Expression of cyclooxygenase-2 in intestine of pigs of different ages and hygiene status

Prostaglandins (PG) are bioactive lipids derived from the metabolism of membrane polyunsaturated fatty acids (PUFA), and play important roles in a number of biological processes including cell division, immune responses and wound healing. Cyclooxygenase (COX) is the key enzyme in PG synthesis from arachidonic acid. The hypothesis of the present study was that expression of COX-2 in porcine intestine was dependent on the microbial load and the age of piglets. Piglets were obtained from sows raised either on outdoor free-range farms or on indoor commercial farms, and littermates were divided into three treatments: One group of piglets suckled the sow, a second group was put into an isolator and fed a milk formula, and a third group was put into the isolator fed milk formula and injected with broad spectrum antibiotics. Samples were collected from the 75% level of the small intestine at day 5, 28 and 56 of age. Tissue section from four piglets from each of these six treatment groups was analysed by immunofluorescence for COX-2 and type-IV collagen (basement membrane, defining lamina propria (LP)). Image analysis was used to determine the number of positive pixels expressing LP and epithelial COX-2. COX-2 expressing cells were observed in LP and epithelium in all porcine intestinal samples. When analysing images obtained on day 28, injection of antibiotics seemed to reduce the COX-2 expression in intestinal samples of piglets when compared to other treatments (P=0.053). No significant effect of farm, treatments or age of piglets was observed on COX-2 expressing data when analysing all data of images obtained at day 28 and 56. By double-labelling experiments, COX-2 was found not to be expressed on cell co-expressing CD45, CD16, CD163 or CD2, thus indicating that mucosal leukocytes, including dendritic cells, macrophages and NK cells did not express COX-2. Future research should investigate the role of COX-2 expression in the digestive tract in relation to pig health.

Immunohistochemical, tomographic and histological study on onlay bone graft remodeling. Part II: calvarial bone

Objectives Little information is available on the molecular events that occur during graft incorporation over time. The calvarial bone (Cb) grafts have been reported to produce greater responses compared with other donor regions in maxillofacial reconstructions, but the scientific evidences for this are still lacking. The objectives of this study are (1) to study the morphological pattern of Cb onlay bone grafts and compare them with the biological events through immunohistochemical responses and (2) to establish the effects of perforations in maintaining the volume and bone density of the receptor bed. Material and methods Sixty New Zealand White rabbits were submitted to Cb onlay bone grafts on the mandible. In 30 rabbits, the receptor bed was perforated (perforated group), while for the remaining animals the bed was kept intact (non-perforated group). Six animals from each group were sacrificed at 5, 7, 10, 20 and 60 days after surgery. Histological sections from the grafted area were prepared for immunohistochemical and histological analyses. Immuno-labeling was found for proteins Osteoprotegerin (OPG), receptor activator of nuclear factor-kappa beta ligand (RANKL), alkaline phosphatase (ALP), osteopontin (OPN), vascular endothelial growth factor (VEGF), tartrate-resistant acid phosphatase (TRAP), Type I collagen (COL I) and osteocalcin (OC). The tomography examination [computerized tomography (CT) scan] was conducted just after surgery and at the sacrifice. Results The histological findings revealed that the perforations contributed to higher bone deposition during the initial stages at the graft-receptor bed interface, accelerating the graft incorporation process. The results of the CT scan showed lower resorption for the perforated group (P < 0.05), and both groups showed high bone density rates at 60 days. This set of evidences is corroborated by the immunohistochemical outcomes indicating that proteins associated with revascularization and osteogenesis (VEGF, OPN, TRAP and ALP) were found in higher levels in the perforated group. Conclusions These findings indicate that the bone volume of calvarial grafts is better maintained when the receptor bed is perforated, probably resulting from more effective graft revascularization and greater bone deposition. The process of bone resorption peaked between 20 and 60 days post-operatively in both groups although significantly less in the perforated group. To cite this article:Pedrosa Jr WF, Okamoto R, Faria PEP, Arnez MFM, Xavier SP, Salata LA. Immunohistochemical, tomographic and histological study on onlay bone grafts remodeling. Part II: calvarial bone.Clin. Oral Impl. Res. 20, 2009; 1254-1264.doi: 10.1111/j.1600-0501.2009.01747.x.

