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AIM: Compare the displacement resistance of AH Plus, Ad Seal and Real Seal on dentine discs (DDs) treated with 10% citric acid, 17% EDTA or 2.5% NaOCl, through a new push-out test method. METHODOLOGY: From the middle third of the roots of 15 maxillary central incisors, three dentine discs 1 ± 0.1 mm thick were obtained. On the axial surface of each dentine disc, three 1.2-mm-wide holes were drilled. In the third dentine disc from the same root, each hole was treated with one of three irrigating solutions for 30 s, rinsed with distilled water and dried. Each hole of the same dentine disc was filled with sealer, and the discs were maintained at 37 °C for 7 days. The dentine discs were fixed on a testing machine to test the shear strength. Data were compared using the univariate anova test with a 5% significance level. The Tukey test was used for multiple comparisons. RESULTS: The irrigating solutions did not affect the adhesion of the sealers (P > 0.05). There was no significant difference between the Ad Seal and AHPlus (P > 0.05); however, Real Seal had a significantly lower shear bond strength (P < 0.05). CONCLUSION: The use of different irrigating solutions did not affect resistance to the displacement of resin sealers. Real Seal sealer was less resistant than Ad Seal and AHPlus.

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INTRODUCTION: Psoriasis is a disease with proinflammatory state that has been associated with an increased risk of cardiovascular disease. METHOD: This is a cross-sectional, observational study, with analysis of 98 patients being treated for psoriasis. Several variables were analysed: gender, age, weight, height, Psoriasis Area and Severity Index (PASI), blood pressure, blood glucose, blood lipids, obesity, metabolic syndrome (MS) and Framingham score. RESULTS: Ninety-eight patients were analysed (51 men; 52.0%). Age ranged from 12 years to 98 years. About 67% of the patients had dyslipidaemia, 14.3% had type 2 diabetes mellitus and 59.2% had systemic arterial hypertension. Forty-four percentage of patients had the MS and 27.6% had a body mass index >30 kg/m(2). The Psoriasis Area and Severiy Index were equal or over 10 in 27 patients, 18 of whom had dyslipidaemia (P = 0.929) and 14 of whom had the MS (P = 0.327). Seventy-four patients were over the age of 40 years and, of these, 56 had dyslipidaemia (P = 0.002) and 41 had the MS (P < 0.001). For 89 patients, whose cardiovascular risk was calculated according to the Framingham criteria, 71.4% at high or moderate risk had a PASI below 10 (P = 0.945) and 100% at high or moderate risk were over the age of 40 years (P = 0.001). CONCLUSION: There was strong evidence that psoriasis patients have an increased cardiovascular risk, chiefly those over the age of 40 years, probably because of the chronic inflammatory state. It seems that the severity of the cutaneous manifestations was not associated with a higher risk in these patients.

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A series of Co/x%Nb2O5/Al2O3 catalysts were prepared by anchoring niobia on an Al2O3 support at different niobia concentrations. Characterization of the structure and nature of surface active sites was attempted in order to correlate the CO hydrogenation activity of these systems with those of the Co/Al2O3 and Co/Nb2O5 catalysts. The effect of the reduction temperature on the CO hydrogenation activity and selectivity was studied, showing that interaction of cobalt and niobia surface species favored the selectivity for hydrocarbon chain growth. However, this effect is less pronounced on the niobia-promoted Co/Al2O3 compared to Co/Nb2O5 catalysts. X-ray photoelectron spectroscopy (XPS) and diffuse reflectance spectroscopy (DRS) results on Co/x%Nb2O5/Al2O3 showed prevailing amounts of Co2+ and Co3+ after calcination and reduction at 573 K, while, after reduction at 773 K, besides metallic cobalt, the Co2+ species still remains in contact with alumina, even for higher niobia loading. It seems that during this process formation and destruction of new interfaces involving Co0-NbOx sites takes place. Results suggest that Co0, Co0-Co2+, and Co0-NbOx are the active sites at the surface. The relative abundance of Co2+ species affects greatly the performance of the catalysts. DRIFTS and selectivity results suggest that these sites might be responsible for the reaction chain growth and therefore for the drastic change in the selectivity of CH4 and C5+ hydrocarbons mainly on the Co/Nb2O5 catalyst. DRIFTS results on Co/Nb2O5/Al2O3 showed the formation of -C=C- and -CH3- besides CHxO species. With increasing reduction temperature, the -C=C- species disappear while -CH3- fragments increased markedly, suggesting the formation of increasing amounts of hydrocarbons with higher chain length.

