Lipopolysaccharides induce changes in the visceral pigmentation of Eupemphix nattereri (Anura: Leiuperidae)

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

In vitro effects of calcium hydroxide and polymyxin B on endotoxins in root canals

Objectives. To evaluate the effects of intracanal medicaments on endotoxins in root canals.Methods. Seventy-five freshly extracted maxillary incisors were used in this study. The crowns of teeth were sectioned near the CEJ in order to standardize the root length to 14 mm. The root canals were instrumented to an apical size #50 file and irrigated with 1% sodium hypochlorite solution and sterilized with 60 Co gamma irradiation. Standardized suspension containing Escherichia coli endotoxin was inoculated into the 60 root canals. The specimens were randomly assigned to 5 groups (n=15), according to the intracanal medicament used: (G1) calcium hydroxide; (G2) polymyxin B; (0) combination neomycin-potymyxin B-hydrocortisone; (G4) positive control (no intracanal medicament); (G5) negative control (no endotoxin and no intracanal medicament). After 7 days, the detoxification of endotoxin was evaluated by Limulus lysate assay and antibody production in B-tymphocytes culture.Results. Groups 1, 2 and 5 presented the best results by Limulus lysate and were significantly different to groups 3 and 4 (p<0.05). Stimulation of antibodies production in cell culture by groups 1 and 6 was smaller and statistically different than groups 2, 3, 4 and 5 (p<0.05). Groups 2 and 5 induced a small increase in the antibodies production in relation to the groups 1 and 6. Groups 3 and 4 induced a significant increase of antibodies production (p<0.05).Conclusions. The calcium hydroxide and polymyxin B intracanal medicaments detoxified endotoxin in root canals and altered the properties of LPS to stimulate the antibody production by B-Lymphocytes. The combination neomycin-polymyxin B-hydrocortisone did not detoxified endotoxin. (C) 2004 Elsevier Ltd. All rights reserved.

Effects of lidocaine on lipopolysaccharide-induced synovitis in horses

Injetou-se lidocaína (100mg 2%) na articulação do carpo para avaliar a resposta inflamatória induzida pela injeção (1,5ng) intra-articular de lipopolissacarídeo (LPS) de E. coli. Utilizaram-se 17 cavalos Mangalarga não castrados, entre dois e três anos, divididos em três grupos. No carpo esquerdo (CE) administrou-se solução fisiológica a 0,9% (SAL) e no carpo direito (CD) uma das seguintes combinações: grupo A (n=6) LPS mais SAL, grupo B (n=6) LPS mais lidocaína e grupo C (n=5) lidocaína mais SAL. Amostras do líquido sinovial e de sangue foram colhidas imediatamente antes da injeção de LPS (T0) e às 1,30 (T1), 3 (T2), 6 (T3), 12 (T4) e 36 horas (T5) após a injeção. Variáveis clínicas e físicas, e características bioquímicas e celulares do líquido sinovial foram avaliadas nos mesmos tempos. A resposta inflamatória local e sistêmica foi mensurada pela concentração do TNF-alfa no soro e líquido sinovial. Observou-se aumento da concentração do TNF-alfa nas articulações injetadas com LPS às 3h no grupo A e de 1,30 às 3h no grupo B. Concluiu-se que o LPS induziu o processo inflamatório e que a lidocaína não inibiu nem atenuou a sinovite induzida pelo LPS, nem a síntese e liberação de TNF-alfa .

Histological study of the effect of some irrigating solutions on bacterial endotoxin in dogs

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

Polymerization shrinkage: effects of boundary conditions and filling technique of resin composite restorations

