A New Mouse Model for Marfan Syndrome Presents Phenotypic Variability Associated with the Genetic Background and Overall Levels of Fbn1 Expression

Marfan syndrome is an autosomal dominant disease of connective tissue caused by mutations in the fibrillin-1 encoding gene FBN1. Patients present cardiovascular, ocular and skeletal manifestations, and although being fully penetrant, MFS is characterized by a wide clinical variability both within and between families. Here we describe a new mouse model of MFS that recapitulates the clinical heterogeneity of the syndrome in humans. Heterozygotes for the mutant Fbn1 allele mg Delta(loxPneo), carrying the same internal deletion of exons 19-24 as the mg Delta mouse model, present defective microfibrillar deposition, emphysema, deterioration of aortic wall and kyphosis. However, the onset of a clinical phenotypes is earlier in the 129/Sv than in C57BL/6 background, indicating the existence of genetic modifiers of MFS between these two mouse strains. In addition, we characterized a wide clinical variability within the 129/Sv congenic heterozygotes, suggesting involvement of epigenetic factors in disease severity. Finally, we show a strong negative correlation between overall levels of Fbn1 expression and the severity of the phenotypes, corroborating the suggested protective role of normal fibrillin-1 in MFS pathogenesis, and supporting the development of therapies based on increasing Fbn1 expression.

Nd:YAG Laser Improves Biocompatibility of Human Dental Root Surfaces

Objective: Our goal was to compare the in vivo biocompatibility of dental root surfaces submitted to four different treatments after tooth avulsion followed by implantation into rat subcutaneous tissue. Background Data: Dental root surface preparation prior to replanting teeth remains a challenge for endodontists. Root surface changes made by Nd:YAG irradiation could be an alternative preparation. Methods: Forty-eight freshly extracted human dental roots were randomly divided into four treatment groups prior to implantation into rat subcutaneous tissue: G1, dry root, left in the environment up to 3 h; G2, the same treatment as G1, followed by a soaking treatment in a 2.4% sodium fluoride solution (pH 5.5); G3, root soaked in physiologic saline after avulsion for 72 h; G4, the same treatment as G1, followed by Nd:YAG laser irradiation (2.0 W, 20 Hz, 100 mJ, and 124.34 J/cm(2)). The animals were sacrificed 1, 7, and 45 d later. Histological and scanning electron microscopy analyses were done. Results: All dental roots were involved and in intimate contact with connective tissue capsules of variable thicknesses. Differences were observed in the degree of inflammation and in connective tissue maturation. In G3 the inflammatory infiltrate was maintained for 45 d, whereas the Nd:YAG laser irradiation (G4) led to milder responses. The overall aspects of the root surfaces were similar, except by the irradiated roots, where fusion and resolidification of the root surface covering the dentinal tubules were observed. Conclusion: Nd:YAG laser irradiation improves the biocompatibility of dental root and thus could be an alternative treatment of dental root prior to replantation.

Investigation of mast cells in human gingiva following low-intensity laser irradiation

Objective: The aims of the present study were to investigate the effect of low-intensity laser irradiation on the total number of mast cells as well as the percentage of degranulation in human gingiva. Blood vessel dilation was also evaluated. Background Data: It has been proposed that low-intensity laser irradiation can ameliorate pain, swelling, and inflammation. In periodontal tissue, mast cells may influence either the destructive events or the defense mechanism against periodontal disease via secretion of cytokines and through cellular migration to improve the healing process. Mast cells play an important role in the inflammatory process. Methods: Twenty patients with gingival enlargement indicated for gingivectomy were selected. Gingival fragments were obtained from each patient and divided into three different groups before surgery. One fragment was removed without any irradiation. The two others were submitted to punctual irradiation with an energy density of 8 J/cm(2) at an output power of 50 mW at 36 Hz for 36 sec before gingivectomy. Nondegranulated and degranulated mast cells were counted in five areas of the gingival fragment connective tissue. Major and minor diameters of the blood vessels were also measured. Results: Both red and infrared radiation promoted a significant increase in mast cell degranulation compared to controls; however, no statistically significant differences (p > 0.05) were observed between the irradiated groups. No significant differences among the groups were observed regarding blood vessel size. Conclusion: The results suggests that red and infrared wavelengths promote mast cell degranulation in human gingival tissue, although no dilation of blood vessels was observed. The effects of premature degranulation of mast cells in human tissue and the laser radiation protocol applied in this study encourage further investigations to extend these results into clinical practice.

