Fibrinolytic activity is associated with presence of cystic medial degeneration in aneurysms of the ascending aorta

Fibrinolytic activity is associated with presence of cystic medial degeneration in aneurysms of the ascending aorta Aims: Thoracic ascending aortic aneurysms (TAA) are characterized by elastic fibre breakdown and cystic medial degeneration within the aortic media, associated with progressive smooth muscle cell (SMC) rarefaction. The transforming growth factor (TGF)-beta/Smad2 signalling pathway is involved in this process. Because the pericellular fibrinolytic system activation is able to degrade adhesive proteins, activate matrix metalloproteinase (MMP), induce SMC disappearance and increase the bioavailability of TGF-beta, the aim was to investigate the plasminergic system in TAA. Methods and results: Ascending aortas [21 controls and 19 TAAs (of three different aetiologies)] were analysed. Immunohistochemistry showed accumulation of t-PA, u-PA and plasmin in TAAs, associated with residual SMCs. Overexpression of t-PA and u-PA was confirmed by reverse transcription-polymerase chain reaction (RT-PCR), immunoblotting and zymography on TAA extracts and culture medium conditioned by TAA. Plasminogen was present on the SMC surface and inside cytoplasmic vesicles, but plasminogen mRNA was undetectable in the TAA medial layer. Plasmin-antiplasmin complexes were detected in TAA-conditioned medium and activation of the fibrinolytic system was associated with increased fibronectin turnover. Fibronectin-related material was detected immunohistochamically in dense clumps around SMCs and colocalized with latent TGF-beta binding protein-1. Conclusions: The fibrinolytic pathway could play a critical role in TAA progression, via direct or indirect impact on ECM and consecutive modulation of TGF-beta bioavailability.

Wide Dispersal and Possible Multiple Origins of Low-Copy-Number Plasmids in Rickettsia Species Associated with Blood-Feeding Arthropods

Plasmids are mobile genetic elements of bacteria that can impart important adaptive traits, such as increased virulence or antibiotic resistance. We report the existence of plasmids in Rickettsia (Rickettsiales; Rickettsiaceae) species, including Rickettsia akari, ""Candidatus Rickettsia amblyommii,"" R. bellii, R. rhipicephali, and REIS, the rickettsial endosymbiont of Ixodes scapularis. All of the rickettsiae were isolated from humans or North and South American ticks. R. parkeri isolates from both continents did not possess plasmids. We have now demonstrated plasmids in nearly all Rickettsia species that we have surveyed from three continents, which represent three of the four major proposed phylogenetic groups associated with blood-feeding arthropods. Gel-based evidence consistent with the existence of multiple plasmids in some species was confirmed by cloning plasmids with very different sequences from each of two ""Ca. Rickettsia amblyommii"" isolates. Phylogenetic analysis of rickettsial ParA plasmid partitioning proteins indicated multiple parA gene origins and plasmid incompatibility groups, consistent with possible multiple plasmid origins. Phylogenetic analysis of potentially host-adaptive rickettsial small heat shock proteins showed that hsp2 genes were plasmid specific and that hsp1 genes, found only on plasmids of ""Ca. Rickettsia amblyommii,"" R. felis, R. monacensis, and R. peacockii, were probably acquired independently of the hsp2 genes. Plasmid copy numbers in seven Rickettsia species ranged from 2.4 to 9.2 per chromosomal equivalent, as determined by real-time quantitative PCR. Plasmids may be of significance in rickettsial evolution and epidemiology by conferring genetic plasticity and host-adaptive traits via horizontal gene transfer that counteracts the reductive genome evolution typical of obligate intracellular bacteria.

Avian Metapneumovirus Subtypes Circulating in Brazilian Vaccinated and Nonvaccinated Chicken and Turkey Farms

Avian metapneumovirus (AMPV) causes turkey rhinotracheitis and is associated with swollen head syndrome in chickens, which is usually accompanied by secondary infections that increase mortality. AMPVs circulating in Brazilian vaccinated and nonvaccinated commercial chicken and turkey farms were detected using a universal reverse transcriptase (RT)-PCR assay that can detect the four recognized subtypes of AMPV. The AMPV status of 228 farms with respiratory and reproductive disturbances was investigated. AMPV was detected in broiler, hen, breeder, and turkey farms from six different geographic regions of Brazil. The detected viruses were subtyped using a nested RT-PCR assay and sequence analysis of the G gene. Only subtypes A and B were detected in both vaccinated and nonvaccinated farms. AMPV-A and AMPV-B were detected in 15 and 23 farms, respectively, while both subtypes were simultaneously found in one hen farm. Both vaccine and field viruses were detected in nonvaccinated farms. In five cases, the detected subtype was different than the vaccine subtype. Field subtype B virus was detected mainly during the final years of the survey period. These viruses showed high molecular similarity (more than 96% nucleotide similarity) among themselves and formed a unique phylogenetic group, suggesting that they may have originated from a common strain. These results demonstrate the cocirculation of subtypes A and B in Brazilian commercial farms.