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The aim of the present study was to analyse the clinical and epidemiological characteristics of bronchiolitis caused by respiratory syncytial virus (RSV) in 225 children observed in a paediatric hospital in Lisbon, Portugal, and to determine the clinical, epidemiological, or laboratory parameters that correlate with greater severity of the disease. This prospective study included hospitalised and ambulatory children younger than 36 months of age with a diagnosis of bronchiolitis and was conducted during two consecutive RSV epidemiological seasons (November-March 2000/01 and 2001/02). The median age of the patients was 5 months, and the male-to-female ratio was 1.6:1. RSV was isolated in 60.9% of patients, predominantly in the hospitalised group. The subtype A:B ratio was 7.4:1 and was similar in both seasons. RSV-positive patients were younger, had more severe clinical forms of bronchiolitis, and fewer changes in leucocyte total and differential counts. Among infected patients, higher clinical severity scores occurred in association with first wheezing episodes, overcrowded households, attendance at day-care centres, or prematurity (<36 weeks). This first prospective study of RSV epidemiology in Portugal provides a foundation for appropriate surveillance programmes of RSV infection in this country. A multicentre study is desirable in order to delineate optimal prophylactic and therapeutic guidelines for RSV infection in Portugal.

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We have previously shown that both promastigotes and amastigotes of Leishmania amazonensis contain a lytic protein that damages erythrocytes and nucleated cells, including macrophages (F. S. M. Noronha, F. J. Ramalho-Pinto, and M. F. Horta, Infect. Immun. 64:3975-3982, 1996). Using the patch-clamp technique, we show here that cell damage by parasite extracts is mediated by the formation of nonselective pores on the target membrane. This demonstrates that L. amazonensis cytolysin is a pore-forming protein (PFP), here named leishporin. We show that the diameters of the pores formed by parasite extracts are heterogeneous, varying from approximately 1.6 to >6.1 nm according to cytolysin concentration or time. We also show that pore formation involves the binding of the PFP to the target cell membrane, a temperature-independent event that is necessary but not sufficient to lyse cells. This is followed by a temperature-dependent step that triggers lysis, probably the insertion and the polymerization of protein subunits in the lipid bilayer. We provide evidence that suggests that polymerization of single subunits must occur for pore formation. We show, in addition, that L. amazonensis expresses molecules antigenically homologous to other PFPs.

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We retrospectively studied seventeen cases of acute mastoiditis admitted to the Paediatric Ward of S. Francisco Xavier Hospital during 8 years and 8 months. Seven patients were admitted in 1995. We found no reason for this increase. Eleven children were male and 5 were under 2 years of age. All cases, except one of post-traumatic mastoiditis, occurred after acute otitis media. Only 5 children referred symptoms for more than seven days before admission. Seven patients had surgery. The surgical group of children had a longer period of illness when compared to those only treated medically (7.6 versus 3.9 days). Tympanocentesis was performed in 6 patients, but it did not affect the outcome of the illness. In three children there were complications: Bezold abscess, labyrinthitis and cholesteatoma. The latter was the only patient in our series with permanent hearing loss.

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American tegumentary leishmaniasis is caused by Leishmania of the subgenera Leishmania and Viannia. In this paper, we demonstrate that promastigotes of these two subgenera display distinct characteristic patterns of complement sensitivity during growth in vitro. Using fresh normal human serum in lytic assays, we show that while promastigotes of two species of the subgenus Leishmania differentiate into forms that are more resistant to the lytic action of complement, promastigotes of three species of the subgenus Viannia remain sensitive to complement mediated lysis during all stages of their growth in vitro. Complement resistance of the subgenus Leishmania is temporary, reaching its peak at the beginning of the stationary phase of growth, and decreasing thereafter. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) we detected in L. amazonensis (subgenus Leishmania), but not in L. guyanensis (subgenus Viannia), three polypeptides whose expression parallels the resistance of promastigotes to complement-mediated lysis.

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We describe here that parasites of the genus Leishmania contain a cytolytic activity which acts optimally at pH 5.0 to 5.5 and at 37 degrees C in vitro. or the four species examined, Leishmania (Leishmania) amazonensis and Leishmania (Leishmania) major presented considerable hemolytic activity, whereas Leishmania (Viannia) panamensis and Leishmania (Viannia) guyanensis showed little and no hemolytic activity, respectively. The cytolytic factor of L. amazonensis promastigotes was characterized as a protein with no protease-, phospholipase-, or detergent-like activity, probably localized inside membranous vesicles. The use of osmotic protectants revealed the colloid-osmotic nature of hemolysis, which is indicative of pore formation in the membranes of target cells. This putative pore-forming protein also damaged nucleated cells, including macrophages, causing an increase in their membrane permeability with leakage of cytoplasmic proteins. Both promastigotes and amastigotes express this lytic activity, suggesting that the cytolysin may have a function in both stages of this parasite. The pH and temperature required for optimal activity indicate that it might be more effective within the mammalian host, particularly inside the macrophage parasitophorous vacuole. In promastigotes of L. amazonensis, the expression of lytic activity seems to be regulated during their growth in vitro, being maximal at the early stationary phase.

