Ex vivo and in vivo biological behavior of Leishmania (Viannia) shawi

Since the first description of Leishmania (Viannia) shawi, few studies were performed with this parasite. In the present work, the in vivo and ex vivo behavior of L. (Viannia) shawi infection was studied using murine model. Peritoneal macrophages from BALB/c and C57BL/6 mice were infected with promastigotes in the stationary phase of growth; after 24 h, the infection index and nitric oxide (NO) levels in the supernatant of the cultures were analyzed. BALB/c and C57BL/6 mice were infected into the hind footpad, and at each 2 weeks, mice were sacrificed, and the histological changes of the skin inoculation site, parasitism, and humoral immune responses were evaluated during 8 weeks. Ex vivo experiments showed that macrophages of BALB/c presented higher infection index and lesser NO levels than macrophages of C57BL/6. In vivo experiments showed that BALB/c presented higher lesion size than C57BL/6 mice; similarly, the histopathological changes and the parasitism in skin were more exacerbate in BALB/c mice. In draining lymph nodes, the main change was increase of germinative centers, and parasites were detected from 6 weeks pi onwards in both mice strain. IgG was detected in BALB/c mice from 4 weeks, while in C57BL/6, from 6 weeks pi onwards. Taken together, these results indicate that BALB/c showed a classical behavior of susceptibility when compared to C57BL/6 mice.

Ex Vivo Lung Perfusion: Early Report of Brazilian Experience

Introduction. Only about 15% of the potential candidates for lung donation are considered suitable for transplantation. A new method for ex vivo lung perfusion (EVLP) can be used to evaluate and recondition ""marginal,"" nonacceptable lungs. We have herein described an initial experience with ex vivo perfusion of 8 donor lungs deemed nonacceptable. Materials and Methods. After harvesting, the lungs were perfused ex vivo with Steen Solution, an extracellular matrix with high colloid osmotic pressure. A membrane oxygenator connected to the circuit received gas from a mixture of nitrogen and carbon dioxide, maintaining a normal mixed venous blood gas level in the perfusate. The lungs were gradually rewarmed, reperfused, and ventilated for evaluation through analyses of oxygenation capacity, pulmonary vascular resistance (PVR), lung compliance (LC), and biopsy. Results. The arterial oxygen pressure (with inspired oxygen fraction of 100%) increased from a mean of 206 mm Hg in the organ donor at the referring hospital to a mean of 498 mm Hg during the ex vivo evaluation. After 1 hour of EVLP, PVR varied from 440-1454 dynes/sec/cm(5); LC was in the range of 26-90 mL/cmH(2)O. There was no histological deterioration after 10 hours of cold ischemia and 1 hour of EVLP. Conclusions. The ex vivo evaluation model can improve oxygenation capacity of ""marginal"" lungs rejected for transplantation. It has great potential to increase lung donor availability and, possibly, reduce time on the waiting list.

Lower cytokine secretion ex vivo by natural killer T cells in HIV-infected individuals is associated with higher CD161 expression

Objective: Natural killer T (NKT) cells are efficiently targeted by HIV and severely reduced in numbers in the circulation of infected individuals. The functional capacity of the remaining NKT cells in HIV-infected individuals is poorly characterized. This study measured NKT cell cytokine production directly ex vivo and compared these responses with both the disease status and NKT subset distribution of individual patients. Methods: NKT cell frequencies, subsets, and ex-vivo effector functions were measured in the peripheral blood mononuclear cells of HIV-infected patients and healthy controls by flow cytometry. We measured cytokines from NKT cells after stimulation with either a-galactosyl ceramide-loaded CD1d dimers (DimerX-alpha GalCer) or phorbol myristate acetate and ionomycin. Results: The frequencies of NKT cells secreting interferon-gamma and tumor necrosis factor-alpha were significantly lower in HIV-infected patients than healthy controls after DimerX-alpha GalCer treatment, but responses were similar after treatment with phorbol myristate acetate and ionomycin. The magnitude of the interferon-gamma response to DimerX-alpha GalCer correlated inversely with the number of years of infection. Both interferon-gamma and tumor necrosis factor-alpha production in response to DimerX-alpha GalCer correlated inversely with CD161 expression. Conclusion: The ex-vivo Th1 responses of circulating NKT cells to CD1d-glycolipid complexes are impaired in HIV-infected patients. NKT cell functions may be progressively lost over time in HIV infection, and CD161 is implicated in the regulation of NKT cell responsiveness. (C) 2009 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins

