Suggestive linkage of the parathyroid receptor type 1 to osteoporosis

We have investigated the role of 23 candidate genes in the control of bone mineral density (BMD) by linkage studies in families of probands with osteoporosis (lumbar spine [LS] or femoral neck [FN] BMD T score < -2.5) and low BMD relative to an age- and gender-matched cohort (Z score < -2.0). One hundred and fifteen probands (35 male, 80 female) and 499 of their first- or second-degree relatives (223 males and 276 females) were recruited for the study. BMD was measured at the LS and FN using dual-energy X-ray absorptiometry and expressed as age- and gender-matched Z scores corrected for body mass index. The candidate genes studied were the androgen receptor, type I collagen A1 (COLIA1), COLIA2, COLIIA1, vitamin D receptor (VDR), colony-stimulating factor 1, calcium-sensing receptor, epidermal growth factor (EGF), estrogen receptor 1 (ESR1), fibrillin type 1, insulin-like growth factor 1, interleukin-1 alpha (IL-1α), interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-11 (IL-11), osteopontin, parathyroid hormone (PTH), PTH-related peptide, PTH receptor type 1 (PTHR1), transforming growth factor-beta 1, and tumor necrosis factors alpha and beta. Sixty-four microsatellites lying close to or within these genes were investigated for linkage with BMD. Using the program MapMaker/Sibs there was suggestive evidence of linkage between BMD and PTHR1 (maximum LOD score obtained [MLS] 2.7-3.5). Moderate evidence of linkage was also observed with EGF (MLS 1.8), COLIA1 (MLS 1.7), COLIIA1/VDR (MLS 1.7), ESR1 (MLS 1.4), IL-1α (MLS 1.4), IL-4 (MLS 1.2), and IL-6 (MLS 1.2). Variance components analysis using the program ACT, correcting for proband-wise ascertainment, also showed evidence of linkage (p ≤0.05) at markers close to or within the candidate genes IL- 1α, PTHR1, IL-6, and COLIIA1/VDR. Further studies will be required to confirm these findings, to refine the location of gene responsible for the observed linkage, and to screen the candidate genes targeted at these loci for mutations.

Competitive adsorption between bovine serum albumin and collagen observed by atomic force microscope

Atomic force microscopy (AFM) was used to study the competitive adsorption between bovine serum albumin (BSA) and type I collagen on hydrophilic and hydrophobic silicon wafers. BSA showed a grain shape and the type I collagen displayed fibril-like molecules with relatively homogeneous height and width, characterized with clear twisting (helical formation). These AFM images illustrated that quite a lot of type I collagen appeared in the adsorption layer on hydrophilic surface in a competitive adsorption state, but the adsorption of BSA was more preponderant than that of type I collagen on hydrophobic silicon wafer surface. The experiments showed that the influence of BSA on type I collagen adsorption on hydrophilic surface was less than that on hydrophobic surface.

Efeito do Sistema de Secreção do Tipo VI de Pseudomonas aeruginosa em modelo murino de pneumonia aguda