Evaluation of Protective Effect of a Water-In-Oil Microemulsion Incorporating Quercetin Against UVB-Induced Damage in Hairless Mice Skin

Purpose. Histological aspects were considered in order to evaluate the in vivo photoprotective effect of a w/o microemulsion containing quercetin against UVB irradiation-induced dermal damages. The toxicity in cell culture and the potential skin irritation resulting from topical application of this formulation were investigated. Methods. Mouse dorsal surfaces were treated topically with 300 mg of the unloaded and quercetin-loaded (0.3%, w/w) microemulsions before and after exposure to UVB (2.87 J/cm(2)) irradiation. The untreated control groups irradiated and non-irradiated were also evaluated. UVB-induced histopathological changes as well as the photoprotective effect of this formulation were evaluated considering the parameters of infiltration of inflammatory cells, epidermis thickening (basale and spinosum layers) and collagen and elastic fiber contents. The cytotoxicity of the reported formulation was evaluated in L929 mice fibroblasts by MTT assay and the skin irritation was investigated after topical application of both unloaded and quercetin-loaded microemulsions once a day for 15 days. Results. The results demonstrated that the w/o microemulsion containing quercetin reduced the incidence of histological skin alterations, mainly the connective-tissue damage, induced by exposure to UVB irradiation. This suggests that protective effects of this formulation against UV-induced responses are not secondary to the interference of UV transmission (i.e., blocking the UVB radiation from being absorbed by the skin), as is usually implied with UVB absorbers and sunscreens, but is instead due to different biological effects of this flavonoid. Furthermore, by evaluating the cytotoxic effect on L929 cells and histological aspects such as infiltration of inflammatory cells and epidermis thickness of hairless mice, the present study also demonstrated the lack of toxicity of the proposed system. Conclusion. Based on these mice models, a detailed characterization of the w/o microemulsion incorporating quercetin effects as a photochemoprotective agent on human skin is presented.

Hybrid layer-by-layer assembly based on animal and vegetable structural materials: multilayered films of collagen and cellulose nanowhiskers

Layer-by-layer (LBL) assembly was used to combine crystalline rod-like nanoparticles obtained from a vegetable source, cellulose nanowhiskers (CNWs), with collagen, the main component of skin and connective tissue found exclusively in animals. The film growth of the multilayered collagen/CNW was monitored by UV-Vis spectroscopy and ellipsometry measurements, whereas the film morphology and surface roughness were characterized by SEM and AFM. UV-Vis spectra showed the deposition of the same amount of collagen, 5 mg m(-2), in each dipping cycle. Ellipsometry data showed an increment in thickness with the number of layers, and the average thickness of each bilayer was found to be 8.6 nm. The multilayered bio-based nanocomposites were formed by single layers of densely packed CNWs adsorbed on top of each thin collagen layer where the hydrogen bonding between collagen amide groups and OH groups of the CNWs plays a mandatory role in the build-up of the thin films. The approach used in this work represents a potential strategy to mimic the characteristics of natural extracellular matrix (ECM) which can be used for applications in the biomedical field.