Human visceral leishmaniasis expresses Th1 pattern in situ liver lesions

The architectural and infiltrate pattern of liver human visceral leishmaniasis (HVL) have been systematically classified as typical, fibrogenic or nodular. Despite this histopathological classification, the immune response based on cytokines and cellular phenotypes have never been performed. The aim of this study was to determine the immunophenotypic pattern and cytokine profile of the nodular involvement of the Liver in HVL. We evaluated nine cases of the nodular form of HVL. In situ immune response was studied through cytokine analysis and immunohistochemical study for phenotype markers: IL-1, IL-4, IL-1 0, TNF-alpha, IFN-gamma, CD4+ T cells, CD8+ T cells, CD20, CD68, CD57 and macrophage activation was determined by evaluation of iNOS activity. HVL seems to be related to a better immune response. Amastigotes were rarely found on liver sections. Leishmania antigen expression was also rare and located in the inflammatory nodules. The lower expression of IL-4 and IL-10, moderate expression of TNF-alpha and IFN-gamma demonstrate a panorama of Th1 phenotype. The increased expression of NK cells could help in sustaining this model of response. This pattern of immune response is probably responsible for improvement in the parasite`s clearance from liver tissue and it is a prognostic marker of human visceral leishmaniasis. (C) 2008 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Reappraisal of the immunopathogenesis of disseminated leishmaniasis: in situ and systemic immune response

Disseminated leishmaniasis (DL) is an emerging form of Leishmania braziliensis infection characterised by multiple cutaneous lesions on different parts of the body and a high rate of mucosal involvement. Systemic production of TNF alpha and IFN-gamma in DL patients is lower than in cutaneous leishmaniasis (CL) caused by L braziliensis, which may account for parasite dissemination due to the decreased ability to control parasite growth. In this study, the systemic and in situ immune response of DL and CL patients was characterised through evaluation of chemokine and cytokine production. In situ evaluation showed similar production of IFN gamma, TNF alpha, IL-10, transforming growth factor-beta (TGF beta), chemokine (C-C motif) ligand 2 (CCL2), CCL3, CCL11 and chemokine (C-X-C motif) ligand 10 (CXCL10) in papular and ulcerative lesions from DL as well as in ulcerated lesions from CL. Serum levels of CXCL9, a chemokine that attracts 1-cells, was higher in serum from DL than from CL These data indicate that a decrease in the type 1 immune response in peripheral blood of DL patients is due to attraction of Leishmania antigen-activated T-cells to the multiple cutaneous lesions. This may account for the absence of or few parasites in the lesions and for the development of ulcers similar to those observed in CL (C) 2011 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Serotonergic neurotransmission in the dorsal raphe nucleus recruits in situ 5-HT2A/2C receptors to modulate the post-ictal antinociception