Protection levels of vaccinated pigeons (Columba livia) against a highly pathogenic newcastle disease virus strain

The purposes of this study were to model a vaccination regimen for Newcastle disease virus (NDV) in pigeons, and to evaluate the susceptibility and behavior of vaccinated birds against a highly pathogenic NDV Brazilian strain. Antibody response was assessed by means of hemagglutination inhibition test (HI), and viral genome excretion by means of RT-PCR. Vaccinal strains (La Sota and Ulster) induced high antibody titers without any adverse effects, both in inoculated and in sentinel birds. A viral strain pathogenic for chickens did not produce clinical signs of the disease in experimentally infected pigeons. Only 4 out of 10 vaccinated pigeons shed NDV genome, and just for two days. Results confirmed the high infectivity of the vaccinal strains used, as all nonvaccinated pigeons showed antibody titers as high as those of vaccinated birds.

Detection and Molecular Characterization of Gene 3 and 5 of Turkey Coronavirus from Turkeys with Severe Enteritis in Brazil

Turkey coronavirus (TCoV) is a causative agent associated with poult enteritis and mortality syndrome (PEMS) in turkeys worldwide. The disease is an acute, highly contagious enteric disease that is characterized by depression, anorexia, diarrhea, and high mortality in commercial turkey flocks. The presence of TCoV in 12 intestinal-content samples, from turkey flocks aged between 10 and 104 days and exhibiting severe enteritis, was monitored during the period of 2004 to 2006. TCoV detection was accomplished by a reverse transcriptase-polymerase chain reaction (RT-PCR) through amplification of the 3` UTR region, followed by amplification of genes 3 and 5. Molecular characterization of the viruses was done through amplification of genes 3 and 5 and showed evidence of genetic similarity between them, although they differed from sequences of other TCoVs described in the literature. In relation to gene 3, samples showed a greater relationship with chicken infectious bronchitis virus (IBV), while gene 5 showed greater identity with pheasant coronavirus (PhCoV). Our results suggest that the strategy of amplification of the 3` UTR region, followed by sequencing of genes 3 and 5, has proven to be an effective means of detecting TCoV in intestinal contents.

Expression of Extracellular Matrix Proteins in Human Dental Pulp Stem Cells Depends on the Donor Tooth Conditions

Introduction: Stem cells are characterized by the ability to renew themselves through mitotic cell division and differentiating into a diverse range of specialized cell types. An important source of adult stem cells is the dental pulp. In dentistry, regenerative strategies are of importance because of hard dental tissue damage especially as result of caries lesions, trauma, or iatrogenic procedures. The regeneration of dental tissues relies on the ability of stem cells to produce extracellular (ECM) proteins encountered in the dental pulp tissue. Thus, the aim of this study was to analyze the expression and distribution of proteins encountered in dental pulp ECM (type I collagen, fibronectin, and tenascin) in stem cells. Methods: Human immature dental pulp stem cells (hIDPSCs) from deciduous (DL-1 and DL-4 cell lines) and permanent (DL-2) teeth were used. The distribution of ECM proteins was observed using the immunofluorescence technique. The gene expression profile was evaluated using reverse transcription polymerase chain reaction (RT-PCR) analysis. Results: Positive reactions for all ECM proteins were observed independently of the hIDPSCs analyzed. Type I collagen appeared less evident in DL-2 than in other hIDPSCs. Fibronectin and tenascin were less clear in DL-4. The RT-PCR reactions showed that type I collagen was lesser expressed in the DL-2 cells, whereas fibronectin and tenascin were similarly expressed in all hIDPSCs. Conclusions: The distribution and expression of ECM proteins differ among the hIDPSCs. These differences seemed to be related to the donor tooth conditions (deciduous or permanent, retained or erupted, and degree of root reabsorption). (J Endod 2010;36:826-831)

