Effects of Microabrasion Prior to In-office Bleaching on Hydrogen Peroxide Permeability, Color Change, and Enamel Morphology.

PURPOSE: This study evaluated hydrogen peroxide (HP) diffusion within the pulp chamber, as well as color change and the surface morphology of teeth subjected to various microabrasion (MA) protocols associated or not with in-office (IO) bleaching. METHODS: Forty sound premolars were randomly divided into the following four groups (n=10): no treatment (NC); IO bleaching only; IO immediately after MA (IMA), and IO seven days after MA (7MA). After treatments, the HP concentration (µg/mL) within the pulp chamber was determined using ultraviolet-visible (UV-Vis) spectrophotometry. The color change (ΔE*) was evaluated using the digital spectrophotometer before and 1 week after bleaching. The surface morphology was evaluated by scanning electron microscope (SEM). Data from each test were submitted to one-way ANOVA and Tukey tests (α=0.05). RESULTS: All experimental groups exhibited higher HP concentrations compared to the NC group (p<0.00001). However, higher amounts of HP were observed for the IMA group compared to the IO and 7MA groups (p<0.00001). No significant difference in color change was observed among the groups (p<0.001). Pronounced grooves in enamel were found in the IMA and 7MA groups. However, enamel erosion areas were observed only in the 7MA group. CONCLUSIONS: The association between MA and IO bleaching could significantly affect the amount of HP inside the pulp chamber. Therefore, it is highly recommended to wait for 1 week after MA procedures before performing IO bleaching.

Efficient duplex solid-phase fluorescent assay (dFISA) for the simultaneous detection of specific anti-T. gondii IgG and IgM due to refined conjugates.

Toxoplasma gondii infections are very common, causing occasional central nervous system and eye diseases, and must be screened in prenatal care for efficient therapy. Here, we developed a duplex solid-phase fluorescent assay (dFISA) for the simultaneous detection of anti-T. gondii IgG and IgM antibodies in prenatal care screening for toxoplasmosis. Assays using commercially available ion-exchange purified conjugates yielded poor results and high background fluorescence. Same-well IgG/IgM dFISA with refined conjugates was used to test 140 samples from university students, 120 samples from pregnant women and 24 samples from adult volunteers at a large public hospital. We found that dFISA offers high concordance, specificity and reproducibility for IgG (Kappa=0.883) and IgM (Kappa=0.918), which is useful in high-throughput applications for antenatal care.

Saliva as a source of anti-Toxoplasma gondii IgG for enzyme immunoassay in human samples.

Toxoplasmosis, a highly prevalent disease, is mainly diagnosed by serology. Incidence studies could be feasible in children, but ethical concerns restrict blood sampling in this group. Saliva contains small amounts of crevicular fluid IgG. Dot-ELISA and a protein A IgG capture immunoassay were standardized for anti-Toxoplasma gondii IgG in paired saliva and serum samples from 20 adult volunteers. A frequency of toxoplasmosis of 19% (95% CI 12-28) was found in 100 saliva samples from university graduates using both assays. Toxoplasmosis immunoassays using saliva IgG are a promising tool for the investigation of the epidemiology of this disease in children and other vulnerable groups.

The effects of human herpesvirus 8 infection and interferon-gamma response in cutaneous lesions of Kaposi sarcoma differ among human immunodeficiency virus-infected and uninfected individuals.

BACKGROUND: Kaposi sarcoma (KS) is associated with human herpesvirus 8 (HHV-8). The cutaneous immune response in this tumour is not well established and a better understanding is necessary. OBJECTIVES: To evaluate the HHV-8 expression and immune response in cutaneous lesions of classic KS (CKS) and AIDS-associated KS (AIDS-KS). METHODS: We performed a quantitative immunohistochemical study of cells expressing HHV-8 latency-associated nuclear antigen (LANA), CD4, CD8 and interferon (IFN)-gamma in skin lesions from patients with CKS and AIDS-KS (with or without highly active antiretroviral therapy, HAART). RESULTS: CKS showed higher LANA expression compared with AIDS-KS, regardless of HAART. We also found higher LANA expression in nodules compared with patch/plaque lesions. The tissue CD4+ cell proportion was lower in AIDS-KS patients without HAART than in patients with CKS. In CKS lesions, CD4+ and CD8+ cells expressed IFN-gamma, as shown by double immunostaining. AIDS-KS presented low numbers of IFN-gamma-expressing cells. CD8+ cell numbers were similar in all groups, which appeared unrelated to the clinical or epidemiological type of KS. CONCLUSIONS: Our quantitative data on the pattern of KS lesions in selected groups of patients, as shown by in situ immune response, demonstrated a CD4+ T-cell involvement associated with IFN-gamma, an environment of immune response-modified human immunodeficiency virus (HIV) infection. In our sample, the promotion of KS in patients without HIV appears to be related to higher HHV-8 load or virulence than in those with AIDS. This higher resistance may be explained by a sustained immune response against this herpesvirus, that is only partially restored but effective after HAART.

