The inhibitory effect of MK886 on the inflammatory process caused by Paracoccidioidomycosis brasiliensis infection in genetically selected mice

Leukotrienes are classic inflammatory response mediators considered chemotactic agents and microbicidal activity regulators in cells of the innate immune system, playing a protective role against different infectious agents. In this study, we investigated the involvement of leukotrienes in the course of murine paracoccidioidomycosis based on the following immunologic parameters: cell influx, mieloperoxydase activity, NO production, cytokine production, and fungal recovery in lungs of mice selected according to the intensity of their low (AIRmin) and high (AIRmax) acute inflammatory response. Infection by P. brasiliensis induced considerable production of IL-6, IL-10, IFN-gamma and TNF-alpha cytokines, and led to cell recruitment, as well as NO production in lungs at different study periods. In animals treated with MK886, a leukotriene biosynthesis inhibitor, IFN-gamma, IL-6 and TNF-alpha production was lower, while neutrophil influx and NO production decreased. These results may explain the higher fungal load in lungs of animals in which leukotriene synthesis was inhibited, suggesting that leukotrienes have a possible protective role in experimental paracoccidioidomycosis. AIRmax animals had lower fungal load in comparison with AIRmin ones, which can be related to the AIR phenotype regarding neutrophil migration, besides lower production of NO and pro-inflammatory cytokines. Thus, mice presenting AIRmax background are more resistant to infection by P. brasiliensis.

Biological properties of medicinal plants: a review of their antimicrobial activity

Plants have been used for thousands of years to flavor and conserve food, to treat health disorders and to prevent diseases including epidemics. The knowledge of their healing properties has been transmitted over the centuries within and among human communities. Active compounds produced during secondary vegetal metabolism are usually responsible for the biological properties of some plant species used throughout the globe for various purposes, including treatment of infectious diseases. Currently, data on the antimicrobial activity of numerous plants, so far considered empirical, have been scientifically confirmed, concomitantly with the increasing number of reports on pathogenic microorganisms resistant to antimicrobials. Products derived from plants may potentially control microbial growth in diverse situations and in the specific case of disease treatment, numerous studies have aimed to describe the chemical composition of these plant antimicrobials and the mechanisms involved in microbial growth inhibition, either separately or associated with conventional antimicrobials. Thus, in the present work, medicinal plants with emphasis on their antimicrobial properties are reviewed.

Ectopic mineralization of articular cartilage in the bullfrog Rana catesbeiana and its possible involvement in bone closure

Mineralization of the articular cartilage is a pathological condition associated with age and certain joint diseases in humans and other mammals. In this work, we describe a physiological process of articular cartilage mineralization in bullfrogs. Articular cartilage of the proximal and distal ends of the femur and of the proximal end of the tibia-fibula was studied in animals of different ages. Mineralization of the articular cartilage was detected in animals at 1 month post-transformation. This mineralization, which appeared before the hypertrophic cartilage showed any calcium deposition, began at a restricted site in the lateral expansion of the cartilage and then progressed to other areas of the epiphyseal cartilage. Mineralized structures were identified by von Kossa's staining and by in vivo incorporation of calcein green. Element analysis showed that calcium crystals consisted of poorly crystalline hydroxyapatite. Mineralized matrix was initially spherical structures that generally coalesced after a certain size to occupy larger areas of the cartilage. Alkaline phosphatase activity was detected at the plasma membrane of nearby chondrocytes and in extracellular matrix. Apoptosis was detected by the TUNEL (TDT-mediated dUTP-biotin nick end-labeling) reaction in some articular chondrocytes from mineralized areas. The area occupied by calcium crystals increased significantly in older animals, especially in areas under compression. Ultrastructural analyses showed clusters of needle-like crystals in the extracellular matrix around the chondrocytes and large blocks of mineralized matrix. In 4-year-old animals, some lamellar bone (containing bone marrow) occurred in the same area as articular cartilage mineralization. These results show that the articular cartilage of R. catesbeiana undergoes precocious and progressive mineralization that is apparently stimulated by compressive forces. We suggest that this mineralization is involved in the closure of bone extremities, since mineralization appears to precede the formation of a rudimentary secondary center of ossification in older animals.