The post-ictal immobility syndrome is followed by a significant increase in the nociceptive thresholds in animals and humans. The aim of this study was to assess the involvement of the dorsal raphe nucleus (DRN) in the post-ictal antinociception. The second aim was to study the role of serotonergic intrinsic mechanisms of the DRN in this hypo-algesic phenomenon. Pentylenetetrazole (PTZ), an ionophore GABA-mediated Cl- influx antagonist, was peripherally used to induce tonic-clonic seizures in Wistar rats. The nociceptive threshold was measured by the tail-flick test. Neurochemical lesions of the DRN, performed with microinjection of ibotenic acid (1.0 mu g/0.2 mu L), caused a significant decrease of tonic-clonic seizure-induced antinociception, suggesting the involvement of this nucleus in this antinociceptive Process. Microinjections of methysergide (1.0 and 5.0 mu g/0.2 mu L), a non-selective serotonergic receptor antagonist, into DRN caused a significant decrease in the post-ictal antinociception in seizing animals, compared to controls, in all post-ictal periods Presently studied. These findings were corroborated by microinjections of ketanserin (at 1.0 and 5.0 mu g/0.2 mu L) into DRN. Ketanserin is an antagonist with large affinity for 5-HT2A/2C serotonergic receptors, which, in this Case, Caused a significant decrease in the tail-flick latencies in seizing animals, compared to controls after the first 20 min following tonic-clonic convulsive reactions. These results indicate that serotonergic neurotransmission of the DRN neuronal clusters is involved in the organization of the post-ictal hypo-algesia. The 5-HT2A/2C receptors of DRN neurons seem to be critically involved in the increase of nociceptive thresholds following tonic-clonic seizures. (c) 2008 Elsevier Inc, All rights reserved.

Diazepam decreases leukocyte-endothelium interactions in situ

High doses of diazepam reduce the inflammatory paw edema in rats. This effect was attributed to an action of diazepam on the Translocator Protein (TSPO). We evaluated the effects of diazepam (10 mg/kg, intraperitoneally) on leukocyte rolling and migration. In carrageenan-induced acute inflammation, diazepam decreased the interaction of leukocytes with endothelial cells (rolling) and the number of leukocytes in the mesentery (migration). RU486 (antagonist of glucocorticoid receptors) reduced the effects of diazepam on leukocyte rolling and migration, suggesting a participation of endogenous corticosteroids. We also showed that the effects of diazepam on leukocyte-endothelium interactions are mediated by nitric oxide (NO), since prior treatment with l-arginine (precursor of NO) partially precludes the inhibitory effects of diazepam; conversely, pretreatment with L-NAME (false substrate of the NO synthase) somewhat potentiates the effects of diazepam. The pathways that underlie the effects of diazepam remain to be further elucidated, but we believe that both local and systemic mechanisms may overlap to explain the influence of diazepam on leukocyte-endothelium interactions.

Influence of Cavity Preparation with Er:YAG Laser on Enamel Adjacent to Restorations Submitted to Cariogenic Challenge In Situ: A Polarized Light Microscopic Analysis

Background and Objectives: Er:YAG laser has been used for caries removal and cavity preparation, using ablative parameters. Its effect on the margins of restorations submitted to cariogenic challenge has not yet been sufficiently investigated. The aim of this study was to assess the enamel adjacent to restored Er:YAG laser-prepared cavities submitted to cariogenic challenge in situ, under polarized light microscopy. Study Design/Materials and Methods: Ninety-one enamel slabs were randomly assigned to seven groups (n = 13): I, II, III-Er:YAG laser with 250 mJ, 62.5 J/cm(2), combined with 2, 3, and 4 Hz, respectively; IV, V, VI-Er:YAG laser with 350 mJ, 87.5 J/cm(2), combined with 2, 3, and 4 Hz, respectively; VII-High-speed handpiece (control). Cavities were restored and the restorations were polished. The slabs were fixed to intra-oral appliances, worn by 13 volunteers for 14 days. Sucrose solution was applied to each slab six times per day. Samples were removed, cleaned, sectioned and ground to polarized light microscopic analysis. Demineralized area and inhibition zone width were quantitatively assessed. Presence or absence of cracks was also analyzed. Scores for demineralization and inhibition zone were determined. Results: No difference was found among the groups with regard to demineralized area, inhibition zone width, presence or absence of cracks, and demineralization score. Inhibition zone score showed difference among the groups. There was a correlation between the quantitative measures and the scores. Conclusion: Er:YAG laser was similar to high-speed handpiece, with regard to alterations in enamel adjacent to restorations submitted to cariogenic challenge in situ. The inhibition zone score might suggest less demineralization at the restoration margin of the irradiated substrates. Correlation between the quantitative measures and scores indicates that score was, in this case, a suitable complementary method for assessment of caries lesion around restorations, under polarized light microscopy. Lasers Surg. Med. 40:634-643, 2008. (c) 2008 Wiley-Liss, Inc.