Immunohistochemical, tomographic and histological study on onlay iliac grafts remodeling

The information concerning the molecular events taking place in onlay bone grafts are still incipient. The objective of the present study is to correlate the effects of perforation of resident bone bed on (1) the timing of onlay autogenous graft revascularization; (2) the maintenance of volume/density of the graft (assessed through tomography); and (3) the occurrence of bone remodeling proteins (using immunohistochemistry technique) delivered in the graft. Thirty-six New Zealand White rabbits were subjected to iliac crest onlay bone grafting on both sides of the mandible. The bone bed was drill-perforated on one side aiming at accelerating revascularization, whereas on the other side it was kept intact. After grafts fixation and flaps suture all animals were submitted to tomography on both mandible sites. Six animals were sacrificed, respectively, at 3, 5, 7, 10, 20 and 60 days after surgery. A second tomography was taken just before sacrifice. Histological slides were prepared from each grafted site for both immunohistochemistry analysis [osteopontin, osteocalcin, type I collagen and vascular endothelial growth factor (VEGF) anti-bodies] and histometric analysis. The values on bone volume measured on tomography showed no statistic significance (P >= 0.05) between perforated and intact sites. Grafts placed on perforated beds showed higher bone density values compared with non-perforated ones at 3 days (P <= 0.05). This correlation was inverted at 60 days postoperatively. The findings from VEGF labeling revealed a tendency for earlier revascularization in the perforated group. The early revascularization of bone grafts accelerated the bone remodeling process (osteocalcin, type I collagen and osteopontin) that led to an increased bone deposition at 10 days. The extended osteoblast differentiation process at intermediate stages in the perforated group cooperated for a denser bone at 60 days.

Functionally conserved cis-regulatory elements of COL18A1 identified through zebrafish transgenesis

Type XVIII collagen is a component of basement membranes, and expressed prominently in the eye, blood vessels, liver, and the central nervous system. Homozygous mutations in COL18A1 lead to Knobloch Syndrome, characterized by ocular defects and occipital encephalocele. However, relatively little has been described on the role of type XVIII collagen in development, and nothing is known about the regulation of its tissue-specific expression pattern. We have used zebrafish transgenesis to identify and characterize cis-regulatory sequences controlling expression of the human gene. Candidate enhancers were selected from non-coding sequence associated with COL18A1 based on sequence conservation among mammals. Although these displayed no overt conservation with orthologous zebrafish sequences, four regions nonetheless acted as tissue-specific transcriptional enhancers in the zebrafish embryo, and together recapitulated the major aspects of col18a1 expression. Additional post-hoc computational analysis on positive enhancer sequences revealed alignments between mammalian and teleost sequences, which we hypothesize predict the corresponding zebrafish enhancers; for one of these, we demonstrate functional overlap with the orthologous human enhancer sequence. Our results provide important insight into the biological function and regulation of COL18A1, and point to additional sequences that may contribute to complex diseases involving COL18A1. More generally, we show that combining functional data with targeted analyses for phylogenetic conservation can reveal conserved cis-regulatory elements in the large number of cases where computational alignment alone falls short. (C) 2009 Elsevier Inc. All rights reserved.

Morphological and quantitative study of ganglionated plexus of Calomys callosus trachea

Calomys callosus is a wild, native forest rodent found in South America. In Brazil, this species has been reported to harbour the parasitic protozoan Trypanosoma cruzi. The ganglionated plexus of this species was studied using whole-mount preparations of trachea that were stained using histological and histochemical methods. The histological methods were used to determine the position of the ganglia with respect to the trachea muscle and to determine the presence of elastic and collagen fibers. The histochemical method of NADH-diaphorase was used for morphometric evaluations of the plexus. The tracheal plexus lies exclusively over the muscular part of the organ, dorsal to the muscle itself. It varies in pattern and extent between animals. The average number of neurons was 279 and the cellular profile area ranged from 38.37 mu m(2) to 805.89 mu m(2). Acetylcholinesterase (AChE) histochemistry verified that both ganglia and single neurons lie along nerve trunks and are reciprocally interconnected with the plexus. Intensely AChE-reactive neurons were found to be intermingled with poorly reactive ones. Two longitudinal AChE-positive nerve trunks were also observed and there was a diverse number of ganglia along the intricate network of nerves interconnecting the trunks. A ganglion capsule of collagen and elastic fibers surrounding the neurons was observed. Under polarized light, the capsule appeared to be formed by Type I collagen fibers. (C) 2008 Elsevier B.V. All rights reserved.