O Sistema de Secreção do Tipo VI (SST6), o mais recente maquinário de secreção descrito em bactérias Gram-negativas, é amplamente distribuído entre as diversas espécies deste grupo de microrganismos. Esse aparato de secreção é capaz de injetar efetores proteicos em células alvo, eucarióticas e procarióticas. Estudos sobre o papel do SST6 na virulência microbiana revelaram que este sistema secretório participa ativamente do estabelecimento de infecções, contribuindo para a sobrevivência das bactérias no interior de fagócitos. O genoma da cepa PAO1 de Pseudomonas aeruginosa apresenta três loci que codificam aparatos de SST6, denominados de H1-SST6, H2-SST6 e H3-SST6, Porém, pouco se sabe sobre a participação do SST6 na patogênese de infecções por P. aeruginosa. Assim, o presente estudo investigou o papel de H1-SST6, H2-SST6 e H3-SST6 durante a infecção pulmonar aguda de camundongos. Para isso, camundongos C57/BL6 foram infectados com diferentes doses de bactérias da cepa selvagem PAO1 ou das cepas mutantes PAO1∆H1, PAO1∆H2, PAO1∆H3 ou PAO1∆H1∆H2∆H3. Após 24 horas, os lavados broncoalveolares (LBAs) de animais controle e infectados foram recuperados para a contagem de leucócitos totais e polimorfonucleares e para a quantificação, por ELISA, da quimiocina para neutrófilos, KC, e das citocinas pró-inflamatórias IL-1β e TNF-α. Em outros experimentos, os pulmões, fígados, baços e rins dos animais foram macerados para a pesquisa da carga bacteriana e da disseminação sistêmica das bactérias. A citotoxicidade do SST6 foi determinada, in vitro, em neutrófilos humanos, pela marcação com iodeto de propídeo (PI) e anexina-V seguida da análise em citometria de fluxo. Os resultados mostraram que a inativação dos três SST6 reduziu significativamente a concentração de neutrófilos nos LBAs quando os animais foram infectados com 10⁷ Unidades Formadoras de Colônias de P. aeruginosa. Nesta dose, foi observado que as medianas do número de bactérias detectadas nos animais infectados com as mutantes no SST6 foram menores do que as detectadas nos animais infectados com a cepa parental PAO1. As mutações no SST6 não afetaram a disseminação sistêmica da bactéria. A pesquisa da secreção de citocinas pró-inflamatórias mostrou que, embora tenha sido observada uma redução nas medianas das concentrações de TNF-α nos LBAs de camundongos infectados com a cepa PAO1∆H1∆H2∆H3, em relação aos LBAs de camundongos infectados com a cepa parental, essa diferença não foi significativa. Como a pesquisa de IL-1β e KC não contribuiu para a elucidação dos mecanismos envolvidos na redução da concentração de neutrófilos nos LBAs dos camundongos infectados pela cepa tripla mutante, foi pesquisado o possível efeito do SST6 na morte de neutrófilos humanos. Os resultados mostraram que não houve diferenças significativas quando as diferentes amostras de células infectadas foram comparedas entre si. Em conclusão, os resultados do presente estudo mostraram que o SST6 pode interferir na resposta de neutrófilos durante a pneumonia aguda, mas estudos adicionais são necessários para determinar o papel deste mecanismo de secreção na patogênese de P. aeruginosa.

Development of a bovine collagen-apatitic calcium phosphate cement for potential fracture treatment through vertebroplasty

The aim of this study was to examine the potential of incorporating bovine fibres as a means of reinforcing a typically brittle apatite calcium phosphate cement for vertebroplasty. Type I collagen derived from bovine Achilles tendon was ground cryogenically to produce an average fibre length of 0.96 ± 0.55 mm and manually mixed into the powder phase of an apatite-based cement at 1, 3 or 5 wt.%. Fibre addition of up to 5 wt.% had a significant effect (P = 0.001) on the fracture toughness, which was increased by 172%. Adding =1 wt.% bovine collagen fibres did not compromise the compressive properties significantly, however, a decrease of 39-53% was demonstrated at =3 wt.% fibre loading. Adding bovine collagen to the calcium phosphate cement reduced the initial and final setting times to satisfy the clinical requirements stated for vertebroplasty. The cement viscosity increased in a linear manner (R = 0.975) with increased loading of collagen fibres, such that the injectability was found to be reduced by 83% at 5 wt.% collagen loading. This study suggests for the first time the potential application of a collagen-reinforced calcium phosphate cement as a viable option in the treatment of vertebral fractures, however, issues surrounding efficacious cement delivery need to be addressed. © 2012 Acta Materialia Inc.

Synovial membrane protein expression differs between juvenile idiopathic arthritis subtypes in early disease

Introduction: Juvenile idiopathic arthritis (JIA) is the most common rheumatological disease of childhood with a prevalence of around 1 in 1000. Without appropriate treatment it can have devastating consequences including permanent disability from joint destruction and growth deformities. Disease aetiology remains unknown. Investigation of disease pathology at the level of the synovial membrane is required if we want to begin to understand the disease at the molecular and biochemical level. The synovial membrane proteome from early disease-stage, treatment naive JIA patients was compared between polyarticular and oligoarticular subgroups.