Gels Containing MMP Inhibitors Prevent Dental Erosion in situ

Matrix metalloproteinase (MMP) inhibition has been shown to reduce dentin caries progression, but its role in dental erosion has not yet been assessed. This study tested the hypothesis that gels containing MMP inhibitors (epigallocatechin gallate-EGCG and chlorhexidine) can prevent dental erosion. Volunteers (n = 10) wore palatal devices containing bovine dentin blocks (n = 10/group) treated for 1 min with EGCG at 10 (EGCG10) or 400 mu M (EGCG400), chlorhexidine at 0.012%, F at 1.23% (NaF), and no vehicle (placebo). Erosion was performed with Coca-Cola (R) (5 min) 4X/day during 5 days. The wear, assessed by profilometry (mean +/- SD, mu m), was significantly reduced by the gels containing MMP inhibitors (0.05 +/- 0.02(a), 0.04 +/- 0.02(a), and 0.05 +/- 0.02(a) for EGCG10, EGCG400, and chlorhexidine, respectively) when compared with NaF (0.79 +/- 0.35(b)) and placebo gels (1.77 +/- 0.35(b)) (Friedman and Dunn`s tests, p < 0.01). The use of gels delivering MMP inhibitors was shown to prevent erosion and opens a new perspective for protection against dental erosion.

Effect of Different Fluoride Concentrations of Experimental Dentifrices on Enamel Erosion and Abrasion

It has been suggested that fluoride products are able to reduce erosive tooth wear. Thus, the purpose of this in vitro study was to evaluate the effect of dentifrices with different fluoride concentrations as well as of a low-fluoridated dentifrice supplemented with trimetaphosphate (TMP) on enamel erosion and abrasion. One hundred twenty bovine enamel blocks were assigned to the following experimental dentifrices: placebo, 1,100 mu g F/g, 500 mu g F/g plus 3% TMP and 5,000 mu g F/g. The groups of enamel blocks were additionally subdivided into conditions of erosion (ERO) and of erosion plus abrasion (ERO + ABR). For 7 days, the blocks were subjected to erosive challenges (immersion in Sprite (R) 4 times a day for 5 min each time) followed by a remineralizing period (immersion in artificial saliva between erosive challenges for 2 h). After each erosive challenge, the blocks were exposed to slurries of the dentifrices (10 ml/sample for 15 s). Sixty of the blocks were additionally abraded by brushing using an electric toothbrush (15 s). The alterations of the enamel were quantified using the Knoop hardness test and profilometry (measurements in micrometers). The data were analyzed using a 2-way ANOVA test followed by a Bonferroni correction (p < 0.05). In in vitro conditions, the 5,000 mu g F/g and 500 mu g F/g plus 3% TMP dentifrices had a greater protective effect when compared with the 1,100 mu g F/g dentifrice, under both ERO and ERO + ABR conditions. The results suggest that dentifrices alone are not capable of completely inhibiting tooth wear. Copyright (C) 2010 S. Karger AG, Basel

Effect of Iron on Matrix Metalloproteinase Inhibition and on the Prevention of Dentine Erosion