Effect of bioglass additions on the sintering of Y-TZP bioceramics

The objective of this work was to evaluate the influence of bioglass additions on the sintering and mechanical properties of yttria-stabilized zirconia ceramics, Y-TZP Samples containing different bioglass additions, varying between 0 and 30 wt.%, were cold uniaxial pressed at 80 MPa and sintered in air at 1200 degrees C or 1300 degrees C for 120 min. Sintered samples were characterized by X-ray Diffractometry and Scanning Electron Microscopy. Hardness and fracture toughness were determined using Vickers indentation method. As a preliminary biological evaluation, in vitro cytotoxicity tests by Neutral Red Uptake method (using mouse connective tissue cells, NCTC clone L929 from ATCC bank) were realized to determine the cytotoxicity level of ZrO(2)-bioglass ceramics. The increasing of bioglass amount leads to the decreasing of relative density due to martensitic (tetragonal-monoclinic) transformation during cooling of the sintered samples. Y-TZP samples sintered at 1300 degrees C containing 5 wt.% of bioglass presented the best results. with high relative density, hardness and fracture toughness of 11.3 GPa and 6.1 MPa m(1/2), respectively. Furthermore, the un-cytotoxic behavior was observed in all sintering conditions and bioglass amounts used in this study. (C) 2009 Elsevier B.V. All rights reserved.

LOW-INTENSITY PULSED ULTRASOUND PRODUCED AN INCREASE OF OSTEOGENIC GENES EXPRESSION DURING THE PROCESS OF BONE HEALING IN RATS

The aim of this study was to measure the temporal expression of osteogenic genes during the process of bone healing in low-intensity pulsed ultrasound (LIPUS) treated bone defects by means of histopathologic and real-time polymerase chain reaction (PCR) analysis. Animals were randomly distributed into two groups (n = 30): control group (bone defect without treatment) and LIPUS treated (bone defect treated with LIPUS). On days 7, 13 and 25 postinjury, 10 rats per group were sacrificed. Rats were treated with a 30 mW/cm(2) LIPUS. The results pointed out intense new bone formation surrounded by highly vascularized connective tissue presenting a slight osteogenic activity, with primary bone deposition was observed in the group exposed to LIPUS in the intermediary (13 days) and late stages of repair (25 days) in the treated animals. In addition, quantitative real-time polymerase chain reaction (RT-qPCR) showed an upregulation of bone morphogenetic protein 4 (BMP4), osteocalcin and Runx2 genes 7 days after the surgery. In the intermediary period, there was no increase in the expression. The expression of alkaline phosphatase, BMP4 and Runx2 was significantly increased at the last period. Our results indicate that LIPUS therapy improves bone repair in rats and upregulated osteogenic genes, mainly at the late stages of recovery. (E-mail: a.renno@unifesp.br) (C) 2010 Published by Elsevier Inc. on behalf of World Federation for Ultrasound in Medicine & Biology.

A Brazilian registry of juvenile dermatomyositis: onset features and classification of 189 cases

Objective To describe onset features, classification and treatment of juvenile dermatomyositis (JDM) and juvenile polymyositis (JPM) from a multicentre registry. Methods Inclusion criteria were onset age lower than 18 years and a diagnosis of any idiopathic inflammatory myopathy (IIM) by attending physician. Bohan & Peter (1975) criteria categorisation was established by a scoring algorithm to define JDM and JPM based oil clinical protocol data. Results Of the 189 cases included, 178 were classified as JDM, 9 as JPM (19.8: 1) and 2 did not fit the criteria; 6.9% had features of chronic arthritis and connective tissue disease overlap. Diagnosis classification agreement occurred in 66.1%. Medial? onset age was 7 years, median follow-up duration was 3.6 years. Malignancy was described in 2 (1.1%) cases. Muscle weakness occurred in 95.8%; heliotrope rash 83.5%; Gottron plaques 83.1%; 92% had at least one abnormal muscle enzyme result. Muscle biopsy performed in 74.6% was abnormal in 91.5% and electromyogram performed in 39.2% resulted abnormal in 93.2%. Logistic regression analysis was done in 66 cases with all parameters assessed and only aldolase resulted significant, as independent variable for definite JDM (OR=5.4, 95%CI 1.2-24.4, p=0.03). Regarding treatment, 97.9% received steroids; 72% had in addition at least one: methotrexate (75.7%), hydroxychloroquine (64.7%), cyclosporine A (20.6%), IV immunoglobulin (20.6%), azathioprine (10.3%) or cyclophosphamide (9.6%). In this series 24.3% developed calcinosis and mortality rate was 4.2%. Conclusion Evaluation of predefined criteria set for a valid diagnosis indicated aldolase as the most important parameter associated with de, methotrexate combination, was the most indicated treatment.

