Search

The purpose of this study was to measure the anti-angiogenic effect of N-desulfated Re-N-acetylated, a chemically modified heparin (mHep).In vitro assays (cell tube formation, viability, proliferation, and migration) with endothelial cells were performed after 24 h of treatment with mHep at 10, 100, and 1000 ng/mL or saline. In vivo tests were performed after laser-induced choroidal neovascularization (CNV) in rats, followed by an intravitreal injection (5 µL) of mHep (10, 100, 1000 ng/mL) or balanced salt solution. Immunofluorescence analysis of the CNV was performed after 14 days.mHep produced a statistically significant reduction in cell proliferation, tube formation, and migration, without cell viability changes when compared to saline. Mean measures of CNV area were 54.84 × 10These results suggest that mHep N-DRN is a potent anti-angiogenic, anti-proliferative, and anti-migratory compound with negligible anticoagulant or hemorrhagic action and no cytotoxicity for retina cells. This compound may serve as a candidate for treating choroidal neovascularization.

Cambiamenti strutturali della matrice extracellulare dopo cross-linking del tessuto polipoide nasale.La rinosinusite cronica con poliposi nasale è una malattia caratterizzata da una disfunzione meccanica che facilita la crescita anomala della mucosa nasale, a causa del suo peculiare processo di rimodellamento. Questo studio si propone di valutare gli effetti del cross-linking sulla matrice extracellulare del tessuto del polipo nasale. Sono stati selezionati sei pazienti sottoposti ad intervento chirurgico di polipectomia. È stata applicata la tecnica del cross linking del collagene con riboflavina / UVA alla superficie dei polipi resecati epitelizzati e disepitelizzati del gruppo di pazienti sottoposti ad intervento. I polipi del gruppo di controllo non sono stati sottoposti a procedure di cross linking. Le sezioni ultrasottili (8 μm) di tessuto polipoide sono state analizzate mediante immunofluorescenza con anticorpi anti collagene di tipo I di topo e anticorpi secondari coniugati AlexaFluor 488 con controcolorazione DAPI e analizzate mediante microscopia confocale. L’effetto del cross-linking è risultato visibile al microscopio confocale a fluorescenza. La morfologia quantitativa è stata associata all’analisi di imaging in fluorescenza e sono state valutate la densità e la luminosità dei pixel. La superficie dei polipi trattati mostrava una maggiore densità di fibre di collagene rispetto ai polipi di controllo, come è stato possibile osservare sia visivamente che attraverso misurazioni oggettive delle regioni fluorescenti. L’effetto è stato potenziato sulla superficie dei polipi disepitelizzati. La maggiore densità di fibre di collagene esibita dai polipi trattati disepitelizzati dimostra la fattibilità di questa tecnica per interferire con il processo di rimodellamento e affrontare la disfunzione meccanica riscontrata nella rinosinusite cronica con poliposi nasale.Chronic rhinosinusitis with nasal polyposis is a disease characterised by a mechanical dysfunction that facilitates anomalous growth of the nasal mucosa, due to its unique remodelling process. The development of procedures capable of interfering with this process is thus of the utmost importance. This study deals with the evaluation of the effects of cross-linking upon the nasal polyp tissue extracellular matrix. Six patients undergoing surgery for polypectomy were selected. The riboflavin/UVA collagen cross-linking technique was applied to the surface of epithelialised and de-epithelialised resected polyps of the intervention group. The control group polyps were not submitted to cross-linking procedures. Ultrathin polyp tissue sections (8 μm) were processed for immunofluorescence with mouse anti-type I collagen antibody and AlexaFluor 488 conjugated secondary antibody plus DAPI counterstaining, and analysed by confocal microscopy. The effect of riboflavin/UVA collagen cross-linking was visible on confocal fluorescence microscopy. Quantitative morphology was associated with fluorescence imaging analysis, and pixel density and brightness were evaluated. The surface of treated polyps exhibited a higher density of collagen fibres compared to control polyps, as could be observed both visually and through objective measurements of the fluorescent regions. The effect was enhanced on the surface of the de-epithelialised polyps. The higher density of collagen fibres exhibited by the de-epithelialised treated polyps demonstrates the feasibility of this technique in interfering with the remodelling process and the mechanical dysfunction found in chronic rhinosinusitis with nasal polyposis.

Age-related macular degeneration is the leading cause of vision loss in elderly individuals, as well as a medical and socio-economic challenge. The treatment of dry age-related macular degeneration is based on vitamin supplementation. New treatment studies are focused on preventing the progression of degeneration and repopulating the atrophic macula. Recently, research on the treatment of neovascular age-related macular degeneration experienced a breakthrough with the advent of anti-vascular endothelial growth factor inhibitors. Nevertheless, despite the fact that ranibizumab, aflibercept, and bevacizumab are effective in reducing severe visual impairment, patients usually lose some vision over time. Therefore, the search for new therapies and diagnostic methods is fundamentally important. Current studies are focused on new anti-vascular endothelial growth factor drugs, nucleoside reverse transcriptase inhibitors, antibody against sphingosine-1-phosphate, anti-platelet-derived growth factor, gene therapy, and RNA interference. The results of ongoing clinical studies may improve the therapy of age-related macular degeneration.

Endothelial cells (ECs) are subjected to physical forces such as shear stress (SS) induced by blood flow that leads to significant changes in morphology, physiology and gene expression. The abnormal mechanical forces applied in the cardiovascular system can influence the development of conditions and diseases such as thrombosis, hypertension and atherosclerosis. This study investigated the expression of glycosaminoglycans (GAGs), proteoglycans and extracellular matrix molecules in ECs exposed to normal and altered SS. ECs were exposed to SS of 12 dyn/cm2 (artery physiological condition) and 4 dyn/cm2 (artery pathological condition). Subsequently, ECs were subjected to immunofluorescence, qPCR, GAG biosynthesis analyses and cell-based assays. SS induced changes in ECs morphology. There were other pathological consequences of altered SS, including inhibited adhesion, stimulation of migration and capillary-like tube formation, as well as increases of GAG synthesis. We observed higher expression of syndecan-4, perlecan, decorin, fibronectin and collagen III α1 and growth factors, including VEGF-A and TGFβ-1. ECs exposed to SS displayed extracellular matrix remodeling as well as expression of cell-matrix and cell-cell interaction molecules. This study contributes to the understanding of how vascular biology is affected by mechanical forces and how these molecules can be affected in cardiovascular diseases.

Age-related macular degeneration is the leading cause of vision loss in elderly individuals, as well as a medical and socio-economic challenge. The treatment of dry age-related macular degeneration is based on vitamin supplementation. New treatment studies are focused on preventing the progression of degeneration and repopulating the atrophic macula. Recently, research on the treatment of neovascular age-related macular degeneration experienced a breakthrough with the advent of anti-vascular endothelial growth factor inhibitors. Nevertheless, despite the fact that ranibizumab, aflibercept, and bevacizumab are effective in reducing severe visual impairment, patients usually lose some vision over time. Therefore, the search for new therapies and diagnostic methods is fundamentally important. Current studies are focused on new anti-vascular endothelial growth factor drugs, nucleoside reverse transcriptase inhibitors, antibody against sphingosine-1-phosphate, anti-platelet-derived growth factor, gene therapy, and RNA interference. The results of ongoing clinical studies may improve the therapy of age-related macular degeneration. RESUMO Degeneração macular relacionada à idade (DMRI) é a principal causa de perda de visão em pessoas idosas. É também um desafio médico e socioeconômico. O tratamento da degeneração macular relacionada à idade seca baseia-se na suplementação vitamínica. Novos tratamentos estão focados na prevenção da progressão da degeneração e tentativas de repovoar a mácula atrófica. A degeneração macular relacionada à idade neovascular experimentou um grande avanço com o advento dos inibidores do fator de crescimento endotelial anti-vascular (anti-VEGF); no entanto, apesar do ranibizumab, aflibercept e bevacizumab serem eficazes na redução do comprometimento visual grave, os pacientes geralmente perdem visão ao longo do tempo. Portanto, a busca por novas terapias, tratamentos e diagnósticos é de fundamental importância. Os estudos estão focados em novos fármacos sobre fator de crescimento endotelial anti-vascular, inibidores nucleosideos da transcriptase reversa, anticorpos contra esfingosina-1-fosfato, fator de crescimento derivado de plaquetas, terapia genética e RNA de interferência. A terapia para degeneração macular relacionada à idade está prestes a melhorar como resultado desses estudos clínicos em andamento.