Objective: To evaluate the linear polymerization shrinkage (LPS) and the effect of polymerization shrinkage of a resin composite and resin-dentin bond strength under different boundary conditions and filling techniques.Methods: Two cavities (4 x 4 x 2 MM) were prepared in bovine incisors (n = 30). The teeth were divided into three groups, according to boundary conditions: In group TE, the total-etch technique was used. In group EE, only enamel was conditioned, and in group NE, none of the watts of the cavities were conditioned. A two-step adhesive system was applied to all cavities. The resin composite was inserted in one (B) or three increments (1), and tight-cured with 600 mW/cm(2) (80 s). The LPS (%) was measured in the top-bottom direction, by placing a probe in contact with resin composite during curing. Enamel and total mean gap widths were measured (400 x) in three slices obtained after sectioning the restorations. Then, the slices were sectioned again, either to obtain sticks from the adhesive interface from the bottom of the cavity or to obtain resin composite sticks (0.8 mm(2)) to be tested for tensile strength (Kratos machine, 0.5 mm/min). The data was subjected to a two-way repeated measures ANOVA and Tukey's test for comparison of the means (alpha = 0.05).Results: the highest percentage of LPS was found for the TE when bulk fitted, and the lowest percentage of LPS was found in the Hand NE when incrementally fitted. The resin dentin bond strength was higher and the total mean gap width was tower for TE group; no significant effect was detected for the main factor fitting techniques. No difference was detected for the tensile strength of resin composite among the experimental groups.Conclusions: the filling technique is not able to minimize effects of the polymerization shrinkage, and bonding to the cavity watts is necessary to assure reduced mean gap width and high bond strength values. (C) 2004 Elsevier Ltd. All rights reserved.

In vitro effects of endodontic irrigants on endotoxins in root canals

Objective. The objective of this study was to evaluate the effects of endodontic irrigants on endotoxins in root canals.Study design. Ninety-eight single-root human teeth were used. Escherichia coli endotoxin was inoculated into 84 root canals. All root canals were enlarged and assigned to 7 groups (n = 14), according to solution used. Group 1 (G1): 2.5% NaOCl; G2: 5.25% NaOCl; G3: 2% chlorhexidine; G4: 0.14% calcium hydroxide; G5: polymyxin B; G6: positive control, saline solution; G7: negative control (no endotoxin). Two samplings of root canal were accomplished: immediate and after 7 days. Detoxification of endotoxin was evaluated by Limulus assay and antibody production in B-lymphocyte culture. Results were analyzed by Kruskal-Wallis/Dunn and ANOVA/Tukey.Results. At the immediate and second samplings, groups G4, G5, and G7 presented the best results, significantly different from groups G1, G2, G3, and G6 (P = .05).Conclusions. Calcium hydroxide and polymyxin B detoxified endotoxin in root canals and altered properties of LPS to stimulate the antibody production by B-lymphocytes. Sodium hypochlorite and chlorhexidine did not detoxify endotoxin.

Fever response induced by intravenous and intracerebrovascular injection of pyrogen in thyroidectomised and protein-calorie malnourished rabbits

The development of a fever in response to intravenous (IV, 1.5 μg/kg body mass) and intracerebroventricular (ICV, 1.5 μg/animal) injections of Escherichia coli lipopolysaccharide (LPS) was studied in control, thyroidectomised and protein-calorie malnourished rabbits (New Zealand Whites, n = 55). ICV injection of LPS is control rabbits produced a fever response, the characteristics of which differed from those obtained after IV pyrogen injection. Thyroid deficiency caused an attenuated fever response, irrespective of whether LPS had been administered by IV or ICV injection. Protein-calorie malnourished rabbits showed a smaller fever response after IV or ICV pyrogen injections. Malnourished rabbits, refed over a period of 15 days, showed a typical biphasic fever response, but with lower magnitude than controls. The results of these experiments suggest that ICV injection of LPS is not an appropriate model for the study of fever mechanisms in disease states, and that the attenuated fever response observed in protein-calorie malnourished rabbits may be related, at least in part, to a decreased ability to produce the endogenous pyrogen interleukin-1.

Use of lipopolysaccharides to characterize Bradyrhizobium spp.

