Human T-Lymphotropic Virus Type 1 and 2 Seroprevalence among first-time blood donors in Chile, 2011-2013.

Infection with human T-lymphotropic virus type 1/2 (HTLV-1/2) is a major health problem. HTLV-1/2 infection is endemic in Chile but representative donor prevalence data are lacking. Data on all blood donors in a large network of Chilean blood centers were examined during 2011-2013. Screening of HTLV-1/2 antibodies were measured by enzyme immunoassay (EIA) at all blood banks. Blood samples with anticoagulants from initially reactive blood donors were analyzed by serological confirmation tests (immunofluorescence or recombinant immunoblot) at the HTLV National Reference Laboratory of the Public Health Institute of Chile. Additionally, detection of HTLV-1 and HTLV-2 provirus in peripheral blood mononuclear cells (PBMCs) was performed in all blood donors as confirmatory test. Prevalence rates were calculated. Among 694,016 donors, 706 were seropositive for HTLV-1 (prevalence, 1.02 cases per 1,000; 95% confidence interval [CI], 0.94-1.09), and 97 were seropositive for HTLV-2 (prevalence, 0.14 cases per 1,000; 95%CI, 0.11-0.17). Prevalence of HTLV-1 differed considerably by region, from 0.51 to 1.69 per 1,000. Prevalence of HTLV-2 was similar across the country (0.12-0.16). HTLV-1 prevalence was associated with female sex, older age, and residence in the north of Chile. HTVL-2 prevalence was associated with older age. The HTLV-1 prevalence among Chilean blood donors was relatively high and could be reduced by improving donor recruitment and selection in high prevalence areas. Blood center data may contribute to surveillance for HTLV-1 and HTLV-2 infections.

Matrix metalloproteinases regulate extracellular levels of SDF-1/CXCL12, IL-6 and VEGF in hydrogen peroxide-stimulated human periodontal ligament fibroblasts.

Periodontitis is a highly prevalent infectious disease characterized by the progressive inflammatory destruction of tooth-supporting structures, leading to tooth loss. The underling molecular mechanisms of the disease are incompletely understood, precluding the development of more efficient screening, diagnostic and therapeutic approaches. We investigated the interrelation of three known effector mechanisms of the cellular response to periodontal infection, namely reactive oxygen species (ROS), matrix metalloproteinases (MMPs) and cytokines in primary cell cultures of human periodontal ligament fibroblast (hPDLF). We demonstrated that ROS increase the activity/levels of gelatinolytic MMPs, and stimulate cytokine secretion in hPDLF. Additionally, we proved that MMPs possesses immune modulatory capacity, regulating the secreted levels of cytokines in ROS-stimulated hPDLF cultures. This evidence provides further insight in the molecular pathogenesis of periodontitis, contributing to the future development of more effective therapies.

Proteolytic roles of matrix metalloproteinase (MMP)-13 during progression of chronic periodontitis: initial evidence for MMP-13/MMP-9 activation cascade.

AIM: Matrix metalloproteinases (MMP)-13 can initiate bone resorption and activate proMMP-9 in vitro, and both these MMPs have been widely implicated in tissue destruction associated with chronic periodontitis. We studied whether MMP-13 activity and TIMP-1 levels in gingival crevicular fluid (GCF) associated with progression of chronic periodontitis assessed clinically and by measuring carboxy-terminal telopeptide of collagen I (ICTP) levels. We additionally addressed whether MMP-13 could potentiate gelatinase activation in diseased gingival tissue. MATERIALS AND METHODS: In this prospective study, GCF samples from subjects undergoing clinical progression of chronic periodontitis and healthy controls were screened for ICTP levels, MMP-13 activity and TIMP-1. Diseased gingival explants were cultured, treated or not with MMP-13 with or without adding CL-82198, a synthetic MMP-13 selective inhibitor, and assayed by gelatin zymography and densitometric analysis. RESULTS: Active sites demonstrated increased ICTP levels and MMP-13 activity (p<0.05) in progression subjects. The MMP-9 activation rate was elevated in MMP-13-treated explants (p<0.05) and MMP-13 inhibitor prevented MMP-9 activation. CONCLUSIONS: MMP-13 could be implicated in the degradation of soft and hard supporting tissues and proMMP-9 activation during progression of chronic periodontitis. MMP-13 and -9 can potentially form an activation cascade overcoming the protective TIMP-1 shield, which may become useful for diagnostic aims and a target for drug development.

Matrix metalloproteinase-13 is highly expressed in destructive periodontal disease activity.