Mesenchymal Stem Cells Attenuate Renal Fibrosis Through Immune Modulation and Remodeling Properties in a Rat Remnant Kidney Model

Mesenchymal stem cells (MSCs) have regenerative properties in acute kidney injury, but their role in chronic kidney diseases is still unknown. More specifically, it is not known whether MSCs halt fibrosis. The purpose of this work was to investigate the role of MSCs in fibrogenesis using a model of chronic renal failure. MSCs were obtained from the tibias and femurs of male Wistar-EPM rats. Female Wistar rats were subjected to the remnant model, and 2 vertical bar x vertical bar 10(5) MSCs were intravenously administrated to each rat every other week for 8 weeks or only once and followed for 12 weeks. SRY gene expression was observed in female rats treated with male MSCs, and immune localization of CD73(+)CD90(+) cells at 8 weeks was also assessed. Serum and urine analyses showed an amelioration of functional parameters in MSC-treated animals at 8 weeks, but not at 12 weeks. Masson`s trichrome and Sirius red staining demonstrated reduced levels of fibrosis in MSC-treated animals. These results were corroborated by reduced vimentin, type I collagen, transforming growth factor beta, fibroblast specific protein 1 (FSP-1), monocyte chemoattractant protein 1, and Smad3 mRNA expression and alpha smooth muscle actin and FSP-1 protein expression. Renal interleukin (IL)-6 and tumor necrosis factor alpha mRNA expression levels were significantly decreased after MSC treatment, whereas IL-4 and IL-10 expression levels were increased. All serum cytokine expression levels were decreased in MSC-treated animals. Taken together, these results suggested that MSC therapy can indeed modulate the inflammatory response that follows the initial phase of a chronic renal injury. The immunosuppressive and remodeling properties of MSCs may be involved in the decreased fibrosis in the kidney. STEM CELLS 2009;27:3063-3073

Deiodinase-mediated thyroid hormone inactivation minimizes thyroid hormone signaling in the early development of fetal skeleton

Thyroid hormone (TH) plays a key role on post-natal bone development and metabolism, while its relevance during fetal bone development is uncertain. To Study this, pregnant once were made hypothyroid and fetuses harvested at embryonic days (E) 12.5, 14.5, 16.5 and 18.5. Despite a marked reduction in fetal tissue concentration of both T4 and T3, bone development, as assessed at the distal epiphyseal growth plate of the femur and vertebra, was largely preserved Lip to E16.5. Only at E18.5, the hypothyroid fetuses exhibited a reduction in femoral type I and type X collagen and osteocalcin mRNA levels, in the length and area of the proliferative and hypertrophic zones, in the number of chondrocytes per proliferative column, and in the number of hypertrophic chondrocyres, in addition to a slight delay in endochondral and intramembranous ossification. This Suggests that LIP to E 16.5, thyroid hormone signaling in bone is kept to a minimum. In fact, measuring the expression level of the activating and inactivating iodothyronine deiodinases (D2 and D3) helped understand how this is achieved. D3 mRNA was readily detected as early as E14.5 and its expression decreased markedly (similar to 10-fold) at E18.5, and even more at 14 days after birth (P14). In contrast. D2 mRNA expression increased significantly by E18.5 and markedly (similar to 2.5-fold) by P14. The reciprocal expression levels of D2 and D3 genes during early bone development along with the absence of a hypothyroidism-induced bone phenotype at this time Suggest that coordinated reciprocal deiodinase expression keeps thyroid hormone signaling in bone to very low levels at this early stage of bone development. (c) 2008 Elsevier Inc. All rights reserved.

Excessiva atividade de remodelamento ventricular sinaliza limitada resposta terapêutica ao manejo agressivo da insuficiência cardíaca avanaçada