Resumo Objetivo Quantificar a concentração urinária do biomarcador telopeptídeo C de ligação cruzada de colágeno de tipo II (C-terminal cross-linked telopeptide of type-II collagen, CTX-II) em casos de lesão isolada do ligamento cruzado anterior (LCA), e comparar as concentrações observadas nessa população com um grupo controle composto por pacientes sem alterações metabólicas no joelho que possam levar à degeneração da cartilagem. Métodos Este é um estudo piloto transversal com dois grupos: pacientes com ruptura do LCA e grupo controle (cada grupo era composto por 10 indivíduos do sexo masculino, com 18 a 35 anos de idade, e índice de massa corporal inferior a 30 kg/m2). Nos dois grupos, as concentrações urinárias de um biomarcador relacionado à degradação do colágeno de tipo II (CTX-II) foram medidas. No grupo com ruptura do LCA, a relação entre o tempo pós-lesão e a quantidade do biomarcador também foi analisada. Resultados Houve diferenças significativas nas concentrações urinárias de CTX-II entre o grupo LCA e o grupo controle (p = 0,009). Não foi observada relação significativa entre o tempo de lesão e a quantidade do biomarcador. Conclusões Pacientes com lesão do LCA apresentaram maiores concentrações urinárias do biomarcador CTX-II do que aqueles sem lesão do LCA (p = 0,009). No entanto, não houve correlação entre a concentração desse biomarcador e o tempo decorrido após a lesão (p> 0,05).

Age-related macular degeneration is the leading cause of vision loss in elderly individuals, as well as a medical and socio-economic challenge. The treatment of dry age-related macular degeneration is based on vitamin supplementation. New treatment studies are focused on preventing the progression of degeneration and repopulating the atrophic macula. Recently, research on the treatment of neovascular age-related macular degeneration experienced a breakthrough with the advent of anti-vascular endothelial growth factor inhibitors. Nevertheless, despite the fact that ranibizumab, aflibercept, and bevacizumab are effective in reducing severe visual impairment, patients usually lose some vision over time. Therefore, the search for new therapies and diagnostic methods is fundamentally important. Current studies are focused on new anti-vascular endothelial growth factor drugs, nucleoside reverse transcriptase inhibitors, antibody against sphingosine-1-phosphate, anti-platelet-derived growth factor, gene therapy, and RNA interference. The results of ongoing clinical studies may improve the therapy of age-related macular degeneration.

Introduction The importance of our study lies in the fact that we have demonstrated the occurrence of mechanical dysfunction within polypoid tissues, which promotes the development of polyps in the nasal cavity. Objective To change the paradigm of nasal polyposis (NP). In this new conception, the chronic nasal inflammatory process that occurs in response to allergies, to pollution, to changes in the epithelial barrier, or to other factors is merely the trigger of the development of the disease in individuals with a genetic predisposition to an abnormal tissue remodeling process, which leads to a derangement of the mechanical properties of the nasal mucosa and, consequently, allows it to grow unchecked. Data Synthesis We propose a fundamentally new approach to intervening in the pathological process of NP, addressing biomechanical properties, fluid dynamics, and the concept of surface tension. Conclusion The incorporation of biomechanical knowledge into our understanding of NP provides a new perspective to help elucidate the physiology and the pathology of nasal polyps, and new avenues for the treatment and cure of NP.

Aims Cardiovascular diseases such as hypertension, thrombosis and atherosclerosis are responses to mechanical forces applied to the endothelium. Endothelial cells respond to hemodynamic mechanical forces such as cellular mechanical stretching. We investigated the expression of glycosaminoglycans, proteoglycans and other extracellular matrix molecules in endothelial cells subjected to various mechanical stimuli. Main methods Endothelial cells were subjected to mechanical stretch in a vacuum system FlexCell™ to 5% (physiological condition) and 15% (pathological condition), for 4 h or 24 h. Culture plates not subjected to strain were used as controls. Subsequently, ECs were subjected to immunofluorescence, real-time PCR, PCR array, glycosaminoglycans biosynthesis using metabolic radiolabeling with 35S-sulfate and cell behavior assays (adhesion, migration and capillary tube formation). Key findings Mechanical stretch induced changes in endothelial cell morphology. Pathological consequences of mechanical stretch included inhibited migration in 2-fold and capillary-like tube formation in 2-fold, when compared to physiological condition after 4 h of ECs exposure; it also reduced total sulfated glycosaminoglycans synthesis thereabout 1.5-fold. Pathological mechanical stretch conditions induced higher expression after 24 h of ECs exposure to mechanical stretch of syndecan-4 (3.5-fold), perlecan (9.1-fold), decorin (5.7-fold), adhesive proteins as fibronectin (5.6-fold) and collagen III α1 (2.2-fold) and growth factors, including VEGF-A (7.3-fold) and TGFβ-1 (14.6-fold) and TGFβ-3 (4.3-fold). Significance Exposure of endothelial cells to mechanical stretch influenced remodeling of the extracellular matrix as well as cell-matrix interactions. These studies improve understanding of how vascular biology is affected by mechanical forces and how these molecules behave in cardiovascular diseases.

Purpose Reconstruction of the ocular surface is challenging. As an alternative to mucosal and limbal epithelial, we study the feasibility of cultivated human conjunctival epithelial (HCjE) cells of patients with total limbal stem cell deficiency (LSCD). Methods We studied superior forniceal conjunctival biopsies harvested from 9 living donors with total LSCD of several etiologies who underwent surgery for ocular surface reconstruction. The conjunctival explants were cultivated on serum and growth factor supplemented DMEM/F12 under submerged conditions on denuded human amniotic membrane and tissue culture dishes. The area of cell growth was assessed. Cell morphology was analyzed by light microscopy, impression cytology, and transmission electron microscopy. Cultures were evaluated for epithelial cytokeratins (CK3, CK19), proliferation marker (Ki-67), and putative stem cells markers (ABCG2 and p63). Confocal immunofluorescence was also performed to assess CK3, CK19, Ki-67, ABCG2, and p63. Results The HCjE cells cultivated ex vivo were successfully expanded on denuded amniotic membrane but with a slower growth than in the tissue culture dish. Transmission electron microscopy showed stratified epithelium with microvilli, desmosomes, and hemidesmosomes. Impression cytology showed PAS+ cells that resembled goblet cells. Immunocytochemical analysis showed positivity for CK3, CK19, Ki-67, ABCG2, and p63. Confocal immunofluorescence was positive for CK3, CK19, Ki-67, ABCG2, and p63. Conclusions Our results showed that it is possible to cultivate HCjE cells ex vivo of patients with ocular surface diseases. This method is important for ocular surface reconstruction in patients with bilateral total LSCD.