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Pelvic endometriosis affects 5 to 10% of all menstruating women, and of these 3% to 34% have intestinal involvement. Commonly, intestinal endometrial lesion affects only the serosa. Although the invading endometrial tissue may extend into the intestinal wall, it does not usually reach the mucosa. The majority of the patients are asymptomatic. The most common symptom is lower recurrent abdominal pain. It has been estimated that 0,9% of women with intestinal endometriosis have obstructive symptoms. Occasionally deeper and more extensive intestinal wall involvement results in cyclic menstrual rectal bleeding. The authors describe a case with obstructive symptoms associated to rectal bleeding. This is an uncommon finding. Histological confirmation of deep muscular and mucosal involvement was obtained. This case led us to some considerations about the diagnostic difficulties and therapeutic possibilities in the present.

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Monoclonal antibodies (mAbs) against Actinobacillus pleuropneumoniae serotype 2 (reference strain Shope 4226 and field isolate F46) were produced. Twelve hybridoma clones were selected against both strains, and all the antibodies secreted were found to be reactive with whole-cell antigen of the homologous strain in ELISA, whereas only one mAb was reactive in slide agglutination test. The predominant antibody classes were IgG2b and IgG3, although IgG1 and IgM were also obtained. Immunoblot assay showed that mAbs could recognize a ladder band profile which is in accordance with the O-antigen of lipopolysaccharide. Most of the epitopes involved were resistant to proteinase K and also to boiling in the presence of sodium dodecyl sulfate and reducing conditions, but they were sensitive to periodic acid. The 12 mAbs recognized neither reference strains of the remaining A. pleuropneumoniae serotypes nor other taxonomically related Gram-negative organisms. The suitability of mAbs for serotyping of field isolates was also examined, and a high correlation (97.4%) was found between the results previously established by indirect hemagglutination with polyclonal rabbit sera and those obtained by ELISA with mAbs. The panel of mAbs described in this study was found to be extremely useful for identifying field isolates belonging to serotype 2 and could be used as a complementary serotyping method.

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Primary woodchuck (Marmota monax) hepatocytes from normal woodchucks and woodchucks with chronic woodchuck hepatitis virus (WHV) infection were cultured in either a conventional serum-containing medium or a serum-free medium. The de novo synthesis of the plasma proteins albumin, transferrin, fibrinogen, and complement C3 were identical under both conditions. However, expression of the WHV and the synthesis of nitric oxide were diminished under serum-free conditions. Primary woodchuck hepatocytes cultured in conventional, serum-containing medium were immortalized utilizing the simian virus 40 T antigen oncogene. Immortalized hepatic cell lines retained differentiated functions of nitric oxide synthesis and expression of complement C3. The woodchuck hepatocyte culture model will supplement current experimental methods, allowing investigation of hepadnaviral pathogenesis, including hepatocarcinogenesis in vitro.

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Several organisms, including the protozoa Entamoeba histolytica and Trypanosoma cruzi, have been shown to contain pore-forming proteins (PFP) thought to play a role in the pathogenesis of the diseases they generate. In the present report, we show that promastigotes of Leishmania amazonensis express a hemolysin that appears to cause colloid-osmotic lysis, typical of pore formation. This hemolysin affects red blood cells of different species at 37 degrees C, but not at 0 degrees C, with maximum activity at pH 5.0. The hemolytic activity is heat-labile, but lysis is not affected by protease inhibitors. These results suggest the involvement of a protein with no proteolytic or detergent activity. Hemolysis is inhibited by polyethyleneglycol, suggesting its colloid-osmotic nature. Hemolytic extracts of the parasite contain a polypeptide that reacts with antibodies to perforin from mouse cytotoxic T lymphocytes or to C9 from human complement. In addition, genomic DNA of L. amazonensis contains a fragment that hybridizes to a perforin cDNA probe. The circumstantial evidence suggests that the L. amazonensis hemolytic activity may be mediated by a PFP homologous to perforin and C9.

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The ability of complement to inactivate feline immunodeficiency virus (FIV) was examined. Treatment of virus with complement plus sub-neutralizing titers of antiserum resulted in a significant reduction in the virus titer compared with treatment of the virus with complement or antibody alone. One of the mechanisms by which cat complement inactivates FIV was shown to be by viral lysis as determined by a reverse transcriptase release assay. Kinetic studies revealed that viral lysis is initiated soon after the addition of complement to a mixture of virus and antiserum. Treatment of FIV with normal non-complement-inactivated human serum resulted in virus inactivation and release of viral RT in the absence of specific antiserum. It appears that FIV activates complement directly through the classical pathway and that integrity of the membrane attack components is a requirement for FIV lysis by human serum. The vulnerability of two distinct isolates of FIV to complement lysis was compared using complement from different species. Oradell isolate was more sensitive to complement lysis than the Petaluma isolate as assessed by reverse transcriptase release. It appears that factors intrinsic to the virus isolate may influence the amplitude of complement-dependent viral lysis.