Increase of gingival matured dendritic cells number in elderly patients with chronic periodontitis

Objective: The purpose of this study was to evaluate the inflammatory cell subset proportions in the upper gingival connective tissue, including mature dendritic cells (DC) in elderly and younger patients with generalized chronic periodontitis in order to further understand the effect of aging on gingival inflammatory phenomenon. Methods: Gingival tissue specimens presenting chronic periodontitis from 8 elderly patients aged >75 (test group, group T) and from 8 younger patients aged 50-60 (considered as controls, group C) were analysed by immunohistochemistry using monoclonal antibodies against CD45RB, CD4, CD8, CD19, CD68, DC-SIGN, DC-LAMP molecules. The number of each immunolabelled cells subset was counted using image analysis. Results: The difference in the number of CD45RB + leucocytes in the upper gingival connective tissue between groups was not significant permitting to use it as reference. As compared. to group C, the lymphocyte subsets/CD45RB + leucocytes ratios tended to decrease in group T but the decrease was significant only for CD4 + T lymphocytes/ CD45RB + cells ratio (p < 0.03). On the opposite, the ratios of antigen-presenting cells DC-SIGN + cells/CD45RB + cells and DC-LAMP + cells/CD45RB + cells were significantly increased;(p < 0.03 and <0.0001, respectively) in group T. Moreover, in group T the DC-LAMP + cells/DC-SIGN + cells ratio was significantly increased (p < 0.05) showing an increased number of matured dendritic cells. Conclusion: During chronic periodontitis in elderly patients, our results show a decrease in the ratio of gingival CD4 + lymphocyte subset associated with an increase in the ratios of antigen-presenting cells subsets and more particularly maturated DC-LAMP + dendritic cells. (C) 2008 Elsevier Ltd. All rights reserved.

Intra-bone marrow injection of mesenchymal stem cells improves the femur bone mass of osteoporotic female rats

The effect of intra-bone injection of differentiated rat bone marrow mesenchymal stem cells (BMMSCs) into the femur of osteoporotic female rats was studied. Osteoporosis was induced in Wistar female rats by bilateral ovariectomy. Then, 0.75 million BMMSCs isolated from healthy rats were injected into the femurs of osteoporotic rats. Histomorphometric analysis and histology clearly revealed improvements in the treated group as compared to untreated group. In 2 months, the femurs of treated rats, unlike untreated rats, showed trabecular bone percentage almost similar to the femurs from control healthy rats. To confirm the origin of newly formed bone, the experiment was repeated with BMMSCs isolated from green fluorescent protein transgenic rats. Confocal microscopy demonstrated green fluorescent protein-positive cells at the surface of trabecular bone of the treated rats. We investigated in vitro osteogenic differentiation of BMMSCs isolated from osteoporotic rats by studying alkaline phosphatase activity, collagen synthesis, and the ability to form mineralized nodules. Osteoporotic BMMSCs showed less differentiation capabilities as compared to those isolated from healthy rats. The results clearly demonstrated the importance of BMMSCs in osteoporosis and that the disease can be treated by injection of BMMSCs.