Development of an enzyme-linked immunosorbent assay for detection of IgM antibodies to Babesia bigemina in cattle

A crude antigenic preparation of Babesia bigemina was used to develop an ELISA for the detection of IgM antibodies. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkerboard titrations. Negative sera from cattle imported from tick-free areas, serum samples collected from infected B. bigemina cattle were used to validate the test. The specificity was 94% and sensitivity of the Elisa 87.5%. Sera from 385 cattle deriving from areas free from tick-borne diseases, which were submitted to a preimmunization process, were screened by this technique. The Elisa detected seroconversion on the 14th day post-inoculation in animals either infested with Boophilus microplus ticks (infected with B. bigemina), or inoculated with B. bigemina infected blood. Antibody titers decreased after day 33; however, all animals remained positive until the end of the experiment (124 days). The ELISA described may prove to be an appropriate serological test for the detection of IgM antibodies against B. bigemina.

Microbial populations and activities in sewage sludge and phosphate fertilizer-amended soil

A greenhouse study was conducted to determine the number of microbial populations and activities in sewage sludge and phosphate fertilizer-amended dark red latosoil for cultivation of tomato plants. Sewage sludge was applied at doses of 0, 10, 20, 40, 80 and 160 t ha(-1), and phosphate (P2O5) at doses of 0, 100, 200, 400 and 800 kg ha(-1). The bacterial populations increased as a function of sewage sludge and phosphate application. Fungal populations were not affected by the application of phosphate alone but were increased by the application of sewage sludge. Phosphate doses higher than 100-200 kg ha(-1) in combination with sewage sludge inhibited both bacterial and fungal growth. The responses determined by microbial counts were reflected in the microbial biomass values, with a more significant effect of sewage sludge than of phosphate or of a combination of both. These results confirm the need for a carbon and energy source (represented here by sewage sludge) for microbial growth in a soil poor in organic matter. Dehydrogenase and urease activities reflected the results of the microbial populations due to the effect of sewage sludge and phosphate, but no satisfactory result was obtained for phosphatase. Urease activity was expressed by a linear regression equation as the result of the effect of sewage sludge, and by a quadratic regression equation as the result of the effect of phosphate. All parameters investigated showed a significant correlation with bacterial counts but not with fungal counts, indicating a greater effect of sewage sludge and phosphate on bacteria than on fungi.

Inflammation-induced modulation of cellular galectin-1 and-3 expression in a model of rat peritonitis

Objective and design: To investigate the effect of galectin-1 (Gal-1) and -3 (Gal-3) on leukocyte migration and analyze the expression of both galectins in inflammatory cells using a model of rat peritonitis.Material or Subjects: Sprague-Dawley rats (n = 4 per group).Treatment: Peritonitis was induced in animals through intraperitoneal injection of carrageenin (1.5 mg/kg) and rat mesenteries were analyzed at different time points (0, 4, 24 and 48h). For pharmacological treatment, rats received intravenous injection of Gal-1 or -3 (3 mu g/kg) followed by carrageenin.Methods: Western blotting and immunoelectron microscopy analysis. Statistical analysis was performed using ANOVA followed by Bonferroni test.Results: Pharmacological treatment with Gal-1, but not Gal-3, inhibited (similar to 50%) leukocyte recruitment into the peritoneal cavity at 4h time-point. In this early phase, immunogold staining of mesenteries showed a diminished Gal-3 expression in degranulated mast cells and Gal-1 in transmigrated neutrophils (similar to 20% reduction compared to intravascular cells). In the later phases (24 and 48 h), leukocyte turnover was associated with augmented Gal-1 expression in neutrophils and macrophages and Gal-3 in mast cells and macrophages.Conclusions: These results point to a balanced expression of cell-associated-Gal-1/Gal-3 and might impact on the development of new therapeutic strategies for inflammatory diseases.

Alloxan-Induced Diabetes Triggers the Development of Periodontal Disease in Rats

Background. Periodontal disease in diabetic patients presents higher severity and prevalence; and increased severity of ligature-induced periodontal disease has been verified in diabetic rats. However, in absence of aggressive stimuli such as ligatures, the influence of diabetes on rat periodontal tissues is incompletely explored. The aim of this study was to evaluate the establishment and progression of periodontal diseases in rats only with diabetes induction. Methodology/Principal Findings. Diabetes was induced in Wistar rats (n = 25) by intravenous administration of alloxan (42 mg/kg) and were analyzed at 1, 3, 6, 9 and 12 months after diabetes induction. The hemimandibles were removed and submitted to radiographical and histopathological procedures. A significant reduction was observed in height of bone crest in diabetic animals at 3, 6, 9 and 12 months, which was associated with increased numbers of osteoclasts and inflammatory cells. The histopathological analyses of diabetic rats also showed a reduction in density of collagen fibers, fibroblasts and blood vessels. Severe caries were also detected in the diabetic group. Conclusions/Significance. The results demonstrate that diabetes induction triggers, or even co-induces the onset of alterations which are typical of periodontal diseases even in the absence of aggressive factors such as ligatures. Therefore, diabetes induction renders a previously resistant host into a susceptible phenotype, and hence diabetes can be considered a very important risk factor to the development of periodontal disease.