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundacao de Apoio a Pesquisa do Estado do Rio Grande do Norte-FAPERN Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Ministerio de Ciencia, Tecnologia e Inovacao-MCTI Fucan is a term that defines a family of homo-and hetero-polysaccharides containing sulfated L-fucose in its structure. in this work, a heterofucan (F2.0v) from the seaweed, Dictyota menstrualis, was evaluated as an antinociceptive and anti-inflammatory agent. F2.0v (20.0 mg/kg) inhibits 100% of leukocyte migration into the peritoneal cavity after chemical stimulation. However, F2.0v does not alter the expression of interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6), as well as tumor necrosis factor alpha (TNF-alpha). F2.0v (20.0 mg/kg) has peripheral antinociceptive activity with potency similar to dipyrone. On the other hand, it had no effect on pain response on the hot plate test. Confocal microscopy analysis and flow cytometry showed that F2.0v binds to the surface of leucocytes, which leads us to suggest that the mechanism of action of anti-inflammatory and antinociceptive F2.0v is related to its ability to inhibit the migration of leukocytes to the site of tissue injury. in summary, the data show that F2.0v compound has great potential as an antinociceptive and anti-inflammatory, and future studies will be performed to further characterize the mechanism of action of F2.0v. Fed Univ Rio Grande Norte UFRN, Dept Biochem, Lab Biotechnol Nat Polymers BIOPOL, BR-59078970 Natal, RN, Brazil Fed Univ Rio Grande Norte UFRN, Grad Program Hlth Sci, BR-59078970 Natal, RN, Brazil Fed Univ São Paulo UNIFESP, Dept Biochem, BR-04044020 São Paulo, Brazil Fed Univ São Paulo UNIFESP, Dept Biochem, BR-04044020 São Paulo, Brazil Web of Science

Summary Objective To investigate the hypothesis that strenuous running is a predisposing factor for osteoarthritis. Design Wistar rats were divided into two groups: a control group (CG) and a trained group (TG). The TG underwent a strenuous treadmill running training regimen of controlled intensity, exhibiting progressively improvement of fitness over 12 weeks, running at least 55 km during this period and finally performing an ultra-endurance running exercise to exhaustion. After this period, rats from both groups were euthanized and their knees removed. The articular cartilage was dissected and submitted to histomorphometrical, histomorphological, and immunohistochemical analyses evaluating cell death pathway (caspase-3 and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL)) and inflammatory cytokines [interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α)]. In addition, the tissues were analyzed regarding the types and the content of glycosaminoglycans. Results The TG knee joints exhibited increase in the number of chondrocytes and chondrocyte clusters, as well as significantly increased levels of caspase-3, a protein involved in apoptosis, and of inflammatory cytokines IL-1α and TNF-α. In addition, histologically higher grades of osteoarthritis (Osteoarthritis Research Society International – OARSI grading), and significantly decreased levels of chondroitin sulfate and hyaluronic acid. Knee cartilage thickness and TUNEL did not significantly differ between the two groups. Conclusions The articular cartilage of rats subjected to a strenuous running regimen of controlled intensity exhibited molecular and histological characteristics that are present in osteoarthritis.

The importance of monitoring post haematopoietic stem cell transplantation (hSCT) chimerism has been defined in numerous publications. Single-nucleotide polymorphisms (SNPs) are molecular markers that vary significantly among different populations. Allied to a very sensible technique, SNP assays seem to be very sensitive (0.001%) when post hSCT chimerism is measured. However, well known SNP frequencies are limited to certain populations, mainly in countries where there is a high level of diversity in its population, therefore restricting their use worldwide. Amplification by SYBR green based quantitative real time PCR of eight pairs of allele-specific SNPs (MLH-1, PECAM-1, ICAM-1, SUR-1, HA-1, rs715405, rs713503, rs2296600) was conducted in 88 patient/donor pairs, who underwent allogeneic myeloablative or non-myeloablative hSCT. One informative allele was detected in at least 42% (n=37) of the samples; 20% (n=18) had at least two informative alleles; 10% (n=9) had at least three informative alleles; 9% (n=8) had more than three informative alleles and 18% (n=16) showed no informative allele at all. Overall, the frequency of informative alleles for these SNPs in the Brazilian population was very low. Consequently, the amount of information attained reached 9% of those expected, being able to discriminate only eight pairs of donor/recipient samples with more than three informative alleles, making them useless for the quantification of chimerism in our routine.

Syndecans are heparan sulfate proteoglycans characterized as transmembrane receptors that act cooperatively with the cell surface and extracellular matrix proteins. Syn4 knockdown was performed in order to address its role in endothelial cells (EC) behavior. Normal EC and shRNA-Syn4-EC cells were studied comparatively using complementary confocal, super-resolution and non-linear microscopic techniques. Confocal and super-resolution microscopy revealed that Syn4 knockdown alters the level and arrangement of essential proteins for focal adhesion, evidenced by the decoupling of vinculin from F-actin filaments. Furthermore, Syn4 knockdown alters the actin network leading to filopodial protrusions connected by VE-cadherin-rich junction. shRNA-Syn4-EC showed reduced adhesion and increased migration. Also, Syn4 silencing alters cell cycle as well as cell proliferation. Moreover, the ability of EC to form tube-like structures in matrigel is reduced when Syn4 is silenced. Together, the results suggest a mechanism in which Syndecan-4 acts as a central mediator that bridges fibronectin, integrin and intracellular components (actin and vinculin) and once silenced, the cytoskeleton protein network is disrupted. Ultimately, the results highlight Syn4 relevance for balanced cell behavior.

OBJETIVO: Determinar se o epitélio corneano pode impedir ou diminuir o efeito do tratamento com cross-linking (CXL). Por meio de microscopia por imunofluorescência, foi indiretamente analisado o efeito do epitélio como escudo aos raios ultravioleta-A (UVA), assim como barreia à penetração da riboflavina. MÉTODOS: Quinze olhos enucleados de porcos foram divididos em 3 grupos. O epitélio corneano foi mantido intacto em todos os grupos. Cinco olhos serviram como controle (Grupo 1). No grupo 2, os olhos foram instilados com colírio anestésico de tetracaína, assim como colírio de riboflavina 0,1% (10 mg de riboflavina-5-fosfato em 10 ml de dextran 20% T-500). No grupo 3, solução de riboflavina foi injetada na câmara anterior para permitir a penetração da droga através do endotélio. Os grupos 2 e 3 foram então expostos à radiação UVA (365 nm, 3 mW/cm²) por 30 minutos. Subsequentemente, cortes ultrafinos (8 µm) das córneas foram marcados com anticolágeno tipo I e DAPI (4,6-diamidino-2-fenilindole dihydrocloride) e analisados com microscópio de imunofluorescência. RESULTADOS: As córneas que receberam injeção intracameral de riboflavina e foram irradiadas com UVA (Grupo 3) mostraram um padrão maior de organização das fibras de colágeno em relação aos grupos 1 (Controle) e 2 (instiladas com colírio anestésico e de riboflavina). Macroscopicamente, a coloração amarelada do estroma, que representa a difusão da riboflavina, foi apenas observada nos olhos que receberam riboflavina intracameral. CONCLUSÃO: Foi demonstrado, através de microscopia por imunofluorescência em córneas de porcos, que o epitélio corneano íntegro diminui a efetividade do CXL por reduzir a penetração da riboflavina, e não por impedir a penetração dos raios UVA. Uma concentração intraestromal inadequada de riboflavina limita o efeito do tratamento. PURPOSE: To determine if the corneal epithelium prevents the collagen cross-linking effect. Using immunofluorescence microscopy after CXL, we indirectly analyzed the role of the epithelium as ultraviolet-A (UVA) shield as well as a barrier to riboflavin penetration. METHODS: Fifteen freshly enucleated porcine eyes were divided into 3 groups. The corneal epithelium was kept intact in all groups. Five eyes served as control (Group 1). On group 2, eyes received tetracaine anesthetic drops and topical 0.1% riboflavin solution (10 mg riboflavin-5-phosphate in 10 mL 20% dextran-T-500). On Group 3, riboflavin was injected into the anterior chamber to allow penetration of the drug through the endothelium. Groups 2 and 3 were exposed to UVA (365 nm, 3 mW/cm²) for 30 minutes. Ultra-thin sections (8 µm) of the corneas were stained with anti-collagen type I and DAPI (4,6-diamidino-2-fenilindole dihydrocloride) and analyzed with fluorescence microscopy. RESULTS: Corneas treated with UVA irradiation and intracameral injection of riboflavin (Group 3) showed greater pattern of collagen organization compared to groups 1 (Control) and 2 (riboflavin and tetracaine eye drops). A yellow stromal staining, which represents the riboflavin diffusion into the stroma, was only observed in eyes injected with riboflavin into the anterior chamber. CONCLUSION: Using immunofluorescence microscopy in porcine corneas, we demonstrated that the corneal epithelium reduces the effectiveness of CXL by preventing the penetration of the drug and not by limiting the UVA transmittance. An inadequate intrastromal concentration of riboflavin may impair CXL effect. Universidade Federal de São Paulo (UNIFESP) Department of Ophthalmology Universidade Federal de São Paulo (UNIFESP) Department of Biochemistry Molecular Biology Division UNIFESP, Department of Ophthalmology UNIFESP, Department of Biochemistry Molecular Biology Division SciELO