Immunogenicity and safety of the 2009 non-adjuvanted influenza A/H1N1 vaccine in a large cohort of autoimmune rheumatic diseases

Background Despite the WHO recommendation that the 2010-2011 trivalent seasonal flu vaccine must contain A/California/7/2009/H1N1-like virus there is no consistent data regarding its immunogenicity and safety in a large autoimmune rheumatic disease (ARD) population. Methods 1668 ARD patients (systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), ankylosing spondylitis (AS), systemic sclerosis, psoriatic arthritis (PsA), Behcet`s disease (BD), mixed connective tissue disease, primary antiphospholipid syndrome (PAPS), dermatomyositis (DM), primary Sjogren`s syndrome, Takayasu`s arteritis, polymyositis and Granulomatosis with polyangiitis (Wegener`s) (GPA)) and 234 healthy controls were vaccinated with a non-adjuvanted influenza A/California/7/2009(H1N1) virus-like strain flu. Subjects were evaluated before vaccination and 21 days post-vaccination. The percentage of seroprotection, seroconversion and the factor increase in geometric mean titre (GMT) were calculated. Results After immunisation, seroprotection rates (68.5% vs 82.9% p < 0.0001), seroconversion rates (63.4% vs 76.9%, p < 0.001) and the factor increase in GMT (8.9 vs 13.2 p < 0.0001) were significantly lower in ARD than controls. Analysis of specific diseases revealed that seroprotection significantly reduced in SLE (p < 0.0001), RA (p < 0.0001), PsA (p=0.0006), AS (p=0.04), BD (p=0.04) and DM (p=0.04) patients than controls. The seroconversion rates in SLE (p < 0.0001), RA (p < 0.0001) and PsA (p=0.0006) patients and the increase in GMTs in SLE (p < 0.0001), RA (p < 0.0001) and PsA (p < 0.0001) patients were also reduced compared with controls. Moderate and severe side effects were not reported. Conclusions The novel recognition of a diverse vaccine immunogenicity profile in distinct ARDs supports the notion that a booster dose may be recommended for diseases with suboptimal immune responses. This large study also settles the issue of vaccine safety. (ClinicalTrials.gov #NCT01151644)

Updated Clinical Classification of Pulmonary Hypertension

The aim of a clinical classification of pulmonary hypertension (PH) is to group together different manifestations of disease sharing similarities in pathophysiologic mechanisms, clinical presentation, and therapeutic approaches. In 2003, during the 3rd World Symposium on Pulmonary Hypertension, the clinical classification of PH initially adopted in 1998 during the 2nd World Symposium was slightly modified. During the 4th World Symposium held in 2008, it was decided to maintain the general architecture and philosophy of the previous clinical classifications. The modifications adopted during this meeting principally concern Group 1, pulmonary arterial hypertension (PAH). This subgroup includes patients with PAH with a family history or patients with idiopathic PAH with germline mutations (e. g., bone morphogenetic protein receptor-2, activin receptor-like kinase type 1, and endoglin). In the new classification, schistosomiasis and chronic hemolytic anemia appear as separate entities in the subgroup of PAH associated with identified diseases. Finally, it was decided to place pulmonary venoocclusive disease and pulmonary capillary hemangiomatosis in a separate group, distinct from but very close to Group 1 (now called Group 1`). Thus, Group 1 of PAH is now more homogeneous. (J Am Coll Cardiol 2009;54:S43-54) (C) 2009 by the American College of Cardiology Foundation

Clinicopathological evaluation of in vivo epidermal nuclear fluorescence

Nuclear fluorescence in keratinocytes is an occasional phenomenon, often present in autoimmune diseases, especially in connective-tissue disease (CTD); however, its clinical significance remains unclear. To investigate the profile of patients with positive nuclear staining on direct immunofluorescence (DIF) of skin samples. A retrospective analysis of 28 patient records from our immunodermatology laboratory was performed between May 2003 and June 2006. Inclusion criteria were the presence of autoantibodies (IgG, IgA or IgM) or complement (C3) binding keratinocyte nuclei on DIF. The most prevalent diseases related to the nuclear keratinocyte DIF staining were systemic lupus erythematosus (n = 9), mixed CTD (n = 3), overlap syndrome (n = 3), Sjogren`s syndrome (n = 1), and CREST (calcinosis, Raynaud`s phenomenon, oesophageal dysmotility, sclerodactyly and telangiectasia) syndrome (n = 1). Serum antinuclear antibody (ANA) was positive in 20 of 28 patients, with titres varying from 1 : 160 to 1 : 1280. Of the 20 patients with positive anti-nuclear antibodies (ANA), 17 were positive for anti-extractable nuclear antigen antibodies, 12 had anti-SSA/Ro, 11 had anti-SSB/La and 8 had anti-ribonucleoprotein. Eight patients were negative for ANA. Positive predictive value of in vivo ANA for systemic CTDs was 75%. The present data suggest that in vivo ANA evaluation is an additional and feasible auxiliary tool for diagnosing CTDs.