Levels of matrix metalloproteinase-2 and metalloproteinase-9 in the cerebrospinal fluid of dogs with visceral leishmaniasis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Municipal solid waste compost amendment in agricultural soil: changes in soil microbial biomass

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

In vitro Anthelmintic effect of Melia azedarach L. and Triclinia claussenii C. against sheep gastrointestinal nematodes

The control of parasitic diseases in small ruminants is mainly done with the use of synthetic anthelmintics. However, incorrect and indiscriminate use of these products has caused the emergence of parasite resistance. Plants with anthelmintic activity are used in folk veterinary medicine, but it is necessary to investigate and scientifically validate low-cost phytotherapeutic alternatives for future use to control gastrointestinal nematodes in small ruminants by family farmers. Thus, the aim of this study was to evaluate the in vitro anthelmintic effect of plant extracts from Melia azedarach and Trichilia claussenii by the egg hatch test (EHT) and larval development test (LDT) against sheep gastrointestinal nematodes. The hexane extract of M. azedarach fruits was extracted through cold percolation and the methanol extract of T. claussenii leaves was obtained by extraction at room temperature in solvents in order of increasing polarity. The efficacy results were analyzed using the Probit program of SAS. The M. azedarach extract showed a LC50 of 572.2 mu g/mL and LC99 of 1137.8 mu g/mL in the EHT, and LC50 of 0.7 mu g/mL and LC99 of 60.81 mu g/mL in the LDT. In turn, the T. claussenii extract presented a LC50 of 263.8 mu g/mL and LC99 of 522.5 mu g/mL in the EHTand LC50 of 1.11 mu g/mL and LC99 of 26.4 mu g/mL in the LDT. Comparing the extracts of the species from the Meliaceae family, T. claussenii showed greater anti-parasite potential in vitro than M. azedarach. However, studies on the isolated compounds, toxicity and administration forms to animals are also needed to validate low-cost alternative herbal remedies for use to control gastrointestinal nematodes by family farmers. (C) 2011 Elsevier B.V. All rights reserved.

Evaluation of homologous, heterologous, and affinity conjugates for the serodiagnosis of Toxoplasma gondii and Neospora caninum in maned wolves (Chrysocyon brachyurus)

Use of serological tests in the diagnosis of infectious diseases in wild animals has several limitations, primarily the difficulty of obtaining species-specific reagents. Wild canids, such as maned wolves (Chrysocyon brachyurus), are highly predisposed to infection by Toxoplasma gondii and, to a lesser extent, to Neospora caninum. The aim of the present study was to evaluate homologous, heterologous, and affinity conjugates in enzyme-linked immunosorbent assays (ELISAs) and indirect fluorescent antibody tests (IFATs) for detecting immunoglobulin (Ig) G antibodies against T. gondii and N. caninum in maned wolves. Serum samples were obtained from 59 captive animals in Brazil and tested by ELISA for T. gondii serology and IFAT for N. caninum serology using 3 different enzymatic and fluorescent conjugates: homologous (guinea pig anti-maned wolf IgG-peroxidase and -fluorescein isothiocyanate [FITC]), heterologous (rabbit anti-dog IgG-peroxidase and -FITC), and affinity (protein A-peroxidase and -FITC). Seropositivity to T. gondii was comparable among the homologous (69.5%), heterologous (74.6%), and affinity (71.2%) enzymatic conjugates. A significant positive correlation was found between the antibody levels determined by the 3 enzymatic conjugates. The highest mean antibody levels (ELISA index = 4.5) were observed with the protein A-peroxidase conjugate. The same seropositivity to N. caninum (8.5%) was found with the homologous and heterologous fluorescent conjugates, but protein A-FITC was not able to detect or confirm any positive samples with homologous or heterologous conjugates. Our results demonstrate that homologous, heterologous, and affinity conjugates might be used in ELISA for serological assays of T. gondii in wild canids, whereas for N. caninum infection, only the homologous or heterologous fluorescent conjugates have been shown to be useful. © American Society of Parasitologists 2005.