OBJETIVO: Comparar a quantidade do glicosaminoglicano dermatam sulfato entre pacientes homens, portadores de hérnia inguinal tipo II de Nyhus e, indivíduos sem hérnia inguinal, com idade entre 20 e 40 anos. MÉTODOS: Foram constituídos dois grupos. Um de 15 pacientes do sexo masculino com hérnia inguinal tipo II de Nyhus e idade entre 20 e 40 anos, com risco ASA I e II, e um grupo controle com dez indivíduos, também do sexo masculino entre 20 e 40 anos, que morreram em período de até 24 h. Foram excluídos os pacientes do sexo feminino, diabéticos, portadores de doença do tecido conjuntivo, tabagistas e com risco cirúrgico ASA III e IV. Foi retirada uma amostra de 1cm² da fáscia transversal na parte intermediária do trígono inguinal, e 1cm² na bainha anterior do músculo reto abdominal na região inguinal correspondente e quantificados os glicosaminoglicanos dermatam sulfato por densitometria, após eletroforese em gel de agarose. RESULTADOS: A quantidade de dermatam sulfato não apresentou diferença estatisticamente significante entre os pacientes com hérnia inguinal e os indivíduos sem hérnia inguinal, tanto na fáscia transversal (p=0,108) quanto na bainha anterior do músculo reto abdominal (p=0,292). CONCLUSÃO: Não se encontrou diferença na quantidade do glicosaminoglicano dermatam sulfato entre os pacientes portadores de hérnia inguinal tipo II de Nyhus e indivíduos sem hérnia inguinal em homens adultos.

To assess structural stromal modifications after riboflavin and UV-A exposure in edematous human corneas.Fourteen eyes with corneal edema were enrolled in the study. In the cross-linking (CXL) group, 7 corneal buttons were obtained from 6 patients who underwent penetrating keratoplasty (PK) 7-90 days after the CXL treatment. The control group was composed of 7 corneal buttons with bullous keratopathy. After the PK, stromal modifications were investigated using immunofluorescence in all corneal grafts. All patients had at least 3 months of corneal edema and were in the eye bank list waiting for keratoplasty.All corneas in the treated group showed a pronounced lamellar zone of collagen fibers highly organized in the anterior stroma, but there was not complete homogeneity between the samples. Corneas with advanced disease and stromal fibrosis were less compacted than ones with mild disease severity. Similarly, those ones that underwent PK 3 months after CXL also showed a decreased effect compared with those with a reduced time between the CXL and the PK. DAPI staining demonstrated a complete fragmentation of keratocytes nuclei in the anterior stroma in all treated corneas, which were absent in the control group.Our study showed an immediate effect of CXL with a limited long-term sustainability. Cross-linked corneas had a pronounced anterior zone of organized collagen fibers. Even the treated corneas with advanced bullous keratopathy and stromal fibrosis had histological evidence of collagen fibers organization, but this effect seems to be decreased compared with corneas in initial stages of the disease.

Glycosaminoglycans (GAGs) play important roles in cell behavior and have the ability to bind and modulate cytokines. Using primary cultured fibroblasts from hereditary gingival fibromatosis (HGF), normal gingiva (NG), and NG treated with cyclosporin-A (NGc) we show changes in the expression and structural characteristics of GAGs as well as in the expression of enzymes involved in their biosynthesis and degradation. In addition, we show the over-expression of TGF-beta1 and TGF-beta type II receptor in HGF and NGc. There is an increase in the GAGs retained in the cellular fraction, and the fine structure of galactosaminoglycans show a decrease in alpha-l-iduronic acid content in HGF and NGc. Elevated extracellular levels of low molecular weight hyaluronan (HA) are found in HGF due to increase in the expression of HA synthase 3 and hyaluronidases 1 and 2. The results bring new insights to the accumulation of extracellular matrix related to TGF-beta over-expression.

Objective To quantify and study the immunoexpression of hyaluronic acid (HA) in the uterine horns of the mouse throughout the estrous cycle phases. Design Experimental study using an ELISA-like fluorometric assay to quantify HA and an avidin-biotin immunoperoxidase method using biotinylated hyaluronan-binding protein for histochemical studies. Setting University-based laboratory. Animal(s) Forty regularly cycling adult female mice were divided into four groups according to the diagnosed phase of the cycle: proestrus, estrus, metaestrus, and diestrus. Intervention(s) None. Main outcome measure(s) Histologic samples of the uterine horns. Immunohistochemical reaction was evaluated by detection of HA and CD44 distribution within the uterine horn. Tissue HA content was determined through an ELISA-like fluorometric assay with the same hyaluronan-binding protein and with europium-labeled streptavidin. Result(s) The immunohistochemical HA and CD44 reactions were most intense during diestrus, mainly below the luminal epithelium. HA was strongly stained in the connective tissue near the myometrium layer during metaestrus. The biochemical data showed that the highest concentration of HA in uterine horns occurred during diestrus (4053.0 ± 651.4 ng/g dry tissue) compared with other phases. Conclusion(s) Our data show that the expression of HA in mouse uterine horns is highest during the diestrous phase. The fluctuations of HA in the mouse uterus during the estrous phase may be related to the varying estrogen and P levels during the cycle and may be important as far as embryo implantation is concerned.