BACKGROUND: Matrix metalloproteinases (MMPs) participate in extracellular matrix degradation in physiological and pathological conditions. The available evidence suggests that MMP-13 plays a significant role in both the initiation and progress of bone resorption. The aim of our study was to identify the presence of MMP-13 in adult patients with untreated chronic periodontitis. We also determined the activity of MMP-13 present in lesions undergoing episodic attachment loss in gingival crevicular fluid (GCF) samples. METHODS: After monitoring at 2 and 4 months, 21 patients showed destructive periodontitis (periodontally affected sites presenting at least two sites with > or =2 mm clinical attachment loss), and GCF samples were collected both from active and inactive sites (21 GCF samples, each). GCF was collected during a 30-second interval using a paper strip, and an immunofluorescence assay was performed to determine the basal activity of MMP-13 and the relationship between 4-aminophenylmercuric acetate (APMA)-activated total MMP-13 and basal MMP-13 activity. Gingival tissues from five patients were fixed in formalin and MMP-13 expression was demonstrated using immunohistochemistry and in situ hybridization. MMP-13 molecular forms were examined by Western immunoblotting with monoclonal antibodies. RESULTS: MMP-13 was found in 100% of GCF samples from patients with chronic periodontitis. Active sites, associated with tissue destruction, had significantly higher proportions of active MMP-13 and MMP-13 activity levels than their inactive counterparts (1.49 versus 1.17 ng fluorescent product, respectively; P <0.05). Western blot, immunohistochemical staining, and in situ hybridization confirmed the presence of MMP-13 in periodontal disease, with observable differences between periodontitis and healthy subjects. MMP-13 immunoreactivities were seen mainly as 55 and 48 kDa, corresponding to partially and fully activated forms, respectively, and a smaller proportion of 60-kDa proenzyme form. CONCLUSION: MMP-13 activity in GCF samples was significantly increased in active sites from progressive periodontal disease, supporting its role in the alveolar bone loss developed in this disease.

Intravenous apyrase administration reduces arterial thrombosis in a rabbit model of endothelial denudation in vivo.

The role of adenine nucleotides on vascular and platelet functions has long been established. Apyrase (CD39) takes part of a family of ecto-enzymes that hydrolyze adenosine diphosphate and adenosine triphosphate. The participation of apyrase in the thromboregulatory system is under study. An in vivo experimental model of acute arterial thrombosis was used to test the hypothesis that administering a soluble form of potato apyrase could prevent thrombus formation. Twenty-five white New Zealand male rabbits suffered balloon aortic endothelium denudation and, after 15 days, they were submitted to a thrombosis-triggering protocol with a procoagulant (Russel's viper venom) and epinephrine. After the thrombosis-triggering protocol, 12 animals received two soluble apyrase administrations intravenously (with 90 min intervals), while 13 control animals received no apyrase. Three hours after the triggering protocol, the animals were killed and the rate and area of arterial thrombosis were analyzed. The rate of thrombosis in the apyrase group was significantly lower than that of the control group (16.7 versus 69%, respectively; P = 0.015), as was the area of thrombosis (1.7 +/- 4.3 versus 21.7 +/- 37.4 mm2, respectively; P = 0.008). Our results confirm that apyrase participates in homeostasis through a potent anti-thrombotic effect.

Levels of cytokine receptor activator of nuclear factor kappaB ligand in gingival crevicular fluid in untreated chronic periodontitis patients.

BACKGROUND: Receptor activator of nuclear factor kappaB ligand (RANK-L) is a cytokine involved in the regulation of osteoclastogenesis in bone remodeling and inflammatory osteolysis. One of the major causes of tooth loss in humans is bone destruction. The aim of our study was to determine the presence of RANK-L in gingival crevicular fluid (GCF) samples from adult patients with untreated chronic periodontitis and in healthy controls. We also identified the RANK-L present in lesions undergoing episodic attachment loss from GCF. METHODS: GCF samples were collected from two periodontally affected sites (probing depth > or = 5 mm, attachment loss > or = 3 mm) in 20 patients (N = 40). After monitoring for 4 months, seven patients showed active periodontal disease, and GCF samples were collected from one active and one inactive site (N = 14 samples). The comparison with healthy controls was carried out by collecting GCF samples from 12 healthy volunteers (N = 24 samples). GCF was collected using a paper strip, and enzyme-linked immunosorbent assay (ELISA) was performed to determine the total amount of RANK-L. RESULTS: RANK-L was found in a higher proportion (85%) of samples from patients than from controls (46%). The total amount of RANK-L was significantly higher in patients (115.53 +/- 78.18 picograms [pg]) than in healthy subjects (63.08 +/- 55.08 pg) (P = 0.003). Active sites, presumably associated with tissue destruction, had significantly higher levels of RANK-L than their inactive counterparts (125.95 pg versus 91.80 pg, P = 0.007). CONCLUSION: GCF total amount of RANK-L is significantly increased in periodontal disease, supporting its role in the alveolar bone loss developed in this disease.