Antagonic effect of the inhibition of inducible nitric oxide on the mortality of mice acutely infected with Escherichia coli and Bacteroides fragilis.

Sepsis, the leading cause of death in intensive care units, is associated with overproduction of nitric oxide (NO) due to inducible NO synthase (iNOS), responsible for some of the pathologic changes. Aminoguanidine (AG) is a selective iNOS inhibitor with reported inconsistent actions in sepsis. To investigate the influence of iNOS, we studied models of acute bacterial sepsis using acute challenges with aerobic (Escherichia coli) and anaerobic (Bacteroides fragilis) bacteria in the presence of AG. Six-week-old, 23 g, male and female BALB/c and C57Bl/6j mice, in equal proportions, were inoculated (ip) with bacteria in groups of 4 animals for each dose and each experiment in the absence or presence of AG (50 mg/kg, ip, starting 24 h before challenge and daily until day 6) and serum nitrate was measured by chemiluminescence. Both types of bacteria were lethal to mice, with an LD50 of 6 nephelometric units (U) for E. coli and 8 U for B. fragilis. Nitrate production peaked on the second day after E. coli inoculation with 8 and 6 U (P < 0.05), but was absent after non-lethal lower doses. After challenge with B. fragilis this early peak occurred at all tested doses after 24 h, including non-lethal ones (P < 0.05). AG-treated mice challenged with E. coli presented higher survival (P < 0.05) and increased LD50. AG-treated mice challenged with B. fragilis had lower LD50 and higher mortality. Control AG-treated animals presented no toxic effects. The opposite effect of iNOS blockade by AG in these models could be explained by restriction of oxygen for immune cells or an efficient action of NO in anaerobic localized infections. The antagonic role of NO production observed in our bacterial models could explain the reported discrepancy of NO action in sepsis.

Seroprevalence of Toxoplasma gondii antibodies in humans from rural Western Amazon, Brazil.

Antibodies to Toxoplasma gondii were assayed in sera of 266 humans from 71 farms located at Rondônia State, Western Amazon, Brazil, by the modified agglutination test (MAT) and the indirect immunofluorescent antibody test (IFAT). Antibodies were found in 195 humans (73.3%), with MAT titers of 1:25 in 11, 1:50 in 11, 1:100 in 16, 1:200 in 27, 1:400 in 38, 1:800 in 37, 1:1,600 in 22, and 1:3,200 or higher in 33. From the 71 farms visited, 69 had seropositive humans. Prevalence of anti-T. gondii antibodies increased with age of the people (P < 0.05), and no difference was observed in the occurrence by gender (P > 0.05). A sanitary questionnaire was applied in each farm, and statistical association between the serologic status and several variables were analyzed. Home-grown vegetable consumption and origin of drinking water (well or river) were the independent variables that displayed significant association (P = 0.002 and 0.02, respectively). Higher values of occurrence were found in people with consumption of home-grown vegetables (76.1%) and people that drink well water (75.4%) compared with people that did not consume this type of food (61.9%) and drink river water (55.2%). By IFAT (> or = 1:16), 194 of 266 (73%) humans were seropositive and there was a good correlation between MAT and IFAT.

Antiprotozoal activity of Brazilian plant extracts from isoquinoline alkaloid-producing families.