The glycosaminoglycan (GAG) layer is referred to as a bladder protective factor. We reproduced an experimental model of urothelial damage to assess GAG metabolism in the process of injury and recovery of the urothelium.Wistar female rats were bladder catheterized and instilled with either protamine sulfate (PS groups) or sterile saline (control groups). At different days after the procedure, 24-hour urine samples were obtained. The urinary levels of hyaluronic acid (HA) and sulfated glycosaminoglycan were determined in all groups and in nonmanipulated rats (day 0). Additionally, sulfated-GAG synthesis was assessed by the incorporation of [(35)S]-inorganic sulfate. The bladders were analyzed by histochemical staining for HA and immunofluorescence for heparin sulfate and syndecan-4.Urinary HA and sulfated-GAG were elevated after PS injection (P0.05). A greater concentration of [(35)S]-labeled GAG in the PS group animals on the fifth day and, especially, on the seventh day represented increased GAG synthesis at these periods (P0.05). Bladder sections from the PS group animals on day 1 showed a greater amount of HA in the urothelium. PS instillation damaged the urothelium layer of heparin sulfate and syndecan-4 seen in the control animals. On day 5, patchy areas of a restored layer were seen, and, on day 7, this layer had completely regenerated.Urinary GAG cannot differentiate urothelial damage from recovery. Elevated levels of urinary GAG can result from either desquamation of the surface cell GAG layer or increased GAG synthesis to regenerate the damaged urothelium.

In the current study, the ability of ATP to promote apoptosis in myeloblasts at different ages was investigated. We have observed that high concentration of extracellular ATP (>1 mM), which activates P2X 7 receptor, produced cell shrinkage an increase in the number of events in the sub-G 0 /G 1 region of the cellular cycle and annexin-V/propidium iodide label, which characterizes the apoptotic cell death. In addition, BzATP produced apoptosis, but not ADP and UTP. Gr-1 + cells express the P2X 7 receptor and oxidized ATP, a specific P2X 7 inhibitor, blocked the ATP-dependent apoptosis. ATP-dependent apoptosis is decreased by aging in myeloblasts of 12 and 22-month-old mice. Furthermore, P2X 7 expression decrease was observed in older mice, explaining apoptosis decrease. This decrease in apoptosis by aging may be related to some diseases in the myelocyte lineage.

Loxoscelism (the condition produced by the bite of brown spiders) has been reported worldwide, but especially in warmer regions. Clinical manifestations include skin necrosis with gravitational spreading while systemic loxoscelism may include renal failure, hemolysis and thrombocytopenia. The venom contains several toxins, of which the best biochemically and biologically studied is the dermonecrotic toxin, a phospholipase-D. Purified toxin induces cutaneous and systemic loxoscelism, especially necrotic lesions, hematological disturbances and renal failure. Herein, we describe cloning, heterologous expression and purification of two novel dermonecrotic toxins: LiRecDT4 and LiRecDT5. The recombinant proteins stably expressed in Escherichia coli cells were purified from culture supernatants in a single step using Ni2+-chelating chromatography producing soluble proteins of 34 kDa (LiRecDT4) and 37 kDa (LiRecDT5). Circular dichroism analysis evidenced correctly folding for toxins but differences in secondary structures. Both proteins were recognized by whole venom serum antibodies and by a specific antibody to dermonecrotic toxin. Also, recombinant toxins with phospholipase activity induced experimental skin lesions and caused a massive inflammatory response in rabbit skin dermis. Nevertheless, toxins displayed different effects upon platelet aggregation, increase in vascular permeability and not caused death in mice. These characteristics in combination with functional studies illustrates that a family of dermonecrotic toxins exists, and includes two novel members that are useful for future structural and functional studies. They will also be useful in biotechnological ends, for example, as inflammatory and platelet aggregating studies, as antigens for serum therapy source and for lipids biochemical research.

The adenoviral conjunctivitis is one of the biggest causes of conjunctival infection in the world. Conjunctivitis causes relatively nonspecific symptoms, as hyperaemia and chemosis. Even after biomicroscopy, complex laboratory tests, such as viral culture, are necessary to identify the pathogen or its etiology. To contribute to the better understanding of the pathobiology of the adenoviral conjunctivitis, the tear fluids of patients with unilateral acute adenovirus conjunctivitis (UAAC), normal donors (control) and patients with allergic conjunctivitis were analyzed. Tear samples were collected with Schirmer strips from control, allergic conjunctivitis and UAAC patients, diagnosed by clinical signs. UAAC tears were tested positive in viral cultures. After the elution, HA was quantified using an ELISA-like fluorometric assay and the protein profile was determined by SDS-PAGE. A profound increase in the HA tear content in UAAC patients was found when compared to control and ALC. This HA increase in UAAC tears remarkably was not observed in tears from contralateral eyes without clinical signs, nor in allergic conjunctivitis. In addition a distinct profile of UAAC tear proteins was observed in patients with UAAC. The quantification of HA in the tear fluid is a rapid, sensitive and specific test. This molecule might be a biomarker candidate for acute conjunctivitis. A conjuntivite causada por adenovírus é uma das maiores causas de infecção da conjuntiva no mundo. A conjuntivite provoca sintomas relativamente inespecíficos, como hiperemia e quemose. Mesmo depois de biomicroscopia, testes laboratoriais complexos, como cultura viral, são necessários para identificar o patógeno ou sua etiologia. Para contribuir para o melhor entendimento da fisiopatologia da conjuntivite causada por adenovírus, lágrimas de pacientes com conjuntivite aguda unilateral causada por adenovírus (UAAC), de doadores normais (controle) e de pacientes com conjuntivite alérgica foram analisadas. As amostras foram coletadas com tiras de Schirmer de doadores normais, pacientes com conjuntivite alérgica e pacientes com UAAC diagnosticados por sinais clínicos e testes positivos em culturas virais. Após a eluição das lágrimas, o HA foi quantificado utilizando um ensaio fluorométrico semelhante ao ELISA e o perfil da proteína foi determinado por SDS-PAGE. Um aumento profundo no conteúdo de HA em lágrima de pacientes com UAAC foi encontrado quando comparado com o controle ou a conjuntivite alérgica. Este aumento de HA em lágrimas de pacientes com UAAC não foi observado em lágrimas do olho contralateral sem sinais clínicos ou de pacientes com conjuntivite alérgica. Além disso observou-se um perfil distinto de proteínas nas lágrimas de pacientes com UAAC. A quantificação de HA no fluido lacrimal é um ensaio rápido, sensível e específico. Esta molécula pode ser um bom candidato a biomarcador para conjuntivite aguda.

Brown spider (Genus Loxosceles) bites are normally associated with necrotic skin degeneration, gravitational spreading, massive inflammatory response at injured region, platelet aggregation causing thrombocytopenia and renal disturbances. Brown spider venom has a complex composition containing many different toxins, of which a well-studied component is the dermonecrotic toxin. This toxin alone may produce necrotic lesions, inflammatory response and platelet aggregation. Biochemically, dermonecrotic toxin belongs to a family of toxins with 30–35 kDa characterized as sphingomyelinase-D. Here, employing a cDNA library of Loxosceles intermedia venom gland, we cloned and expressed two recombinant isoforms of the dermonecrotic toxin LiRecDT2 (1062 bp cDNA) and LiRecDT3 (1007 bp cDNA) that encode for signal peptides and complete mature proteins. Phylogenetic tree analysis revealed a structural relationship for these toxins compared to other members of family. Recombinant molecules were expressed as N-terminal His-tag fusion proteins in Escherichia coli and were purified to homogeneity from cell lysates by Ni2+ chelating chromatography, resulting in proteins of 33.8 kDa for LiRecDT2 and 34.0 kDa for LiRecDT3. Additional evidence for related toxins containing sequence/epitopes identity comes from antigenic cross-reactivity using antibodies against crude venom toxins and antibodies raised with a purified dermonecrotic toxin. Recombinant toxins showed differential functionality in rabbits: LiRecDT2 caused a macroscopic lesion with gravitational spreading upon intradermal injection, while LiRecDT3 evoked transient swelling and erythema upon injection site. Light microscopic analysis of skin biopsies revealed edema, a collection of inflammatory cells in and around blood vessels and a proteinaceous network at the dermis. Moreover, differential functionality for recombinant toxins was also demonstrated by a high sphingomyelinase activity for LiRecDT2 and low activity for LiRecDT3 as well as greater in vitro platelet aggregation and blood vessel permeability induced by LiRecDT2 and residual activity for LiRecDT3. Cloning and expression of two recombinant dermonecrotic toxins demonstrate an intraspecific family of homologous toxins that act in synergism for deleterious activities of the venom and open possibilities for biotechnological applications for recombinant toxins as research tools for understanding the inflammatory response, vascular integrity and platelet aggregation modulators.