Introdução: Níveis de fator de necrose tumoral–alfa (TNF-α), N-peptídeo do pró-colágeno III (PIIINP) e metaloproteinase de matriz –1 (MMP-1), marcadores biológicos de remodelamento ventricular, estão elevados em pacientes com insuficiência cardíaca (IC), talvez refletindo elevadas pressões de enchimento. A correlação destes marcadores com variáveis clínicas e hemodinâmicas permanece pouco compreendida, particularmente no contexto ambulatorial da IC. Objetivo: Avaliar níveis séricos de marcadores biológicos de remodelamento ventricular em pacientes com IC, comparando tratamento guiado por ecocardiografia (ECO), buscando redução de pressões de enchimento, versus tratamento convencional (CLÍNICO), baseado em sinais e sintomas. Métodos: Ensaio clínico randomizado. Pacientes estáveis com IC e fração de ejeção menor do que 40% foram alocados entre os grupos de tratamento e submetidos a ecocardiograma e coletas de sangue no início do estudo e em 180 dias. TNF-α e MMP- 1 foram medidos por ELISA, e PIIINP, por radioimunoensaio. Resultados: Incluiu-se 80 pacientes, com 59 ± 15 anos e fração de ejeção de 26 ± 7%; 25% isquêmicos e 52% masculinos. Houve redução dos marcadores biológicos intragrupos, não havendo diferença entre os tratamentos. No grupo CLÍNICO, os níveis de TNF-α, MMP-1 e PIIINP apresentaram diferenças estatisticamente significativas entre os momentos basal e final (respectivamente, 3,11 ± 2,90 versus 1,24 ± 0,60 pg/mL p < 0,0003; 2,66 ± 1,00 versus 1,16 ± 0,40 ng/mL p < 0,0001; 6,12 ± 2,60 versus 3,89 ± 1,60 μg/L p < 0,0001). De maneira semelhante, tal diferença também foi observada no grupo ECO para os três marcadores (respectivamente, 3,90 ± 4,90 versus 1,40 ± 1,30 pg/mL p < 0,0001; 2,50 ± 0,90 versus 1,09 ± 0,40 ng/mL p < 0,0001; 6,09 ± 2,60 versus 3,50 ± 1,30 μg/L p<0,0001). Ao final da intervenção, no entanto, não foi observada diferença significativa dos valores de TNF-α , MMP-1 e PIIINP entre os dois grupos de tratamento (p = 0,7; p = 0,8; e p = 0,2; respectivamente). A combinação dos valores basais das variáveis biológicas gerou um escore que se associou significativamente com o comportamento final das pressões atrial direita e sistólica da artéria pulmonar. Pacientes com marcadores biológicos basais no quartil 75% mantiveram níveis superiores de pressões atrial direita (13 mmHg; p = 0,034) e sistólica de artéria pulmonar (60 mmHg; p = 0,007) ao final do seguimento. Conclusão: Independente do tratamento alocado, houve redução dos níveis de marcadores biológicos ao final do seguimento; no entanto, níveis basais mais elevados destes marcadores foram preditores de menor redução das pressões em átrio direito e sistólica da artéria pulmonar. Os dados sugerem que indicativos de intenso processo de remodelamento ventricular se associam à progressão da IC e a pressões de enchimento elevadas.

Tricomas foliares em tomateiro com teores contrastantes do aleloquímico 2-tridecanona

Duas espécies de tomateiro, Lycopersicon esculentum Mill. (Linhagem TOM 556- padrão com baixo teor da metil cetona 2-tridecanona (2-TD) e Lycopersicon hirsutum Dunal var. glabratum Mill. (Linhagem PI 134417- padrão com elevado teor de 2-TD), foram analisadas em conjunto para identificação e quantificação de tricomas foliares. A parte experimental constou da identificação dos tricomas, baseado na presença ou ausência da cabeça secretora na extremidade apical dos tricomas, arranjo e número de células da cabeça, comprimento dos tricomas, e da quantificação do número de tricomas glandulares e não glandulares nos folíolos. O delineamento utilizado para o número de tricomas foi inteiramente casualizado e as médias comparadas pelo teste de Tukey, a 5%. A identificação e quantificação para as espécies foram: a) TOM 556: Tricomas não glandulares do tipo II + III + V (2590 cm-2 de área foliolar); glandular do tipo VI (16 cm-2); glandular do tipo VII (138 cm-2); b) PI 134417: não glandulares do tipo II + III + V (140 cm-2 de área foliolar); glandular do tipo I + IV (843 cm-2); glandular do tipo VI (83 cm-2); glandular do tipo VII (11 cm-2). As maiores concentrações da 2-TD nos folíolos, estão associadas às maiores densidades de tricomas glandulares presentes em ambas espécies.

Binding of extracellular matrix proteins to Paracoccidioides brasiliensis

Adhesion to extracellular matrix (ECM) proteins plays a crucial role in invasive fungal diseases. ECM proteins bind to the surface of Paracoccidioides brasiliensis yeast cells in distinct qualitative patterns. Extracts from Pb18 strain, before (18a) and after animal inoculation (18b), exhibited differential adhesion to ECM components. Pb18b extract had a higher capacity for binding to ECM components than Pb18a. Laminin was the most adherent component for both samples, followed by type I collagen, fibronectin, and type IV collagen for Pb18b. A remarkable difference was seen in the interaction of the two extracts with fibronectin and their fragments. Pb18b extract interacted significantly with the 120-kDa fragment. Ligand affinity binding assays showed that type I collagen recognized two components (47 and 80 kDa) and gp43 bound both fibronectin and laminin. The peptide 1 (NLGRDAKRHL) from gp43, with several positively charged amino acids, contributed most to the adhesion of P. brasiliensis to Vero cells. Synthetic peptides derived from peptide YIGRS of laminin or from RGD of both laminin and fibronectin showed the greatest inhibition of adhesion of gp43 to Vero cells. In conclusion, this work provided new molecular details on the interaction between P. brasiliensis and ECNI components. (c) 2006 Elsevier SAS. All rights reserved.