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The woodchuck and the woodchuck hepatitis virus (WHV) have been used as a model of hepatitis B virus infection and its disease sequelas. Serologic responses to WHV infection have been described in previous reports from this laboratory by using virus-specific radioimmunoassays (RIAs) for WHV surface antigen, antibody to WHV core antigen, and antibody to WHsAg. In this study, we developed and evaluated new enzyme immunoassays (EIAs) for these WHV serologic markers. Relative to the established RIAs, the EIAs were either improved or comparable in their sensitivity and specificity, and in their utility for monitoring experimental WHV infection and classifying woodchucks into serological diagnostic categories. These EIA systems are amenable to the quantitative titration of antibodies and quantitation of WHV antigens in serum, and ultimately should allow improved resolution of virologic and humoral immune responses of woodchucks to WHV infection.

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Monoclonal antibodies (MAbs) 3B7 and 1C11 were produced against the gag gene products of feline immunodeficiency virus (FIV). These MAbs reacted strongly with FIV p24 in Western blots (immunoblots) and recognized p50 with a lower intensity. They specifically bound antigens in the cytoplasm of FIV-infected cells as determined by indirect immunofluorescence and immunocytochemistry. Although neither MAb inhibited viral replication in vitro, they were useful in a simple assay for the detection and quantification of infectious virus and neutralizing antibody activity. The assay utilizes Crandell feline kidney cells and requires 4 days for completion. Neutralizing antibodies in cats were detected 3 to 4 weeks after experimental infection with FIV. Antibody titres progressively increased during the first year of infection reaching high titres which were maintained 2.5 years post-infection. The MAbs produced should be valuable reagents for the monitoring of viral replication in cells or tissues from FIV-infected cats and for other in vitro applications.

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The present study describes an approach to the development and use of anti-idiotypic antibodies as a possible immunization strategy to prevent retroviral infection. The rationale for using anti-idiotypes (anti-Ids) to try to elicit an antigenic-specific immune response is examined, and the production and characterization of polyclonal and monoclonal anti-Ids are described. Several techniques were used to determine antigenic mimicry and anti-Id subtypes. The potential use of anti-Ids in feline leukemia virus (FeLV) receptor studies and vaccine trials in vivo were investigated. Results from these studies suggest that the anti-Id strategy is feasible for the FeLV model. Polyclonal Ab2 reagents were developed that blocked virus-receptor binding and thus inhibited viral infection in vitro and induced humoral immune responses in 6- to 8-week old kittens characterized by production of Ab3 with the ability to bind the original FeLV envelope protein gp70 as assessed by Western blot analysis.

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The antibody response in cats to feline immunodeficiency virus (FIV) reverse transcriptase (RT) was followed for 3 years. Eight of the nine cats used in this study produced reverse transcriptase-inhibiting (RTI) antibodies. Relative inhibitory means of 2.9%, 18.4%, 33%, and 47% were found 6, 12, 24, and 36 months, respectively, after infection with FIV. The enzyme activity was suppressed by greater than or equal to 78% with the use of 100 micrograms of FIV-associated IgG. The RTI antibodies were FIV-specific, as they did not inhibit other mammalian retroviral polymerases, including feline leukemia virus RT. An RT-inhibition assay with sera in the presence of protein A and immunoblot analysis showed that antibody binding to FIV RT protein p62 is independent of antibody ability to block enzyme activity. Viral RT released by detergent-treated virus was stable for more than 6 weeks at 4 degrees C, whereas its activity was reduced by 50% after 2 weeks at 37 degrees C. Because significant concentrations of RTI antibodies are detected only at 1 to 2 years after infection, they can be used to determine the approximate time of virus infection and as a marker for disease progression.

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Lymphocytes from normal cats or cats experimentally infected with feline immunodeficiency virus (FIV) were stimulated with phytohemagglutinin, pokeweed mitogen, or concanavalin A. Lymphocytes from infected cats had lower responses than those from uninfected cats. These results support the hypothesis that FIV induces immunosuppression.

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In the present communication we evaluate the feline leukemia virus (FeLV) infection of cats as a model for antiretroviral chemotherapy studies. Additionally, we report the results of testing the antiviral effect of the compounds, 3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxycytidine, 2',3'-dideoxyinosine and 2',3'-dideoxyadenosine against FeLV replication in vitro. Cumulative data from experiments in which FeLV-infected cats were treated with AZT at different stages of experimental infection are also evaluated.

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