The prorenin receptor [(P)RR] is upregulated in the diabetic kidney and has been implicated in the high glucose (HG)-induced overproduction of profibrotic molecules by mesangial cells (MCs), which is mediated by ERK1/2 phosphorylation. The regulation of (P)RR gene transcription and the mechanisms by which HG increases (P)RR gene expression are not fully understood. Because intracellular levels of angiotensin II (AngII) are increased in MCs stimulated with HG, we used this in vitro system to evaluate the possible role of AngII in (P)RR gene expression and function by comparing the effects of AT1 receptor blockers (losartan or candesartan) and (P)RR mRNA silencing (siRNA) in human MCs (HMCs) stimulated with HG. HG induced an increase in (P)RR and fibronectin expression and in ERK1/2 phosphorylation. These effects were completely reversed by (P)RR siRNA and losartan but not by candesartan (an angiotensin receptor blocker that, in contrast to losartan, blocks AT1 receptor internalization). These results suggest that (P)RR gene activity may be controlled by intracellular AngII and that HG-induced ERK1/2 phosphorylation and fibronectin overproduction are primarily induced by (P)RR activation. This relationship between AngII and (P)RR may constitute an additional pathway of MC dysfunction in response to HG stimulation.

To evaluate the retinal penetration and toxicity of two doses of intravitreal infliximab in primates.Ten marmosets (Callithrix jacchus) were given intravitreal injection of 100 μg or 400 μg of infliximab, and balanced salt solution served as control. At baseline and after 24 hours (5 animals) and 7 days (the other 5), the eyes were examined by electroretinography. They were then killed (at 24 hours and 7 days) and assessed by light microscopy and transmission electron microscopy for toxicity and immunohistochemistry, using a biotinylated anti-human immunoglobulin G, to evaluate retinal penetration.There was no difference over 50% of the electroretinography b-wave between baseline and the time points studied in all animals. Light and electron microscopy, and electroretinography analysis, showed no signs of toxicity in any of the animals. Strong presence of infliximab was observed in all retinal layers 7 days after intravitreal injection at both doses (100 and 400 μg).Infliximab at doses of 100 and 400 μg seemed to cause no damage to the retina 24 hours and 7 days after its intravitreal injection, and deeply penetrated all its layers, in primates. These results encourage future perspectives for the treatment of chronic inflammatory diseases of the retina in humans.

Summary. Background: Choroidal neovascularization (CNV) is the main cause of severe visual loss in age-related macular degeneration (AMD). Heparin/heparan sulfate are known to play important roles in neovascularization due to their abilities to bind and modulate angiogenic growth factors and cytokines. Previously, we have isolated from marine shrimp a heparin-like compound with striking anti-inflammatory action and negligible anticoagulant and hemorrhagic activities. Objectives: To investigate the role of this novel heparin-like compound in angiogenic processes. Methods and Results: The anti-angiogenic effect of this heparinoid in laser-induced CNV and in vitro models is reported. The compound binds to growth factors (FGF-2, EGF and VEGF), blocks endothelial cell proliferation and shows no cytotoxic effect. The decrease in proliferation is not related to cell death either by apoptosis or secondary necrosis. The results also showed that the heparinoid modified the 2-D network organization in capillary-like structures of endothelial cells in Matrigel and reduced the CNV area. The effect on CNV area correlates with decreases in the levels of VEGF and TGF-β1 in the choroidal tissue. The low content of 2-O-sulfate groups in this heparinoid may explain its potent anti-angiogenic effect. Conclusions: The properties of the shrimp heparinoid, such as potent anti-angiogenic and anti-inflammatory activities but insignificant anticoagulant or hemorrhagic actions, point to this compound as a compelling drug candidate for treating neovascular AMD and other angioproliferative diseases. A mechanism for the anti-angiogenic effect of the heparinoid is proposed.

The continuing threat to biodiversity lends urgency to the need of identification of sustainable source of natural products. This is not so much trouble if there is a microbial source of the compound. Herein, violacein, a natural indolic pigment extracted from Chromobacterium violaceum, was evaluated for its antitumoral potential against the Ehrlich ascites tumor (EAT) in vivo and in vitro. Evaluation of violacein cytotoxicity using different endpoints indicated that EAT cells were twofold (IC(50)=5.0 microM) more sensitive to the compound than normal human peripheral blood lymphocytes. In vitro studies indicated that violacein cytotoxicity to EAT cells is mediated by a rapid (8-12h) production of reactive oxygen species (ROS) and a decrease in intracellular GSH levels, probably due to oxidative stress. Additionally, apoptosis was primarily induced, as demonstrated by an increase in Annexin-V positive cells, concurrently with increased levels of DNA fragmentation and increased caspase-2, caspase-9 and caspase-3 activities up to 4.5-, 6.0- and 5.5-fold, respectively, after 72 h of treatment. Moreover, doses of 0.1 and 1.0 microg kg(-1) violacein, administered intraperitoneally (i.p.) to EAT-bearing mice throughout the lifespan of the animals significantly inhibited tumor growth and increased survival of mice. In view of these results, a 35-day toxicity study was conducted in vivo. Complete hematology, biochemistry (ALT, AST and creatinine levels) and histopathological analysis of liver and kidney indicated that daily doses of violacein up to 1000 microg kg(-1) for 35 days are well tolerated and did not cause hematotoxicity nor renal or hepatotoxicity when administered i.p. to mice. Altogether, these results indicate that violacein causes oxidative stress and an imbalance in the antioxidant defense machinery of cells culminating in apoptotic cell death. Furthermore, this is the first report of its antitumor activity in vivo, which occurs in the absence of toxicity to major organs.

Purpose To determine effects of intravitreous anti-TNF-alpha (infliximab) in a laser-induced choroidal neovascularization (CNV) model by fluorescein angiogram (FA), immunofluorescence, ELISA, and glycosaminoglycan analyses. Methods CNV induction was performed using argon laser. Rats were divided into eight groups (no-laser no-infliximab; laser; laser with 10, 20, 40, 80, or 320 microg infliximab; and isotype-matched IgG). After 3 weeks, CNV area was measured by FA and von Willebrand factor (vWF) immunofluorescence. VEGF, TGF-beta, and syndecan-4 were evaluated by ELISA and immunofluorescence. Glycosaminoglycan expression was determined in retina and choroid of animals metabolically labeled with [(35)S]-sulfate. Cytotoxicity was investigated using ARPE-19 and endothelial cells. Results FA showed significant reduction in the low-dose infliximab groups (10-40 microg), confirmed by vWF immunofluorescence that showed 49% decrease in the CNV. VEGF and TGF-beta decreased expression detected by ELISA and immunofluorescence paralleled these results. Similar data were observed for syndecan-4. The expression of these molecules in the neovascularization area using 320 microg was similar to the no-infliximab laser group or laser with isotype-matched IgG. Heparan sulfate expression in retina and choroid paralleled the observed effects on angiogenesis. Increased expression of chondroitin sulfate in retina and dermatan sulfate in choroid reflects the effects of injury and fibrosis using high doses of anti-TNF-alpha. Infliximab showed no cytotoxic effect in ARPE-19 cells, whereas high doses led to 20% decrease in endothelial cell viability. Conclusions Intravitreal infliximab shows dual effect on the development of laser-induced CNV. It reduces angiogenesis and glycosaminoglycan expression at low doses, whereas opposite effects are observed at high doses.