MHC class I and II expression in juvenile dermatomyositis skeletal muscle

Objective To assess MHC I and II expressions in muscle fibres of juvenile dermatomyositis (JDM) and compare with the expression in polymyositis (PM), dermatomyositis (DM) and dystrophy. Patients and methods Forty-eight JDM patients and 17 controls (8 PM, 5 DM and 4 dystrophy) were studied. The mean age at disease onset was 7.1 +/- 3.0 years and the mean duration of weakness before biopsy was 9.4 +/- 12.9 months. Routine histochemistry and immunohistochemistry (StreptABComplex/HRP) for MHC I and II (Dakopatts) were performed on serial frozen muscle sections in all patients. Mann-Whitney, Kruskal Wallis, chi-square and Fisher`s exact statistical methods were used. Results MHC I expression was positive in 47 (97.9%) JDM cases. This expression was observed independent of time of disease corticotherapy previous to muscle biopsy and to the grading of inflammation observed in clinical, laboratorial and histological parameters. The expression of MHC I was similar on JDM, PM and DM, and lower in dystrophy. On the other hand, MHC II expression was positive in just 28.2% of JDM cases was correlated to histological features as inflammatory infiltrate, increased connective tissue and VAS for global degree of abnormality (p < 0.05). MCH II expression was similar in DM/PM and lower in JDM and dystrophy, and it was based on the frequency of positive staining rather than to the degree of the MCH II expression. Conclusions MHC I expression in muscle fibres is a premature and late marker of JDM patient independent to corticotherapy, and MHC II expression was lower in JDM than in PM and DM.

Ultrastructural evaluation of gingival glycosaminoglycans in ovariectomized rats: Effects of steroid hormone therapy

Background/Aims: To evaluate the behavior of glycosaminoglycans (GAGs) in rat gingiva and the effects of lack of sexual steroids and the hormonal therapy with estrogen and dexamethasone (DEX). Methods: 40 female rats were divided into four groups: GI: animals in permanent estrus; GII: ovariectomized (OVX) animals + vehicle; GIII: OVX animals treated with 17 beta-estradiol benzoate (10 mu g/kg), and GIV: OVX animals treated with 17 beta-estradiol benzoate (10 mu g/kg) + DEX (3 mg/kg). After treatment, the gingiva was removed and its GAGs content was evaluated by electronic microscopy after stained by cuprolinic blue technique. Results: The electron-microscopic data showed that low values of chondroitin sulfate were found in castrated animals (35.05 +/- 3.58%) compared to other groups (GI: 41.17 +/- 1.13; GIII: 48.04 +/- 2.60; GIV: 49.09 +/- 2.68%). In contrast, the amount of dermatan sulfate in GII (57.70 +/- 2.50%) was higher than in the other groups (GI: 46.12 +/- 1.30; GIII: 42.65 +/- 2.98; GIV: 42.68 +/- 5.43%). Conclusions: GAGs may be influenced by estradiol, and DEX did not seem to antagonize the role of estradiol in the GAGs of gingiva. The histotypical structure of gingiva is related to the amount of chondroitin sulfate. Consequently, the estrogen therapy may be important for gingival health. Copyright (C) 2007 S. Karger AG, Basel.