Effect of rotary instrumentation and of the association of calcium hydroxide and chlorhexidine on the antisepsis of the root canal system in dogs

This study aimed at evaluating the antisepsis of the root canal system (RCS) and periapical region (PR) provided by rotary instrumentation associated with chlorhexidine + calcium hydroxide as intracanal medicament. Chronic periapical lesions were induced in 26 pre-molar roots in two dogs. After microbiological sampling, automatic instrumentation using the Profile system and irrigation with 5.25% sodium hypochlorite solution, with a final rinse of 14.3% EDTA followed by profuse irrigation with physiological saline were carried out in 18 root canals. After drying the canals, a paste based on calcium hydroxide associated with a 2% chlorhexidine digluconate solution was placed inside them. After 21 days, the medication was removed, leaving the root canals empty and coronally sealed. After 96 hours, a final microbiological sample was obtained, followed by histomicrobiological processing by the Brown & Brenn method. Eight untreated root canals represented the control group (C-G). Based on the Mann-Whitney test at a confidence level of 5% (p < 0.05), the procedures of antisepsis used offered significant efficacy (p < 0.05) resulting in 100.0% of the canals free of microorganisms. In the C-G, an elevated incidence of various microbial morphotypes was confirmed in all sites of the RCS, with the presence of microbial colonies in the periapical region. In contrast, the experimental group showed a similar pattern of infection in the RCS, although less intense and a reduced level of periapical infection (p < 0.05). It was concluded that adequate instrumentation followed by the application of calcium hydroxide + chlorhexidine offered significant elimination of microorganisms.

Detection and neutralization of venom by ovine antiserum in experimental envenoming by Bothrops jararaca

In this study we optimized an enzyme-linked immunosorbent assay (ELISA) to evaluate bothropic venom levels in biological samples. These samples were obtained by two distinct protocols. In the first one, Swiss mice were injected with 1 LD 50 of Bothrops jararaca (B. jararaca) venom and 15 minutes later, animals were treated with ovine antibothropic serum. Blood and spleen homogenate samples were obtained 6 hours after antiserum therapy. Ovine antibothropic serum significantly neutralized venom levels in serum and spleen. In the second protocol, BALB/c mice were injected with 1 LD 50 of bothropic venom by either intraperitoneal (IP) or intradermal (ID) route and venom levels were evaluated 1, 3 and 6 hours after, in blood, spleen homogenates and urine. Serum and splenic venom levels were significantly higher in animals envenomed by IP route comparing with animals envenomed by ID route. Higher venom levels were also detected in urine samples from animals envenomed by IP route. However, these differences were not statistically significant. These results demonstrated that the optimized ELISA was adequate to quantify venom levels in different biological samples. This assay could, therefore, substitute the in vivo neutralizing assay and also be useful to evaluate the severity of human and experimental envenomations.

Periapical tissue healing after post space preparation with or without use of a protection plug and root canal exposure to the oral environment. Study in dogs

The purpose of this study was to evaluate the influence of coronal leakage on the healing of dogs' periapical tissues after root canal filling, post space preparation and protection or not with a temporary sealer plug. Forty root canals of dogs' teeth were instrumented and filled by the lateral condensation technique with gutta-percha points and Endomethasone or CRCS sealers. After post space preparation, the remaining filling material was protected or not with a plug of temporary Coltosol sealer and exposed to the oral environment for 90 days. Thereafter, the animals were sacrificed and the specimens were removed and prepared for histomorphological and histobacteriological analysis. The findings revealed 35% of microbial leakage in the groups without plugs and 15% of leakage in the groups with plugs. Statistical analysis showed that the use of a Coltosol plug improved significantly the histomorphological results regardless of the type of root canal sealer (p=0.05) and that CRCS and Endomethasone sealers showed similar results (p>0.05).

Advances in sickle cell disease treatment: From drug discovery until the patient monitoring

Sickle Cell Disease (SCD) is one of the most prevalent hematological diseases in the world. Despite the immense progress in molecular knowledge about SCD in last years few therapeutical sources are currently available. Nowadays the treatment is performed mainly with drugs such as hydroxyurea or other fetal hemoglobin inducers and chelating agents. This review summarizes current knowledge about the treatment and the advancements in drug design in order to discover more effective and safe drugs. Patient monitoring methods in SCD are also discussed. © 2011 Bentham Science Publishers Ltd.