Leishmaniasis and Chagas disease afflict the poorest countries in the world. The Brazilian flora represents a rich source for the screening of potential antiparasitic compounds. In this work, we tested the total alkaloid and ethanol extracts of nine different plants from Brazilian families which produce isoquinoline alkaloids, to determine their in vitro antiparasitic effect against L. chagasi and T. cruzi parasites. Promastigotes of L. chagasi were shown to be susceptible only to the total alkaloid extracts of A. crassiflora (EC50 value = 24.89 microg/ml), A. coriacea (EC50 value = 41.60 microg/ml), C. ovalifolia (EC50 value = 63.88 microg/ml) and G. australis (EC50 value = 37.88 microg/ml). Except for the G. australis total alkaloids, all the three extracts presented a considerable activity when tested against intracellular amastigotes. The most effective alkaloid extracts were those from A. crassiflora and C. ovalifolia, which reduced the number of infected macrophages at 25 microg/ml by 86.1% and 89.8%, respectively. Among the 18 tested extracts, 16 showed anti-Trypanosoma activity. Eight extracts (A. crassiflora, A. coriacea, C. ovalifolia, D. furfuracea, D. lanceolata, S. guianensis, X. emarginata and G. australis) were the most effective against the trypomastigotes, killing approximately 100% of the parasites at the maximal concentration of 100 microg/ml. Cytotoxicity against mammalian cells was evaluated for all extracts, but potential ones showed little or no cytotoxicity and a considerable antiparasitic effect, including D. furfuracea, D. lanceolata, G. australis, S. guianensis and X. emarginata. Plants are a rich source of natural compounds, and a powerful tool for the development of new arsenals for the therapy of protozoan diseases.

Toxoplasma gondii spreading in an urban area evaluated by seroprevalence in free-living cats and dogs.

Infection by the protozoan parasite, Toxoplasma gondii is widely prevalent in humans and animals throughout the world. Transmission takes place mainly by ingestion of raw or undercooked meat that contains parasite cysts or by ingestion of oocysts excreted in cat faeces, which can contaminate water and raw vegetables. The incidence of toxoplasmosis in urban areas can thus be also related to environmental contamination with oocysts. A direct measure of this environmental contamination by oocyst counting is unfeasible for technical reasons. An interesting alternative for measuring T. gondii urban spreading is the seroprevalence in free-living urban animals, used as sentinels, once they are exposed to similar risks of Toxoplasma infection-like humans. With this aim, we tested serum samples from stray cats and dogs for antibodies to T. gondii by indirect haemagglutination assay (IHA) and enzyme-linked immunosorbent assay (ELISA). Antibodies to T. gondii were found in 40% (40 of 100) of the cats, less than the 50.5% (101 of 200) found in dogs by ELISA (P < 0.05). Haemagglutination showed low resolution and concordance, precluding their use for diagnosis of T. gondii infection compared with ELISA. The prevalence of T. gondii was lower among stray cats probably due to their selective alimentary habits and lower water and food intake. Toxoplasma gondii seroprevalence in stray dogs and cats could be an indirect indicator of the parasite spreading in urban areas.

Characterization of cellular phenotypes and cytokine expression in balt from children with congenital heart diseases.

UNLABELLED: The present study was performed to target and call attention to the bronchial associated lymphoid tissue (BALT), part of our immune system, from which, we believe, several forms of prophylactic and therapeutic approaches can be developed. The characterization of its immune components, cells, and cytokines, in absence of antigenic stimuli, is pioneer in literature. Eighteen cases of necropsies were chosen and selected the paraffin-embedded lungs. The ages of 11 females and 7 males varied from 5 to 31 months. Cause of death: congenital heart diseases. EXCLUSION CRITERIA: lung infection at necropsy and/or arterial hypertrophy greater than Heath-Edwards' 1st degree. Immunohistochemical technique was applied to identify the cell phenotypes and the cytokines in situ. BALT was identified in all cases in this study. The main cellular phenotypes in BALT were T helper (TH) and B lymphocytes surrounded by T cytotoxic lymphocytes, natural killer cells, and dendritic cells in less quantities. Interleukin 10 and Tumor Necrosis Factor alpha were the predominant cytokines in BALT without antigenic stimuli. BALT is an important structure of the lung immune system in infants, with a tendency to maintain an environment favorable to the Th2 arm of immune response. It needs more exploration to define its behavior in front of infections, especially those with pulmonary tropism.