Methods: Protein was extracted from 15 newly diagnosed, treatment naive JIA synovial membrane biopsies and separated by two dimensional fluorescent difference in-gel electrophoresis. Proteins displaying a two-fold or greater change in expression levels between the two subgroups were identified by matrix assisted laser desorption ionization-time of flight mass spectrometry with expression further verified by Western blotting and immunohistochemistry.

Results: Analysis of variance analysis (P <= 0.05) revealed 25 protein spots with a two-fold or greater difference in expression levels between polyarticular and oligoarticular patients. Hierarchical cluster analysis with Pearson ranked correlation revealed two distinctive clusters of proteins. Some of the proteins that were differentially expressed included: integrin alpha 2b (P = 0.04); fibrinogen D fragment (P =0.005); collagen type VI (P = 0.03); fibrinogen gamma chain (P = 0.05) and peroxiredoxin 2 (P = 0.02). The identified proteins are involved in a number of different processes including platelet activation and the coagulation system.

Conclusions: The data indicates distinct synovial membrane proteome profiles between JIA subgroups at an early stage in the disease process. The identified proteins also provide insight into differentially perturbed pathways which could influence pathological events at the joint level.

A biocomposite of collagen nanofibers and nanohydroxyapatite for bone regeneration

This work aims to design a synthetic construct that mimics the natural bone extracellular matrix through innovative approaches based on simultaneous type I collagen electrospinning and nanophased hydroxyapatite (nanoHA) electrospraying using non-denaturating conditions and non-toxic reagents. The morphological results, assessed using scanning electron microscopy and atomic force microscopy (AFM), showed a mesh of collagen nanofibers embedded with crystals of HA with fiber diameters within the nanometer range (30 nm), thus significantly lower than those reported in the literature, over 200 nm. The mechanical properties, assessed by nanoindentation using AFM, exhibited elastic moduli between 0.3 and 2 GPa. Fourier transformed infrared spectrometry confirmed the collagenous integrity as well as the presence of nanoHA in the composite. The network architecture allows cell access to both collagen nanofibers and HA crystals as in the natural bone environment. The inclusion of nanoHA agglomerates by electrospraying in type I collagen nanofibers improved the adhesion and metabolic activity of MC3T3-E1 osteoblasts. This new nanostructured collagen–nanoHA composite holds great potential for healing bone defects or as a functional membrane for guided bone tissue regeneration and in treating bone diseases.

The role of Microsomal prostaglandin synthase-1 (mPGES-1) and Ephrin B2 in Scleroderma