End-to-end arterial anastomosis with fibrin glue in larger arteries: histology, hydroxyproline concentration and tensile strength study in carotids of rabbits

A anastomose arterial término-terminal é demorada, requer tempo prolongado de oclusão vascular e esta associada a necrose focal, infiltração leucocitária e, conseqüentemente, à fibrose e calcificação da parede arterial. A cola de fibrina é uma alternativa para a anastomose microvascular e pode evitar estas alterações com menor aderência aos tecidos vizinhos e melhor coaptação das bordas arteriais. OBJETIVO: Comparar o processo cicatricial de anastomoses convencionais com anastomoses feitas com cola de fibrina em artérias maiores. MÉTODOS: em 22 coelhos, ambas carótidas foram seccionadas transversalmente e reconstruídas por meio de anastomose término-terminal com 4 pontos simples de reparo e cola de fibrina de um lado (G1), e com 8 pontos separados do outro lado (G2). Após 3 e 15 dias, os animais foram destinados aleatoriamente para estudo de força tênsil concentração de hidroxiprolina (8 animais) e avaliação histológica das anastomoses (3 animais). As lâminas histológicas foram coradas pelo HE Masson e Picrossirius polarização (PSP). RESULTADOS: Após 3 e 15 dias a força tênsil aumenta em ambos os grupos, de 280,0± 32,6g para 432,2± 131,2g no Grupo 1 e de 221,4± 72,4g para 452,2± 132,0g no Grupo 2; sem diferença estatística entre os grupos em cada período. A concentração de hidroxiprolina expressa como razão hidroxiprolina/proteína, variou de 0,0816± 0,0651 para 0,0622± 0,0184 no Grupo 1 e de 0,0734± 0,0577 para 0,0460± 0,0271 no Grupo 2; sem diferença estatística entre os períodos e grupos. Os estudos histológicos mostraram discreto aumento das reações de inflamação e reparação no Grupo 2. A técnica PSP mostrou predomínio do colágeno tipo I em relação do colágeno tipo II nas anastomoses de ambos os grupos, sem diferença expressiva entre esses grupos. CONCLUSÃO: A anastomose com a cola de fibrina foi menos lesiva para a parede arterial do que a anastomose convencional. Mesmo usando menos pontos, as características de força tênsil e de cicatrização da anastomose com cola de fibrina foram similares em ambos os grupos. Os tempos de realização das anastomoses foram significativamente maiores do que na anastomose convencional.

Edema de Reinke: estudo da imunoexpressão da fibronectina, da laminina e do colágeno IV em 60 casos por meio de técnicas imunoistoquímicas

Edema de Reinke é doença crônica da laringe na qual a camada superficial da lâmina própria é expandida por muco espesso conferindo-lhe aspecto gelatinoso. Relaciona-se ao tabagismo e acomete, preferencialmente mulheres, as quais apresentam a voz mais grave. Suas características histológicas nem sempre conseguem diferenciá-lo das demais lesões benignas da laringe, havendo necessidade de técnicas histológicas adicionais. OBJETIVOS: Estudar a imunoexpressão da fibronectina, do colágeno IV e da laminina no edema de Reinke por meio de técnicas imunoistoquímicas. Estudo prospectivo. MATERIAL E MÉTODOS: Blocos histológicos de 60 casos cirúrgicos de edema de Reinke foram resgatados, submetidos a novos cortes e às reações imunoistoquímicas para fibronectina, laminina e colágeno IV pelo método da Avidina Biotina Peroxidase. Todos os pacientes eram fumantes e adultos, sendo 50 mulheres e 10 homens. RESULTADOS: As análises da imunoexpressão da fibronectina, do colágeno IV e da laminina foram mais expressivas no endotélio dos vasos (68,33%, 76,66%, 73,33%, respectivamente), e menos relevantes na membrana basal (25,0%, 5,0% e 3,3%, respectivamente). CONCLUSÕES: No edema de Reinke, a imunoexpressão da fibronectina, da laminina e do colágeno IV na membrana basal não apresentam relevância, havendo predomínio desses anticorpos no endotélio do vasos.

Implante de condrócitos homólogos em defeitos osteocondrais de cães: padronização da técnica e avaliação histopatológica

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Marcadores do metabolismo ósseo em gatos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Use of Adipose Tissue-Derived Mesenchymal Stem Cells for Experimental Tendinitis Therapy in Equines

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)