. These integrated subsystems form InCor's Electronic Patient Record (EPR). Since InCor is one of the six institutes of the University of São Paulo Medical School Hospital (HC) and each institute has its own information system, exchanging information among the institutes is also a very important issue. This work describes the experience in the effort to develop a functional and comprehensive EPR, which includes access control, lab exams, images (static, dynamic and 3D), clinical reports, documents and even real-time vital signals. This paper addresses also the design and prototype for integration of distributed and heterogeneous EPR. Currently, more than 2.5 TB of DICOM images, have been stored using the proposed architecture. The EPR stores more than 5 GB/day of data and presents more than 300 hits per day. The proposed storage subsystem allow six months of visibility for rapid retrieval (online mode) and more than two years for automatic retrieval using the jukebox

Assembly of high molecular weight kininogen and activation of prekallikrein on cell matrix.

Investigations determined if extracellular matrix of endothelial cells (EC) is a platform for HK assembly and PK activation. In buffers containing bovine serum albumin, biotin-HK binding to ECV304 cells or their matrix requires > or = 50 microM added Zn2+. Ortho-phenanthroline or a HK domain 5 peptide blocks HK binding. Binding to umbilical vein EC or matrix, but not ECV304 cells or matrix, is mediated by cytokeratin 1. Biotin-HK binds to ECV304 cells or matrix with a Kd of 15.8 or 9.0 nM and a Bmax of 2.6 x 10(7) or 2.4 x 10(7) sites/cell, respectively. PK activation on ECV304 cells or matrix is blocked by antipain or SBTI and corn trypsin inhibitor partially inhibits kallikrein formation. PK activation occurs on ECV304 cells or matrix prepared without serum or in human factor XII deficient serum, indicating that the PK activator is not factor XIIa. EC matrix promotes plasminogen activation after the assembly of HK, PK and pro-urokinase. These studies indicate that matrix of various EC has the ability to assemble HK allowing for PK activation and subsequent activities.

Prevalence of celiac disease in Argentina: screening of an adult population in the La Plata area.

OBJECTIVES: Up to now, the epidemiological characteristic of celiac disease among adults in South America remains unknown. The present prospective screening was designed to determine the prevalence of celiac disease in adults from the general population in an urban area of Argentina. METHODS: Between January. 1998, and May, 2000, all couples attending a centralized laboratory for an obligatory prenuptial examination in the La Plata area were offered participation in a screening program for celiac disease. The study included 2000 subjects (996 women; median age 29 yr, range 16-79 yr). All individuals completed a clinical questionnaire at the time that serum samples were obtained. A three-step screening protocol was used, as follows: 1) all samples were tested for antigliadin antibodies (AGAs) (type IgA and IgG); 2) samples that were IgA AGA positive were tested for antiendomysial antibody (EmA type IgA); samples that were positive for AGA-G but negative for IgA AGAs were tested for total IgA serum levels and EmA type IgG; and 3) subjects who were EmA-positive were referred for intestinal biopsy. RESULTS: At the end of the screening we detected 10 subjects who were EmA-A positive and two others who were IgA-deficient (both were EmA-G positive). Up to now, 11 of the 12 subjects (including nine EmA-positive and two IgA-deficient subjects) had endoscopic intestinal biopsies showing the characteristic celiac histology. The remaining EmA-positive individual was considered to be affected by celiac disease. The overall prevalence assessed was 1:167 (6.0 x 1000 subjects; 95% CI = 3.1-10.5). Eight of the 12 (67%) subjects were female (1:124; 8.0 x 1000; 95% CI = 3.5-15.8) and four (33%) were male (1:251; 4.0 x 1000; 95% Cl = 1.1-10.2). Although eight new patients were considered to be asymptomatic, three presented with a subclinical course and one was classically symptomatic. Only one patient had been previously diagnosed with celiac disease. CONCLUSIONS: Our screening protocol showed a very high prevalence of celiac disease for an urban area of Argentina that is ethnically similar to 90% of the general population of the country. The prevalence among women was double that for men, and the heterogeneous clinical picture of new patients showed predominance of asymptomatic cases.

Human plasma kallikrein processing: proteolysis as an alternative control.