It is known that some metal salts can inhibit matrix metalloproteinase (MMP) activity, but the effect of iron has not been tested yet. On the other hand, it has recently been suggested that MMP inhibition might influence dentine erosion. Based on this, the aims of this study were: (1) to test in vitro the effect of FeSO(4) on MMP-2 and -9 activity, and (2) to evaluate in situ the effect of FeSO(4) gel on dentine erosion. MMP-2 and -9 activities were analysed zymographically in buffers containing FeSO(4) in concentrations ranging between 0.05 and 1.5 mmol/l or not. Volunteers (n = 10) wore devices containing bovine dentine blocks (n = 60) previously treated with the following gel treatments: FeSO(4) (1 mmol/l FeSO(4)), F (NaF 1.23%; positive control) and placebo (negative control). The gels were applied once and removed after 1 min. Erosion was performed extraorally with Coca-Cola 4 times per day for 5 min over 5 days. Dentine wear was evaluated by profilometry. The data were analysed by Kruskal-Wallis and Dunn`s tests (p < 0.05). FeSO(4) inhibited both MMP-2 (IC(50) = 0.75 mmol/l) and MMP-9 (IC(50) = 0.50 mmol/l) activities. In the in situ experiment, the mean wear (+/- SD) found for the F gel (0.79 8 +/- 0.08 mu m) was significantly reduced in more than 50% when compared to the placebo gel (1.77 +/- 0.33 mu m), but the FeSO(4) gel completely inhibited the wear (0.05 +/- 0.02 mu m). Since FeSO(4) was able to inhibit MMP in vitro, it is possible that the prevention of dentine wear by the FeSO(4) gel in situ might be due to MMP inhibition, which should be investigated in further studies. Copyright (C) 2010 S. Karger AG, Basel

Preventive Effect of Commercial Desensitizing Toothpastes on Bovine Enamel Erosion in vitro

This study evaluated in vitro commercial desensitizing toothpastes with respect to the prevention of erosion and explored the effect of their agents alone or in combination with fluoride. Bovine enamel blocks were randomly allocated to five groups of 20 and exposed to: Sensodyne ProNamel (1,425 ppm F as NaF, 5% KNO(3)), Sensodyne Original (no fluoride, 10% SrCl(2)), Colgate Sensitive (1,450 ppm F as sodium monofluorophosphate, 5% K citrate), Crest (fluoride-only toothpaste, 1,100 ppm F as NaF) and water (negative control). A second experiment was conducted with experimental dentifrices containing fluoride (NaF, 1,100 ppm F), 10% SrCl(2), 5% KNO(3) or 5% K citrate alone or the latter three combined with F. The samples were submitted to four cycles, alternating demineralization (cola, 10 min) and remineralization (artificial saliva, 1 h). Before and between cyclic de- and remineralization, blocks were treated with slurries of the respective toothpastes or water (1 min). Erosive tissue loss was analyzed by profilometry. Data were analyzed by Kruskal-Wallis and Dunn`s tests (p < 0.05). The mean erosion depth (+/- SE, mu m) was significantly less for Colgate Sensitive (0.04 +/- 0.00), Sensodyne Original (0.06 +/- 0.01) and Crest (0.07 +/- 0.01) than for Sensodyne ProNamel (2.36 +/- 0.25) or water (2.92 +/- 0.24), which did not significantly differ from each other. Both F and the desensitizing agents alone reduced erosion, but no additive effect was found. In addition, the combination of F and KNO(3) did not reduce erosion. These in vitro results suggest that the presence of fluoride or desensitizing substances in toothpastes, alone or in combination, can reduce erosion of enamel, but this is not valid for all the formulations. Copyright (C) 2010 S. Karger AG, Basel