Three methods of extraction of lipopolysaccharide (LPS) were compared-the conventional hot phenol-water method with 40% phenol, a modified form of this method using 10% phenol, and the hot saline method. Good recovery of LPS was achieved by each of the three methods, with the LPS found in the aqueous phase with the two phenol-based procedures. The application of SDS-PAGE to the LPS extracts, followed by silver staining, showed similar banding with all three methods of extraction. When the hot saline extraction LPS fraction from eight strains of Bradyrhizobium spp. and eight strains of Bradyrhizobium japonicum was compared with SDS-PAGE, characteristic profiles were achieved. Serological analysis of eight strains of Bradyrhizobium spp., using antisera prepared against whole cells in agglutination reactions, showed extensive sharing of antigens. When antisera was prepared using outer membrane LPS, extracted by the hot saline method, the amount of cross-reaction was reduced greatly. The results indicated that LPS provide an efficient means of obtaining monospecific antisera to be used for serological identification of strains of Bradyrhizobium spp. and that the hot saline extraction method is recommended for a fast, simple and efficient way to obtain LPS and characterize this bacterium.

Juvenile Hormone Effect on the Venom Gland Secretory Cycle in Workers of Apis mellifera (Hymenoptera, Apidae)

The present investigation analyzed the influence of Juvenile Hormone (JH) on the venom glands of Apis mellifera workers through protein dosage and electrophoresis of venom gland extracts of newly emerged workers which were treated with 1 μl JH dissolved in hexane, in concentration of 2μg/μl. Newly emerged workers non-treated and treated with 1 μl hexane were the controls. Both JH and hexane provoke quantitative changes on the gland protein titre and on the protein electrophoretic profile. The disappearance of protein bands in the venom gland extracts of 14 day-old treated workers, a situation normally found only in 35 day-old non-treated workers, suggests that the JH treatment induces a precocious maturation of the worker venom gland.

Solubilization of proteins from human lymph node tissue and two-dimensional gel storage.

In the present study, we compared six different solubilization buffers and optimized two-dimensional electrophoresis (2-DE) conditions for human lymph node proteins. In addition, we developed a simple protocol for 2-D gel storage. Efficient solubilization was obtained with lysis buffers containing (a) 8 M urea, 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 40 mM Tris base, 65 mM DTT (dithiothreitol) and 0.2% carrier ampholytes; (b) 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10 (N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), 40 mM Tris base, 65 mM DTT and 0.2% carrier ampholytes or (c) 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT and 0.2% carrier ampholytes. The optimal protocol for isoelectric focusing (IEF) was accumulated voltage of 16,500 Vh and 0.6% DTT in the rehydration solution. In the experiments conducted for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), best results were obtained with a doubled concentration (50 mM Tris, 384 mM glycine, 0.2% SDS) of the SDS electrophoresis buffer in the cathodic reservoir as compared to the concentration in the anodic reservoir (25 mM Tris, 192 mM glycine, 0.1% SDS). Among the five protocols tested for gel storing, success was attained when the gels were stored in plastic bags with 50% glycerol. This is the first report describing the successful solubilization and 2D-electrophoresis of proteins from human lymph node tissue and a 2-D gel storage protocol for easy gel handling before mass spectrometry (MS) analysis.

Description of electrophoretic and chromatographic hemoglobin profile of Rhinoclemmys punctularia

Studies of the hemoglobin pattern in Brazilian reptiles are important for determining ecological and phylogenetic relationships, but they are scarce. Peripheral blood samples were obtained from 7 males and 18 females of Rhinoclemmys punctularia. The hematological profile was based on the total hemoglobin and hematocrit values. The hemoglobin profile was obtained using electrophoretic procedures at different pH, isoelectric focusing, globin chain electrophoresis, and HPLC. The hematocrit (31 ± 2%) and total hemoglobin (7.5 ± 0.2 g/dL) values did not indicate gender variations. Alkaline pH electrophoresis of the total blood samples treated with 1% saponin demonstrated the presence of four well-defined hemoglobin fractions, one major component (fraction I), showing cathodic migration and three others faster than fraction I with anodic migration. When the samples were precipitated with chloroform, only two hemoglobin fractions were observed, similar to fractions I and III from the first procedure. Isoelectric focusing and HPLC showed the same pattern. With acid and neutral pH electrophoresis, two fractions with anodic migration were observed. The globin chain identification at alkaline pH showed two fractions, but four fractions were observed at acidic pH, suggesting that different polypeptide chains are involved in the hemoglobin molecule. The chromatographic separation of the total blood sample demonstrated that the major fraction comprised 81.9% and the minor 18.1%. The results obtained demonstrated a similarity between these hemoglobin components and those of some Chelidae reported in the literature for both land and aquatic animals, reflecting the adaptation to environmental conditions. ©FUNPEC-RP.