La sclérodermie (sclérose systémique, ScS) est une maladie auto-immune du tissu conjonctif caractérisée  par  l’épaississement  de  la  peau,  l’apparition  spontanée de lésions cicatricielles, des maladies   des   vaisseaux   sanguins,   divers   degrés   d’inflammation,   en   association   avec   un   système   immunitaire hyperactif. La pathogénèse exacte de cette maladie est inconnue et aucun traitement approprié   n’est   disponible.   La fibrose est un élément distinctif de la maladie de ScS et est considérée   résulter   d’une   incapacité   à   mettre   fin   de   façon   appropriée   à   la   réponse   normale   de   réparation   des   plaies.   L’analyse   histologique   du   stade   initial   de   la   ScS   révèle   une   infiltration   périvasculaire de cellules mononucléaires dans le derme, associée à une synthèse accrue de collagène dans les fibroblastes environnants. Ainsi, la compréhension des moyens de contrôler le stade inflammatoire de la ScS pourrait être bénéfique pour contrôler la progression de la maladie peu après son apparition. La mPGES-1 est une enzyme inductible qui agit en aval de la cyclo- oxygénase (COX) pour catalyser spécifiquement la conversion de la prostaglandine (PG) H2 en PGE2. La mPGES-1  joue  un  rôle  clé  dans  l’inflammation,  la  douleur  et  l’arthrite;;  toutefois,  le   rôle de la mPGES-1 dans les mécanismes de fibrose, spécifiquement en rapport avec la ScS humaine, est inconnu. Mon laboratoire a précédemment montré que les souris à mPGES-1 nulle sont résistantes à la fibrose   cutanée   induite   par   la   bléomycine,   à   l’inflammation,   à   l’épaississement  cutané,  à  la  production  de  collagène  et  à  la  formation  de  myofibroblastes.  Sur  la   base  de  ces  résultats,  j’ai  formulé  l’hypothèse  que  l’inhibition pharmacologique de la mPGES-1 régulera à la baisse la production de médiateurs pro-inflammatoires et pro-fibreux au cours de la maladie   de   ScS.   Afin   d’explorer   le   rôle   de   la   mPGES-1   dans   l’inflammation   et   la   fibrose   associées  à  la  maladie  de  ScS,  j’ai  d’abord  examiné  l’expression  de  la  mPGES-1 dans la peau normale comparativement à des biopsies de peau extraites de patients atteints de ScS. Mes résultats ont montré que la mPGES-1 est nettement élevée dans la peau de patients atteints de ScS en comparaison avec la peau humaine normale. De plus, les niveaux de PGE2 dérivés de la mPGES-1 étaient également significativement plus élevés dans les fibroblastes cutanés isolés de patients  atteints  de  ScS  comparativement  aux  fibroblastes  isolés  de  témoins  sains.  J’ai  également   étudié  l’effet  de  l’inhibition pharmacologique de la mPGES-1  sur  l’expression  de  marqueurs  pro- fibreux.   Mes   études   ont   montré   que   l’expression   de   médiateurs   pro-fibreux clés (α-SMA, endothéline-1, collagène de type 1 et facteur de croissance du tissu conjonctif (FCTC)) est élevée dans les fibroblastes cutanés ScS en comparaison avec les fibroblastes cutanés normaux. Un traitement avec un inhibiteur de la mPGES-1 a eu pour effet de réduire significativement l’expression  de  l’α-SMA,  de  l’endothéline-1, du collagène de type 1 mais pas du FCTC dans les fibroblastes  ScS,  sans  effet  significatif  sur  les  fibroblastes  normaux.  J’ai  en  outre  examiné  l’effet   de  l’inhibition  de  la  mPGES-1 sur des cytokines pro-inflammatoires clés impliquées dans la pathologie de la ScS, incluant IL-6, IL-8 et MCP-1.  L’inhibition  pharmacologique  de  la  mPGES- 1 a eu pour effet de réduire significativement les niveaux de production de cytokines pro- inflammatoires IL6, IL8 et MCP-1 dans les fibroblastes avec lésion ScS comparativement à des fibroblastes non traités. De plus, les patients atteints de ScS ont présenté des niveaux plus élevés de p-AKT, de p-FAK et de p-SMAD3 en comparaison avec les fibroblastes cutanés normaux. L’inhibiteur  de  la  mPGES-1 a pu réguler à la baisse cette expression accrue de p-AKT et de p- FAK, mais pas de p-SMAD3,  dans  les  fibroblastes  ScS.  