Aims: Experimental retinal research has gained great importance due to the ophthalmic pharmacotherapy era. An increasing number of drugs are constantly released into the market for the treatment of retinal diseases. In this review, animal species, animal models and toxicity assays in retinal research are discussed. Methods: An extensive search of the literature was performed to review various aspects of the methods of investigation of drug toxicity. The different types of animal species, as well as single animal models available for the evaluation of safety and efficacy of retinal pharmacotherapy, were identified. In addition, a large variety of reported laboratory techniques were critically examined. Results: In vitro studies are the first-line experiments for the development of a new drug for retinal diseases, using retinal pigment epithelial cells and other cell lines. The next step involves in vivo animal studies where nonhuman primates are considered the gold standard. However, cost and legal issues make their use difficult. Mice and rats provide genetically controlled models for investigations. Pigs, dogs and cats represent good large-size animal models, while rabbits are one of the most used species for retinal toxicity evaluations. Various laboratory methods were identified, including light microscopy, electron microscopy, electroretinography and new emerging methods, such as optical coherence tomography and scanning laser ophthalmoscopy for experimental purposes. Conclusions: A great number of animal species and models are available that simulate retinal diseases and provide experimental data for further human use. Work with animal models should include properly designed toxicity assays to obtain reliable results for safety and efficacy.

PURPOSETo assess ultrastructural stromal modifications in porcine corneas after riboflavin and ultraviolet A (UVA) exposure using immunofluorescence confocal imaging. METHODSTwenty-five freshly enucleated porcine eyes were enrolled in the study. Five eyes served as control (group I). Twenty eyes had their epithelium removed (groups I, II, IV, and V) and five eyes had their epithelium intact (group III). Groups II and III were cross-linked with riboflavin 0.1% solution (10 mg riboflavin-5-phosphate in 10 mL 20% dextran-T-500) and exposed to UVA (365 nm, 3 mW/cm2) for 30 minutes. Group IV included five eyes soaked with riboflavin without posterior irradiation, and group V included five eyes irradiated, without previous exposure to riboflavin. Ultra-thin sections (8 µm) of the corneas were stained with anti-collagen I and DAPI and their fluorescence was revealed under confocal microscopy. RESULTSOnly the cross-linked corneas (group II) showed a pronounced, highly organized anterior fluorescence zone of 182.5±22.5 µm. Using DAPI staining, an anterior and concentrated displacement of cell nuclei due to collagen compaction was observed after cross-linking (group II). No structural changes were observed in all other groups. CONCLUSIONSThe cross-linking treatment effect can be directly visualized using confocal fluorescence imaging, allowing for a quantitative analysis. Cross-linked corneas showed a pronounced and limited anterior zone of organized collagen fibers, which was not observed in the other groups. Treatment of the cornea with riboflavin and UVA without previous deepithelialization did not induce any cross-linking effect. Consequently, to facilitate diffusion of riboflavin throughout the corneal stroma, the epithelium should be removed as an important initial step in the treatment. [J Refract Surg. 2008;24:S715-S719.] ABOUT THE AUTHORS From the Departments of Ophthalmology (Bottós, Regatieri, Lima-Filho, Schor, Chamon) and Biochemistry (Dreyfuss, Regatieri, Nader), Federal University of São Paulo, Brazil. The authors have no financial interest in the materials presented herein. Presented in part at the International Congress of Corneal Cross-Linking; December 7-8, 2007; Zurich, Switzerland. Correspondence: Wallace Chamon, MD, Av Indianopolis, 1797, São Paulo, SP 04063-003, Brazil. E-mail: visus@pobox.com

Purpose In this study, the antitumoral potential of a novel lectin (CvL) puriWed from the marine sponge Cliona varians was studied in diVerent cancer cell lines. Methods CvL cytotoxicity was evaluated in mammalian tumor cells and in normal human peripheral blood lymphocytes by the MTT assay using the same range of concentrations (1–150 _g ml¡1). The mechanisms involved in K562 cell death were investigated by confocal Xuorescence microscopy, Xow cytometry and immunoblot. Results CvL inhibited the growth of human leukemia cells, with IC50 values of 70 and 100 _gml¡1 for K562 and JURKAT cells, respectively, but it was ineVective on blood lymphocytes and solid tumor cell lines. K562 cell death occurred 72 h after exposure to the lectin and with signs of apoptosis, as analyzed by DAPI and annexin V/PI staining. Investigation of the possible mediators of this process showed that cell death occurred via a caspase-independent pathway. Confocal Xuorescence microscopy indicated a pivotal role for the lysosomal protease cathepsin B in mediating cell death. Accordingly, pre-incubation of K562 cells with the cathepsin inhibitor L-trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64) abolished CvL cytotoxic eVect. Furthermore, we found upregulation of tumor necrosis factor receptor 1 (TNFR1) and down-modulation of p65 subunit of nuclear factor kappa B (NF_B) expression in CvL-treated cells. These eVects were accompanied by increased levels of p21 and reduced expression of pRb, suggesting that CvL can induce cell cycle arrest. Conclusions Collectively, these Wndings indicate an antileukemic eVect for CvL and suggest that cathepsin B acts as a death mediator in CvL-induced cytotoxicity possibly in an uncharacterized connection with the membrane death receptor pathway.

Heparan sulfate proteoglycans are ubiquitously found at the cell surface and extracellular matrix in all the animal species. This review will focus on the structural characteristics of the heparan sulfate proteoglycans related to protein interactions leading to cell signaling. The heparan sulfate chains due to their vast structural diversity are able to bind and interact with a wide variety of proteins, such as growth factors, chemokines, morphogens, extracellular matrix components, enzymes, among others. There is a specificity directing the interactions of heparan sulfates and target proteins, regarding both the fine structure of the polysaccharide chain as well precise protein motifs. Heparan sulfates play a role in cellular signaling either as receptor or co-receptor for different ligands, and the activation of downstream pathways is related to phosphorylation of different cytosolic proteins either directly or involving cytoskeleton interactions leading to gene regulation. The role of the heparan sulfate proteoglycans in cellular signaling and endocytic uptake pathways is also discussed.Proteoglicanos de heparam sulfato são encontrados tanto superfície celular quanto na matriz extracelular em todas as espécies animais. Esta revisão tem enfoque nas características estruturais dos proteoglicanos de heparam sulfato e nas interações destes proteoglicanos com proteínas que levam à sinalização celular. As cadeias de heparam sulfato, devido a sua variedade estrutural, são capazes de se ligar e interagir com ampla gama de proteínas, como fatores de crescimento, quimiocinas, morfógenos, componentes da matriz extracelular, enzimas, entreoutros. Existe uma especificidade estrutural que direciona as interações dos heparam sulfatos e proteínas alvo. Esta especificidade está relacionada com a estrutura da cadeia do polissacarídeo e os motivos conservados da cadeia polipeptídica das proteínas envolvidas nesta interação. Os heparam sulfatos possuem papel na sinalização celular como receptores ou coreceptores para diferentes ligantes. Esta ligação dispara vias de sinalização celular levam à fosforilação de diversas proteínas citosólicas ou com ou sem interações diretas com o citoesqueleto, culminando na regulação gênica. O papel dos proteoglicanos de heparam sulfato na sinalização celular e vias de captação endocítica também são discutidas nesta revisão.