Effect of ion supplementation of a commercial soft drink on tooth enamel erosion

Acidic soft drinks are potentially erosive for dental hard tissues. This in vitro study evaluated the effect of calcium, fluoride, iron and phosphate, supplemented alone or in combination to a commercial citric acid-based carbonated beverage on dental erosion. Ninety enamel samples (4 x 4 x 3 mm) were randomly allocated to nine groups (n = 10): G1 - pure beverage (control); G2 - with 1 mM Ca; G3 - with 0.047 mM F; G4 - with 1 mM Fe; G5 - with 1 mM P; G6 - with 1 mM Ca and 0.047 mM F; G7 - with 1 mM Ca and 1 mM P; G8 - with 1 mM Fe and 0.047 mM F; and G9 - with 1 mM Ca, 1 mM P, 0.047 mM F and 1.0 mM Fe. The samples were subjected to six pH cycles over a 24-h period. In each cycle, the samples were immersed in pure or modified beverage (1 min) and in artificial saliva (59 min). During the remaining period (18 h), the samples were maintained in artificial saliva. Enamel loss was assessed by profilometry (mm). Data were tested using ANOVA and Tukey`s tests (p < 0.05). Highest enamel losses were observed in the control group (G1) and in the groups containing Fe (G4 and G8). The groups containing Ca (G2 and G6) showed significantly less wear compared to control. In conclusion, the modification of an erosive soft drink with low concentrations of Ca with or without F may reduced its erosive potential.

Effect of prolonged erosive pH cycling on different restorative materials

This in vitro study evaluated the effect of a prolonged erosive pH cycling on the superficial microhardness change (SMHC) and the erosive wear of different restorative materials. Eighty enamel specimens with prepared cavities of 1.5 x 1.5 mm were randomly divided into eight groups according to the restorative materials used for the fillings (RMGI - resin-modified glass-ionomer, CGI - conventional glass-ionomer, CR- composite resin, A - amalgam) and immersion media used (ERO - erosive medium or SAL - artificial saliva). During 35 days, half of the specimens were immersed in a cola drink (ERO), for 5 min, three times a day, and they remained in SAL between the erosive cycles. The other half of the specimens was immersed in SAL only, for the entire experimental period (control). Data were tested for significant differences by anova and Tukey`s tests (P < 0.05). Scanning electron microscopy images were made to illustrate the enamel erosive wear and restorative materials alterations. The mean SMHC (%) and mean erosive wear (mu m) of the materials were: RMGI-ERO (30/0.5); CGI-ERO (37/0.5); CR-ERO (-0.3/0.3); A-ERO (-4/0.3); RMGI-SAL (4/0.4); CGI-SAL (-6/0.4); CR-SAL (-3/0.2) and A-SAL (2/0.4). Scanning electron microscopy images showed pronounced enamel erosive wear on groups submitted to erosive pH cycling when compared with groups maintained in saliva. In conclusion, the prolonged pH cycling promoted significantly higher alterations (SMHC and erosive wear) on the glass-ionomer cements than the CR and amalgam.

Comparison of GPX with or without solvent and hand files in removing filling materials from root canals-An ex vivo study

Objective. The aim of this study was to compare GPX instruments and hand files for gutta-percha removal. Study design. Fifty maxillary central incisors with a single straight canal were instrumented and filled. The teeth were divided into 5 groups of 10 specimens each, according to the gutta-percha removal techniques: group 1: GPX (21-mm-long teeth); group 2: GPX and xylol as solvent (21-mm-long teeth); group 3: GPX (25-mm-long teeth); group 4: GPX and xylol as solvent (25-mm-long teeth); and group 5: hand files and xylol as solvent. The amount of time for gutta-percha removal and the number of fractured instruments were evaluated. Radiographs were taken, and the teeth were grooved longitudinally and split. The area of residual debris was measured using Sigma Scan software. Results. The time for filling material removal was significantly shorter when GPX was used (P < .05). Overall, hand files and solvent produced fewer remnants of filling materials (P < .05). In the GPX 25 mm-long teeth group, the filling material was not removed in the apical third. Conclusions. Under the experimental conditions, the GPX instruments proved to be faster than hand instruments in removing root filling materials; however, hand instruments left a smaller amount of residual filling materials on the canal walls. The GPX instruments did not pull the gutta-percha beyond its tip. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2010; 110: 675-680)