Ces  résultats  ont  suggéré  que  l’inhibition   de la mPGES-1 pourrait être une méthode viable pour réduire le développement de sclérose cutanée et constituent une cible thérapeutique potentielle pour contrôler les mécanismes fibreux et inflammatoires associés à la pathophysiologie de la maladie de ScS. L’un   des   autres   processus   critiques   reliés   à   l’évolution de la réponse fibreuse associée à la maladie de ScS est la différenciation des fibroblastes en des cellules activées spécialisées iii iv appelées myofibroblastes, responsables de déclencher une signalisation adhésive excessive et le dépôt excessif de matrice extracellulaire,   conduisant   à   la   destruction   de   l’architecture   de   l’organe.   Ainsi,   l’identification   des   facteurs   endogènes   qui   initient/   favorisent   la   différenciation   fibroblaste-myofibroblaste peut mener à des stratégies thérapeutiques prometteuses pour contrôler  l’excès  de  signalisation  adhésive  et  de  fibrose  associé  à  la  maladie  de  ScS.  Des  études   antérieures  dans  le  domaine  de  la  biologie  du  cancer  ont  suggéré  que  l’éphrine  B2,  une  protéine   transmembranaire appartenant à la famille des éphrines, est impliquée dans la signalisation adhésive   et   le   remodelage   extracellulaire.   Cependant,   son   rôle   dans   la   fibrose   n’a   jamais   été   exploré.   Dans   la   deuxième   partie   de   mon   étude,   j’ai   donc   étudié   le   rôle   de   l’éphrine   B2   dans   la   fibrose.   Mes   études   montrent   que   l’expression   de   l’éphrine   B2   est   significativement   augmentée   dans la peau humaine ScS comparativement à la peau normale. Plus important encore, le traitement in vitro de   fibroblastes   de   la   peau   humaine   normale   avec   de   l’éphrine   B2   recombinante est capable de transformer des fibroblastes en cellules myofibroblastiques manifestant toutes les caractéristiques myofibroblastiques typiques, incluant la formation accrue de  fibres  de  tension,  des  adhérences  focales,  l’activation  accrue  de  la  FAK,  un  accroissement  de   l’expression  et  de  la  migration  de  fibroblastes  et  de  leur  adhérence  à  la  fibronectine  à  la  fois  chez   les   fibroblastes   cutanés   normaux   et   ScS.   En   outre,   j’ai   traité   des   souris   avec   de   l’éphrine   B2   recombinante et montré que ces souris ont développé une fibrose cutanée significative associée à une épaisseur dermique et à une synthèse de collagène augmentées, une teneur en hydroxyproline (teneur en collagène) accrue et un nombre accru de myofibroblastes exprimant de   l’α-SMA, une activation augmentée de la FAK et de marqueurs pro-fibreux incluant le collagène de type 1 et le FCTC. Dans  l’ensemble,  mes  études  ont  identifié  deux  médiateurs  endogènes  cruciaux  impliqués  dans  la   propagation  de  l’inflammation  et  de  la  fibrose  associées  à  la  maladie  de  ScS.  L’inhibition  de  la   mPGES-1   pourrait   représenter   une   bonne   stratégie   alternative   pour   contrer   l’inflammation   et   la   fibrose au moins durant les stades précoces de la maladie de ScS. De plus, une signalisation excessive   de   l’éphrine B2 favorise la signalisation adhésive et fibreuse en déclenchant la différenciation   de   fibroblastes   en   myofibroblastes   par   l’activation   de   la   voie   de   signalisation   de   la  FAK.  Ainsi,  l’inhibition  d’éphrine  B2  bloquera  la  formation  de  fibroblastes-myofibroblastes et régulera à la baisse la fibrose associée à la maladie de ScS. En somme, la mPGES-1  et  l’éphrine   B2 semblent toutes deux des cibles attrayantes pour le traitement de la ScS et des troubles fibreux qui y sont reliés.