In vivo Acid Etching Effect on Bacteria within Caries-Affected Dentin

Acid etching procedures may disrupt residual bacteria and contribute to the success of incomplete caries removal followed by adhesive restoration. This study evaluated the in vivo effect of acid etching on cariogenic bacterial activity within affected dentin after minimally invasive treatment of caries lesions. Twenty-eight carious permanent teeth received standardized selective caries removal and random acid etch treatment (E) or not (NE) prior to adhesive restoration. Baseline and 3-month dentin biopsies were collected. The number of bacteria and activity of total bacterial cells and Streptococcus mutans were determined by quantitative PCR and RT-PCR. No statistically significant differences were observed in total bacterial number and activity between E and NE treatments (p > 0.3008). For NE, however, the residual S. mutans bacterial cells were reduced (p = 0.0027), while the activity per cell was significantly increased (p = 0.0010) after reentry at 3 months after restoration. This effect was not observed in group E. Although no significant differences were found between groups, this study suggests that acid etching of affected dentin prior to adhesive restoration may directly or indirectly have an inhibitive effect on the activity of residual cariogenic bacteria. Further research is required to investigate this potential effect. Copyright (C) 2010 S. Karger AG, Basel

Rat forming incisor requires a rigorous ECM remodeling modulated by MMP/RECK balance

Reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) is a single membrane-anchored MMP-regulator and regulates matrix metalloproteinases (MMP) 2, 9 and 14. In turn, MMPs are endopeptidases that play a pivotal role in remodeling ECM. In this work, we decided to evaluate expression pattern of RECK in growing rat incisor during, specifically focusing out amelogenesis process. Based on different kinds of ameloblasts, our results showed that RECK expression was conducted by secretory and post-secretory ameloblasts. At the secretory phase, RECK was localized in the infra-nuclear region of the ameloblast, outer epithelium, near blood vessels, and in the stellate reticulum. From the transition to the maturation phases, RECK was strongly expressed by non-epithelial immuno-competent cells (macrophages and/or dendritic-like cells) in the papillary layer. From the transition to the maturation stage, RECK expression was increased. RECK mRNA was amplified by RT-PCR from whole enamel organ. Here, we verified the presence of RECK mRNA during all stages of amelogenesis. These events were governed by ameloblasts and by non-epithelial cells residents in the enamel organ. Concluding, we found differential expression of MMPs-2, -9 and RECK in the different phases of amelogenesis, suggesting that the tissue remodeling is rigorously controlled during dental mineralization.

COX-2 inhibition decreases VEGF expression and alveolar bone loss during the progression of experimental periodontitis in rats

Background: Vascular endothelial growth factor (VEGF) is a macromolecule of importance in inflammation that has been implicated in periodontitis. The aims of this study were to investigate VEGF expression during the progression of periodontal disease and to evaluate the effect of a preferential cyclooxygenase (COX)-2 inhibitor meloxicam on VEGF expression and alveolar bone loss in experimentally induced periodontitis. Methods: A total of 120 Wistar rats were randomly separated into groups 1 (control) and 2 (meloxicam, 3 mg/kg/day, intraperitoneally, for 3, 7, 14, or 30 days). Silk ligatures were placed at the gingival margin level of the lower right first molar of all rats. VEGF expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR), Western blot (WB), and immunohistochemical (IHC) analyses. The hemiarcades were processed for histopathologic analysis. RT-PCR and WB results were submitted to analysis of variance, the Tukey test, and Pearson correlation analysis (P<0.05). Results: A reduction in alveolar bone resorption was observed in the meloxicam-treated group compared to the control group at all periods studied. There was a positive correlation between COX-2 mRNA and VEGF mRNA in the gingival tissues and periodontal disease (R = 0.80; P = 0.026). Meloxicam significantly reduced the increased mRNA VEGF expression in diseased tissues after 14 days of treatment (P = 0.023). Some alterations in VEGF receptor I mRNA expression were observed, but these were not statistically significant. VEGF protein expression in WB experiments was significantly higher in diseased sites compared to healthy sites (P<0.05). After 14 days of treatment with meloxicam, an important decrease in VEGF protein expression was detected in diseased tissues (P = 0.08). Qualitative IHC analysis revealed that VEGF protein expression was higher in diseased tissues and decreased in tissues from rats treated with meloxicam. Conclusions: The present data suggest an important role for VEGF in the progression of periodontal disease. Systemic therapy with meloxicam can modify the progression of experimentally induced periodontitis in rats by reducing VEGF expression and alveolar bone loss.