Human plasma kallikrein (HuPK) was detected in normal non-activated and dilution-activated plasma by immunoblotting, using polyclonal antibodies. In non-activated plasma, the predominatly detected protein corresponds to prokallikrein (Mr 80,000-90,000). Activated plasma, besides kallikrein, contains larger proteins (Mr > 130,000) that possibly represent complexes between kallikrein and proteinase inhibitors. Plasma also contains species (Mr 43,000) which corresponds to kallikrein heavy chain. In activated plasma, monoclonal antibodies against kallikrein heavy chain detected, besides these same bands described above, two additional bands (Mr 30,000 and 20,000) possibly correspondent to fragments of kallikrein heavy chain.

Human plasma kallikrein. Immunoreactivity and activity on natural and synthetic substrates.

Human plasma kallikrein (HuPK) is a serine protease found in mammalian plasma. Following limited proteolysis, the enzyme is activated and forms two chains. The light chain occurs with molecular weight 36,000 or 33,000 and contains the active site. The heavy chain occurs with molecular weight of 45,000 and contains the binding site for high-molecular-weight kininogen. Both chains were prepared from active HuPK, following mild reduction with dithiothreitol and carboxymethylation with iodoacetamide. The light chain was isolated in SBTI-Sepharose and its kinetic properties were determined with synthetic derivatives of arginine-p-nitroanilides, to investigate any possible alteration of the active site. These studies showed that substrate modifications affected the hydrolytic activity more than the binding capacity. The ability to cleave high-molecular-weight kininogen, as observed for intact kallikrein, was reduced in beta-kallikrein and absent in the light chain, even when equimolar amounts of both light and heavy chains were tested together. Anti-kallikrein antiserum formed immunoprecipitates not only with kallikrein itself, but also with the separated chains. The immunoreactivity of the light-chain was not identical with that of kallikrein. Immunoselected specific antibodies for both chains, depending on the selectivity, reacted with the heavy chain and kallikrein, or with the light chain and kallikrein. These antibodies were shown to be effective in binding radio-iodinated HuPK in the radioimmunoassay developed for intact kallikrein.

Action of plant proteinase inhibitors on enzymes of the kallikrein kinin system.

Serine proteinase inhibitors, in the seeds of several Leguminosae from the Pantanal region (West Brazil), were studied using bovine trypsin, Factor XIIa and human plasma kallikrein. The inhibitors were purified from Enterolobium contortisiliquum (Mr = 23,000), Torresea cearensis (Mr = 13,000), Bauhinia bauhinioides (Mr = 20,000), Bauhinia mollis (Mr = 20,000) and Bauhinia pentandra (Mr = 20,000). E. contortisiliquum inhibitor inactivates all three enzymes, whereas the T. cearensis inhibitor inactivates trypsin and Factor XIIa, but does not affect plasma kallikrein. B. bauhinioides and B. pentrandra inhibitors, on the other hand, inactivate trypsin and plasma kallikrein but only the B. pentandra inhibitor affects Factor XIIa, and B. mollis inhibitor causes trypsin inactivation only. Calculated Ki values were between 10(-7) and 10(-9) M. Chymotrypsin, like trypsin, is also inhibited, but with lower affinity. The trypsin inhibitors, isolated from E. contortisiliquum, B. pentandra, B. bauhinioides and B. mollis seem to be of the Kunitz type; the inhibitor purified from T. cearensis is of the Bowman-Birk type.

Lysis of red blood cells by extracts from benthic dinoflagellates.

Samples of the cultured benthic dinoflagellates Gambierdiscus toxicus and Ostreopsis lenticularis, both isolated from a shallow back reef habitat in southwestern Puerto Rico, were extracted in methanol, dried and resuspended in distilled water. After centrifugation, aliquots of the supernatant, or dilutions thereof, were added to suspensions of washed human and mouse red blood cells and incubated at different temperatures for different time periods. Further spectrophotometrical examinations of the samples showed a hemolytic activity against mouse and human red blood cells. The hemolytic activity of G. toxicus extract was 3 to 4 times greater than that of O. lenticularis and was less temperature-dependent. Such findings suggest that these two dinoflagellates produce chemically different hemolysins.