Identification et caractérisation des principaux fragments du collagène de type II du cartilage équin, produit in vitro par l'enzyme cathepsine K

La dégradation protéolytique du collagène de type II est considérée comme étant un facteur majeur dans le processus irréversible de dégradation de la matrice cartilagineuse lors d’ostéoarthrose. Outre les collagénases de la famille des métaloprotéinases de la matrice (MMP-1, -8, -13), la cathepsine K est parmi les seules enzymes susceptibles de dégrader la triple hélice intacte du collagène de type II, devenant ainsi un élément pertinent pour les recherches sur l’ostéoarthrose. L’objectif à court terme de notre étude consiste en l’identification et la caractérisation de sites de clivage spécifiques de la cathepsine K sur le collagène de type II équin. La technique d’électrophorèse SDS-PAGE 1D permet la visualisation des produits de digestion et la validation des résultats de la caractérisation moléculaire des fragments protéolytiques. La caractérisation est réalisée en combinant la digestion trypsique précédant l’analyse HPLC-ESI/MS. Les résultats ont permis d’établir les sites, présents sur la carte peptidique de la molécule de collagène de type II équin, des 48 résidus prolines (P) et 5 résidus lysines (K) supportant une modification post-traductionnelle. De plus, 6 fragments majeurs, différents de ceux produits par les MMPs, sont observés par SDS-PAGE 1D puis confirmés par HPLC-ESI/MS, correspondant aux sites suivants : F1 [G189-K190], F2 [G252-P253], F3 [P326-G327], F4 [P428-G429], F5 [P563-G564] et F6 [P824-G825]. Le fragment F1 nouvellement identifié suggère un site de clivage différent de l’étude antérieure sur le collagène de type II bovin et humain. L’objectif à long terme serait le développement d’anticorps spécifiques au site identifié, permettant de suivre l’activité protéolytique de la cathepsine K par immunohistochimie et ÉLISA, dans le cadre du diagnostic de l’ostéoarthrose.

Ingestion of quercetin inhibits platelet aggregation and essential components of the collagen-stimulated platelet activation pathway in humans

Background: Quercetin, a flavonoid present in the human diet, which is found in high levels in onions, apples, tea and wine, has been shown previously to inhibit platelet aggregation and signaling in vitro. Consequently, it has been proposed that quercetin may contribute to the protective effects against cardiovascular disease of a diet rich in fruit and vegetables. Objectives: A pilot human dietary intervention study was designed to investigate the relationship between the ingestion of dietary quercetin and platelet function. Methods: Human subjects ingested either 150 mg or 300 mg quercetin-4'-O-beta-D-glucoside Supplement to determine the systemic availability of quercetin. Platelets were isolated from subjects to analyse collagen-stimulated cell signaling and aggregation. Results: Plasma quercetin concentrations peaked at 4.66 mum (+/-0.77) and 9.72mum (+/-1.38) 30min after ingestion of 150-mg and 300-mg doses of quercefin-4'-O-beta-D-glucoside, respectively, demonstrating that quercetin was bioavailable, with plasma concentrations attained in the range known to affect platelet function in vitro. Platelet aggregation was inhibited 30 and 120 min after ingestion of both doses of quercetin-4'-O-beta-D-glucoside. Correspondingly, collagen-stimulated tyrosine phosphorylation of total platelet proteins was inhibited. This was accorripanied by reduced tyrosine phosphorylation of the tyrosine kinase Syk and phospholipase Cgamma2, components of the platelet glycoprotein VI collagen receptor signaling pathway. Conclusions: This study provides new evidence of the relatively high systemic availability of quercetin in the form of quercetin-4'-O-beta-D-glucoside by supplementation, and implicates quercetin as a dietary inhibitor of platelet cell signaling and thrombus formation.

Analysis of protein networks in resting and collagen receptor (GPVI)-stimulated platelet sub-proteomes.

Proteomics approaches have made important contributions to the characterisation of platelet regulatory mechanisms. A common problem encountered with this method, however, is the masking of low-abundance (e.g. signalling) proteins in complex mixtures by highly abundant proteins. In this study, subcellular fractionation of washed human platelets either inactivated or stimulated with the glycoprotein (GP) VI collagen receptor agonist, collagen-related peptide, reduced the complexity of the platelet proteome. The majority of proteins identified by tandem mass spectrometry are involved in signalling. The effect of GPVI stimulation on levels of specific proteins in subcellular compartments was compared and analysed using in silico quantification, and protein associations were predicted using STRING (the search tool for recurring instances of neighbouring genes/proteins). Interestingly, we observed that some proteins that were previously unidentified in platelets including teneurin-1 and Van Gogh-like protein 1, translocated to the membrane upon GPVI stimulation. Newly identified proteins may be involved in GPVI signalling nodes of importance for haemostasis and thrombosis.