The venom of the brown spider is remarkable because it causes dermonecrotic injury, hemorrhagic problems, hemolysis, platelet aggregation and renal failure. The mechanism by which the venom causes hemorrhagic disorders is poorly understood. Rabbits intradermally exposed to the venom showed a local hemorrhage starting 1 h after inoculation and reaching maximum activity between 2 and 3 days. Biopsies examined by light and transmission electron microscopy showed subendothelial blebs, vacuoles and endothelial cell membrane degeneration in blood vessels, plasma exudation into connective tissue, and fibrin and thrombus formation within blood vessels. Loxosceles intermedia venom incubated with fibrinogen partially degrades Aalpha and Bbeta chains of intact fibrinogen, and significantly cleaves all Aalpha, Bbeta and gamma chains when they were separated or when fibrinogen is denatured by boiling. Proteolytic kinetic studies showed that the Aalpha chain is more susceptible to venom hydrolysis than the Bbeta chain. The fibrinogenolysis is blocked by ethylenediamine tetraacetic acid and 1,10-phenanthroline, but not by other protease inhibitors. Human plasma incubated with the venom had coagulation parameters such as prothrombin time, activated partial thromboplastin time and thrombin time increased. Through molecular sieve chromatography, we isolated a venom toxin of 30 kDa with fibrinogenolytic activity. We propose that the local and systemic hemorrhagic disorders evoked in loxoscelism are consequences of direct venom fibrinogenolysis together with cytotoxicity to subendothelial structures and endothelial cells in blood vessels.

High molecular weight serine-proteases have been identified in Loxosceles intermedia (brown spider) venom. The mechanism by which Loxosceles spp venoms cause dermonecrotic injury (a hallmark of loxoscelism) is currently under investigation, but it seems to be molecularly complex and in some instance proteases might be expected to play a role in this skin lesion. In the present investigation, when we submitted L. intermedia venom to linear gradient 3-20% SDS-PAGE stained by a monochromatic silver method we detected a heterogeneous protein profile in molecular weight, ranging from 850- to 5-kDa. In an attempt to detect zymogen molecules of proteolytic enzymes, venom aliquots were treated with several exogenous proteases. Among them, trypsin activated two gelatinolytic molecules of 85- and 95-kDa in the venom. In experiments of hydrolysis inactivation using different protease inhibitors for four major class of proteases, we detected that only serine-type protease inhibitors were able to inactivate the 85- and 95-kDa enzymes in the venom. An examination of the 85- and 95-kDa gelatinolytic activities as a function of pH showed that these proteases had no apparent activities at pH below 5.0 and higher than 9.0 and displayed little activity at pH 6.0. with the optimal pH for their activities ranging from 7.0 to 8.0. Evaluation of the functional specificities of the 85- and 95-kDa venom proteases showed that these proteases efficiently degrade gelatin (denatured collagen) but have no proteolytic activity on hemoglobin, immunoglobulin, albumin, librinogen or laminin, suggesting specificity of their proteolytic actions. We describe here two serine-proteases activities in L. intermedia venom probably involved in the harmful effects of the venom.

One of the goals of the graduate Program in Molecular Biology from UNIFESP (PrMB -UNIFESP) is to contribute for continuing education of biology teachers from public high schools. A close relation between university and public schools is an important channel for dissemination of scientific knowledge. Thus, a 40h Molecular Biology updating course was offered to 20 high school teachers. The objective was to discuss genomic and proteomic advances and their application. The course was organized by graduate students from PrMB -UNIFESP. Three groups ofstudents were formed, two being responsible for theorical and practical classes and one for global logistic including searching for financial support. The themes presented to the teachers were flow of genetic information, recombinant DNA, gene cloning, transgenic plants and animals, mutation, super bacteria and stem cell. The teachers also had hands-on classes including DNA extraction, PCR, gene cloning and SDS-PAGE. The teachers received an assignment to go back to their s chools and do some activity with their students that would be related to the themes discussed. The students produced videos, discussions, posters, theater, experimental models and pratical classes related to the course themes. After 3 months the teachers r eturned to show their students’ work. We conclude that information was transmitted to the teachers, updating them, and to high school students, that learned science in a entertaining way. Also, the graduate students had an experience on how to organize a c ourse including all its responsibilities.

The Autoimmune Hemolytic Anemia (AIHA) is caused by the destruction of antibody-coated red blood cells, but mechanisms that initiate the production of autoantibodies remains unclear. It had been suggest that decreased production of Th1-type cytokines and production of autoantibodies in AIHA can be secondary to the imbalance between anti- and pro-inflammatory factors. Moreover, the presence of single nucleotide polymorphisms (SNPs) showed association with different production of immunoregulatory factors which may modulate the disease expression in AIHA. OBJECTIVES: To determine the importance of SNPs of pro- and anti-inflammatory factors in the development of AIHA. PATIENTS: We studied 17 patients with AIHA who has been followed in the Hematology and Blood Transfusion Unit at the Federal University of Sao Paulo (UNIFESP/EPM), Brazil. The control group was composed by 40 healthy volunteer blood donors. METHODS: After DNA extraction from peripheral blood samples, the frequency of the SNPs was determinate by PCR-RFLP in patients and healthy individuals. The following SNPs were analyzed: Interleukin 12: IL-12 1188 (A/C), Tumor Necrosis Factor alpha: TNFa-308 (G/A), and Lymphotoxin alpha: Lta +252 (A/G); Interleukin 10: IL-10-592 (C/A), and Cytotoxic T-lymphocyte associated protein 4: CTLA4 exon 1 49 (A/G). RESULTS: The patient group was composed predominantly by female individuals (14 or 82%) and the median of age was 56 years old (18 to 76 years). The frequency observed for each allele studied in the patient group was: allele A of IL-12 = 0.82; allele G of FNTa = 0.85; allele A of Lta = 0.68; allele C of IL-10 = 0.82; allele G of CTLA4 = 0.59. No differences in allele frequency were found between patient and control groups. CONCLUSIONS: Our results suggest that these polymorphisms appear not to contribute for the development of the AIHA.

Background All hematopoietic cells express P2 receptors, however pharmacological characteristics such as expression and affinity in granulocytes are unknown. Methods Pharmacological characteristics of P2 receptors were evaluated by Ca2+ measurements using Fura-2 fluorophore. P2 receptors expression were analyzed by flow cytometry and RT-PCR. P2 interaction were shown by coimmunoprecipitation, western blotting and FRET. Results Granulocytes were responsive to P2Y agonists, whereas P2X agonists were ineffective. Ca2+ increase, elicited by ADP and UTP was dependent on intracellular stocks and sensitive to G-coupled receptor inhibition. Moreover, MRS2179, a specific antagonist of the P2Y1 receptor, abolished ADP response. Interestingly, ADP and UTP exhibited full heterologous desensitization, suggesting that these agonists interact with the same receptor. The heteromeric association between P2Y1 receptor and the P2Y2 and P2Y4 receptors was shown by immunoprecipitation and FRET analysis. Conclusion Clear evidence of heteromeric association of P2Y receptors was found during the evaluation of P2 receptors present in mice granulocytes, which could impact in the classical pharmacology of P2Y receptors in granulocytes.