Induction and secretion of elastinolytic and proteolytic activity in cultures of Paracoccidioides brasiliensis

P. brasiliensis parasitizes various human tissues and proteinases exported by this fungus may allow it to metabolize and invade host tissues. The influence of the culture medium on the production of proteinases by P. brasiliensis isolates was studied and the export of these enzymes was followed as a function of culture time. The fungus was grown in neopeptone, BSA, elastin or collagen medium. The culture medium was assayed for azocollytic, elastinolytic and caseinolytic activity. Proteolytic activity was also analysed by electrophoresis of the culture medium on gelatin and casein substrate gels. P. brasiliensis expressed relatively high levels of azocoll, elastin and casein degrading activity in all types of medium, except in neopeptone medium. Generally, expression of azocollytic activity peaked during the third week of culture and caseinolytic activity during the fourth week of culture. Azocollytic activity was highest at pH 4.0 and caseinolytic activity at pH 8.0. Elastinolytic activity was also highest at pH 8.0. This activity, as well as the others, may provide the fungus with a source of carbon and nitrogen and may also be responsible for the invasion of host tissues, such as pulmonary elastic fiber, by P. brasiliensis.

Role of periodontal pathogenic bacteria in RANKL-mediated bone destruction in periodontal disease

Accumulated lines of evidence suggest that hyperimmune responses to periodontal bacteria result in the destruction of periodontal connective tissue and alveolar bone. The etiological roles of periodontal bacteria in the onset and progression of periodontal disease (PD) are well documented. However, the mechanism underlying the engagement of periodontal bacteria in RANKL-mediated alveolar bone resorption remains unclear. Therefore, this review article addresses three critical subjects. First, we discuss earlier studies of immune intervention, ultimately leading to the identification of bacteria-reactive lymphocytes as the cellular source of osteoclast-induction factor lymphokine (now called RANKL) in the context of periodontal bone resorption. Next, we consider (1) the effects of periodontal bacteria on RANKL production from a variety of adaptive immune effector cells, as well as fibroblasts, in inflamed periodontal tissue and (2) the bifunctional roles (upregulation vs. downregulation) of LPS produced from periodontal bacteria in a RANKL-induced osteoclast-signal pathway. Future studies in these two areas could lead to new therapeutic approaches for the management of PD by down-modulating RANKL production and/or RANKL-mediated osteoclastogenesis in the context of host immune responses against periodontal pathogenic bacteria. © 2010 Mikihito Kajiya et al.

RBPI21 interacts with negative membranes endothermically promoting the formation of rigid multilamellar structures

rBPI21 belongs to the antimicrobial peptide and protein (AMP) family. It has high affinity for lipopolysaccharide (LPS), acting mainly against Gram-negative bacteria. This work intends to elucidate the mechanism of action of rBPI21 at the membrane level. Using isothermal titration calorimetry, we observed that rBPI21 interaction occurs only with negatively charged membranes (mimicking bacterial membranes) and is entropically driven. Differential scanning calorimetry shows that membrane interaction with rBPI21 is followed by an increase of rigidity on negatively charged membrane, which is corroborated by small angle X-ray scattering (SAXS). Additionally, SAXS data reveal that rBPI21 promotes the multilamellarization of negatively charged membranes. The results support the proposed model for rBPI21 action: first it may interact with LPS at the bacterial surface. This entropic interaction could cause the release of ions that maintain the packed structure of LPS, ensuring peptide penetration. Then, rBPI21 may interact with the negatively charged leaflets of the outer and inner membranes, promoting the interaction between the two bacterial membranes, ultimately leading to cell death. © 2013 Elsevier B.V.

Avaliação da liberação de metaloproteinases da matriz (MMP-3 e MMP-8) por macrófagos ativados por diferentes concentrações de endotoxinas (LPS) em variados períodos de tempo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)