Rhinocetin, a venom-derived integrin-specific antagonist inhibits collagen-induced platelet and endothelial cell functions

Snaclecs are small non-enzymatic proteins present in viper venoms reported to modulate haemostasis of victims through effects on platelets, vascular endothelial and smooth muscle cells. In this study, we have isolated and functionally characterised a snaclec which we named rhinocetin from the venom of West African gaboon viper, Bitis gabonica rhinoceros. Rhinocetin was shown to comprise α and β chains with the molecular masses of 13.5 and 13kDa respectively. Sequence and immunoblot analysis of rhinocetin confirmed this to be a novel snaclec. Rhinocetin inhibited collagen-stimulated activation of human platelets in dose dependent manner, but displayed no inhibitory effects on glycoprotein VI (collagen receptor) selective agonist, CRP-XL-, ADP- or thrombin-induced platelet activation. Rhinocetin antagonised the binding of monoclonal antibodies against the α2 subunit of integrin α2β1 to platelets and coimmunoprecipitation analysis confirmed integrin α2β1 as a target for this venom protein. Rhinocetin inhibited a range of collagen induced platelet functions such as fibrinogen binding, calcium mobilisation, granule secretion, aggregation and thrombus formation. It also inhibited integrin α2β1 dependent functions of human endothelial cells. Together, our data suggest rhinocetin to be a modulator of integrin α2β1 function and thus may provide valuable insights into the role of this integrin in physiological and pathophysiological scenarios including haemostasis, thrombosis and envenomation.

Basic aminopeptidase activity is an emerging biomarker in collagen-induced rheumatoid arthritis

The objective of this study was to investigate the catalytic activity of basic aminopeptidase (APB) and its association with periarticular edema and circulating tumor necrosis factor (TNF)-alpha and type II collagen (CII) antibodies (AACII) in a rat model of rheumatoid arthritis (RA) induced by CII (CIA). Edema does not occur in part of CH-treated, even when AACII is higher than in control. TNF-alpha is detectable only in edematous CII-treated. APB in synovial membrane is predominantly a membrane-bound activity also present in soluble form and with higher activity in edematous than in non-edematous CH-treated or control. Synovial fluid and blood plasma have lower APB in non-edematous than in edematous CII-treated or control. In peripheral blood mononuclear cells (PBMCs) the highest levels of APB are found in soluble form in control and in membrane-bound form in non-edematous CII-treated. CII treatment distinguishes two categories of rats: one with arthritic edema, high AACII, detectable TNF-alpha, high soluble and membrane-bound APB in synovial membrane and low APB in the soluble fraction of PBMCs, and another without edema and with high AACII, undetectable TNF-alpha, low APB in the synovial fluid and blood plasma and high APB in the membrane-bound fraction of PBMCs. Data suggest that APB and CIA are strongly related. (C) 2011 Elsevier B.V. All rights reserved.

THERMAL BEHAVIOR OF IN VITRO MINERALIZED ANIONIC COLLAGEN MATRICES

In the present study porcine skin and bovine pericardium were used as a source of type I collagen. Both were submitted to an alkaline treatment and mineralized by the alternate soaking method. Thermal stability and extent of mineralization have been investigated using DSC and TG. After alkaline hydrolysis there is a decrease in thermal stability but mineralization stabilizes collagen structure. Thermogravimetric data have shown that the amount of hydroxyapatite present in bovine pericardium matrix (45%) was greater than on porcine skin matrix (20%). Presence of hydroxyapatite was confirmed by EDX.