Tax posttranslational modifications and interaction with calreticulin in MT-2 cells and human peripheral blood mononuclear cells of human T cell lymphotropic virus type-I-associated myelopathy/tropical spastic paraparesis patients.

The human retrovirus human T cell lymphotropic virus type-I (HTLV-1) is the etiologic agent of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Axonal degeneration in HAM/TSP patients occurs without neuron infection, with the secreted viral Tax protein proposed to be involved. We previously found that Tax secreted into the culture medium of MT-2 cells (HTLV-1-infected cell line) produced neurite retraction in neuroblastoma cells differentiated to neuronal type. To assess the relevance of Tax posttranslational modifications on this effect, we addressed the question of whether Tax secreted by MT-2 cells and peripheral blood mononuclear cells (PBMCs) of HTLV-1-infected subjects is modified. The interaction of Tax with calreticulin (CRT) that modulates intracellular Tax localization and secretion has been described. We studied Tax localization and modifications in MT-2 cells and its interaction with CRT. Intracellular Tax in MT-2 cells was assessed by flow cytometry, corresponding mainly to a 71-kDa protein followed by western blot. This protein reported as a chimera with gp21 viral protein-confirmed by mass spectrometry-showed no ubiquitination or SUMOylation. The Tax-CRT interaction was determined by confocal microscopy and coimmunoprecipitation. Extracellular Tax from HAM/TSP PBMCs is ubiquitinated according to western blot, and its interaction with CRT was shown by coimmunoprecipitation. A positive correlation between Tax and CRT secretion was observed in HAM/TSP PBMCs and asymptomatic carriers. For both proteins inhibitors and activators of secretion showed secretion through the endoplasmic reticulum-Golgi complex. Tax, present in PBMC culture medium, produced neurite retraction in differentiated neuroblastoma cells. These results suggest that Tax, whether ubiquitinated or not, is active for neurite retraction.

Immunodominant role of CCHA subunit of Concholepas hemocyanin is associated with unique biochemical properties.

Hemocyanin, the oxygen transporter metallo-glycoprotein from mollusks, shows strong relationship between its notable structural features and intrinsic immunomodulatory effects. Here we investigated the individual contribution of CCHA and CCHB subunits from Concholepas hemocyanin (CCH) to in vivo humoral immune response and their pre-clinical evaluation as immunotherapeutic agent in a mice bladder cancer model, in relation to their biochemical properties. To this end, subunits were purified and well characterized. Homogeneous subunits were obtained by anionic exchange chromatography, and its purity assessed by electrophoretic and immunochemical methods. While each CCH subunit contains eight functional units showing partial cross reaction, the vibrational spectral analysis showed several spectral differences, suggesting structural differences between them. In addition, we demonstrated differences in the carbohydrate content: CCHA had a 3.6% w/w sugar with both N- and O-linked moieties. In turn, CCHB had a 2.5% w/w sugar with N-linked, while O-linked moieties were nearly absent. Considering these differences, it was not possible to predict a priori whether the immunogenic and immunotherapeutic properties of subunits might be similar. Surprisingly, both subunits by itself induced a humoral response, and showed an antitumor effect in the bladder carcinoma cell line MBT-2. However, when immunologic parameters were analyzed, CCHA showed better efficiency than CCHB. No allergic reactions or any toxic effects were observed in mice treated with CCHA, sustaining its potential therapeutic use. Our study supports that CCHA subunit accounts for the most important features involved in the immunogenicity of CCH, such as better hydrophilicity and higher content of carbohydrates.

MMP-13 and TIMP-1 determinations in progressive chronic periodontitis.

UNLABELLED: Matrix metalloproteinase (MMP)-13 is a collagenase involved in extracellular matrix degradation either by its direct degradative effects or by processing bioactive substrates. The aim of this study was to determine the levels of MMP-13 and tissue inhibitor of metalloproteinase (TIMP)-1 in gingival crevicular fluid (GCF) and gingival biopsies obtained from active and inactive sites during chronic periodontitis progression. MATERIALS AND METHODS: This was a longitudinal study in which chronic periodontitis patients with moderate to severe disease were included and followed until they developed progression determined by the tolerance method. GCF samples were obtained from periodontitis, active, inactive and healthy sites and additional gingival biopsies were taken from active and inactive sites. MMP-13 and TIMP-1 determinations were carried out by immunodot blots and immunowestern blots. RESULTS: In progressive periodontitis, MMP-13 and TIMP-1 remained unchanged between active and inactive sites, but as the TIMP-1 relative levels increased together with MMP-13 elevation in inactive samples, an inverse correlation was observed in active sites. Besides, MMP-13 was undetectable in healthy controls. CONCLUSION: Chronic periodontitis is characterized by increased MMP-13 expression. During disease progression, active sites tended to decrease TIMP-1 levels in association with MMP-13 elevation.