Estudo clínico e bioquímico de 28 pacientes com mucopolissacaridose tipo VI

Realizamos um estudo observacional de pacientes com mucopolissacaridose tipo VI, com o objetivo de determinar o perfil epidemiológico, clínico e bioquímico de um grupo de pacientes sul-americanos a fim de contribuir em estudos futuros de correlação genótipo-fenótipo e de avaliação de protocolos clínicos. Os critérios de inclusão foram: ter 4 anos ou mais e confirmação bioquímica da doença (níveis reduzidos da atividade da ARSB, aumento de GAGs urinários e atividade normal de outra sulfatase). Os critérios de exclusão foram: terapia com reposição enzimática atual ou prévia ou ter realizado transplante de medula óssea. Foram avaliados 28 pacientes por anamnese, exame físico, acocardiograma, eletrocardiograma, avaliação oftalmológica, medidas de glicosaminoglicanos urinários e da atividade da N-acetilgalactosamina-4-sulfatase em leucócitos. A amostra estudada tinha 92,9% de brasileiros, sendo 53,8% da região sudeste. No momento da avaliação, a média de idade foi de 97,1 meses e a média de idade ao diagnóstico foram de 48,4 meses. Em 88% da amostra os sintomas iniciaram com menos de 36 meses e em 27% das famílias houve relato de consangüinidade entre os pais. A média de peso e estatura ao nascimento foi de 3481 gramas e 51,3 centímetros, respectivamente. Da amostra, 57,1% nasceram de parto vaginal. Todos apresentavam alguma alteração ecocardiográfica, bem como opacificação corneana. As manifestações clínicas mais freqüentes foram: baixa estatura, opacificação corneana, facies grosseira, contraturas articulares e mãos em garra. A média da atividade enzimática em leucócitos foi de 5,4 nmoles/h x mg proteína e a excreção urinária de glicosaminoglicanos foi, em média, 7,9 vezes superior ao normal. O número de manifestações clínicas citadas não apresentou correlação significativa com a idade, com a excreção urinária de GAGs ou com a atividade enzimática em leucócitos. Também não houve correlação significativa entre a excreção urinária de GAGS e a atividade enzimática. Concluímos que a MPS VI é uma patologia com alta morbidade e que, comparados com a literatura, os pacientes da nossa amostra têm um diagnóstico tardio e maior freqüência de alterações cardiológicas.

Effects of the basic fibroblast growth factor and its anti-factor in the healing and collagen maturation of infected skin wound

PURPOSE: The infection is one of the main factors that affect the physiological evolution of the surgical wounds. The aim of this work is to evaluate the effects of fibroblast growth factor (FGFâ) and anti-FGFâ in the healing, synthesis and maturation of collagen when topically used on infected skin wounds of rats. METHODS: An experimental study was perfomed in 60 male Wistar rats. All animals were divided in two groups (A and B). Each group was divided in three subgroups A1, B1; A2, B2 and A3, B3. After anesthesia with pentobarbital, two open squared wounds (1cm2), 4cm distant to each other, were done in the dorsal skin of all the rats. In group A (n=30) the wounds were contaminated with multibacterial standard solution, and in group B(n=30) the wounds were maintained sterile. These wounds were named F1 (for inflammation analysis) and F2 (for collagen study). The open wounds of A1 and B1 rats were topically treated with saline solution, A2 and B2 were treated with FGFâ and subgroups A3 and B3 were treated with FGFâ and anti-FGFâ. The rats were observed until complete epitelization of F2 wounds for determination of healing time and the expression of types I and III collagen, using Picro Sirius Red staining. Inflammatory reaction in F1 wounds was studied using hematoxilineosin staining. The three variable was measured by the Image Pro-Plus Média Cybernetics software. The statistical analysis was performed by ANOVA and Tukey test, considering p<0.05 as significant. RESULTS: It was observed that infection retarded significantly (p<0.05) the time of wound scarring and the topical application of FCFb reverted the inhibition of healing caused by bacteria. The inflammatory reaction was greater in the subgroup B2 than in B1 and A3, and the difference was significant (p<0.05). It was observed greater expression of type I collagen in all the subgroups treated with FCFb, when compared with the untreated subgroups. Type III collagen was significantly decreased in wounds of B3 rats, comparing to the other subgroups. CONCLUSIONS: The FCFb accelerated the healing of open infected wounds and contributed with maturation of collagen, enhancing the type I collagen density. The anti-FCFb antibody was able to attenuate the production of both type I and III collagen