Infectivity of cysts of the ME-49 Toxoplasma gondii strain in bovine milk and homemade cheese.

OBJECTIVE: Analyze the infectivity and storage resistance of cysts of the ME-49 strain of Toxoplasma gondii in artificially infected bovine milk and homemade fresh cheese. METHODS: Pasteurized bovine milk was infected with 10 cysts/ml of the ME-49 strain of T.gondii and inoculated in different groups of mice, immediately or after storage at 4 degrees C for 5, 10 and 20 days. Homemade fresh cheese was prepared with artificially infected milk, and also tested in groups of mice, using the same storage process. Infection was identified by the presence of cysts in the brain or serological testing in challenged mice after 5 weeks, confirmed by Western Blot and histology. RESULTS: The infectivity of cysts of the ME-49 strain of T.gondii was maintained in the milk even after storage for 20 days at refrigerator temperatures. Cysts were also able to survive the production process of homemade fresh cheese and storage for a period of 10 days in the same conditions. CONCLUSIONS: These data demonstrated that milk and dairy products could be an important source of T.gondii in human contamination, reinforcing the importance of milk pasteurization before any processing or ingestion.

Bothrops moojeni venom kills Leishmania spp. with hydrogen peroxide generated by its L-amino acid oxidase.

Leishmaniasis is an endemic tropical disease in South America, with few therapeutic approaches. Snake venoms are complex protein mixtures with biological actions that could be used as tools for drug development. Here we show that Bothrops moojeni crude venom presented a killing effect in vitro against Leishmania spp. promastigotes, but not with amastigotes, as determined by a viability assay using the mitochondrial oxidative function. Purification of active fractions from crude venom was performed by molecular exclusion and ion exchange chromatography. Anti-Leishmania and l-amino acid oxidase (L-AAO, EC.1.4.3.2.) activities co-eluted in the same fractions. The molecular weight of the active enzyme was estimated to be 140 kDa by molecular exclusion chromatography, and 69 kDa by SDS--PAGE, with a 4.8 isoelectric point. Using substrate subtraction and catalase for scavenging, the action of L-AAO was demonstrated to be hydrogen-peroxide-dependent.

The prevalence and avidity of Toxoplasma gondii IgG antibodies in pigs from Brazil and Peru.

Raw or inadequately cooked pork is an important source of Toxoplasma gondii infection, and the infection rate in animals used as human food, is an important risk predictor. The prevalence of this infection was estimated in 396 sera from 5-month old pigs obtained at abattoirs in São Paulo, Brazil (300) and Lima, Peru (96). The seroprevalence was higher in pigs from Peru (32.3%) as compared to Brazil (9.6%), as detected by ELISA and Western blot. Hemagglutination gave poor resolution which was not useful for the diagnosis of T. gondii infection. Specific antibody avidity is correlated with infection time, as shown in experimentally infected piglets. Using an arbitrary cut-off of 50% avidity index, Brazilian pigs were found to be more recently infected than Peruvian pigs. Pork should be considered a significant source of human T. gondii infection both in Brazil and Peru. Avidity assays could help in the detection of the time of T. gondii infection in pigs, allowing preventive management.

Santo Inácio revisited: protozoan diseases in an isolated village in northeastern Brazil after twenty years.

The northeastern highlands of Brazil are endemic for several tropical diseases, especially American trypanosomiasis (Chagas' disease) and schistosomiasis. Twenty years ago, we measured the seroprevalence of protozoan diseases in Santo Inácio, a village of approximately 1,000 inhabitants located 1,000 m above sea level. We detected small numbers of sera with antibodies against Trypanosoma cruzi and Toxoplasma gondii, and the area had a low prevalence both of American trypanosomiasis (3.54%) and toxoplasmosis (27.43%) compared with nearby Brazilian areas. This was attributed to a specific triatomine vector and local housing conditions. Twenty years later, we again determined the prevalences of both diseases and compared these results with those from Iraquara, a larger town with the same ethnic and social background but with a higher prevalence of rural activities. The incidence of Chagas' disease in San Inácio showed the same low level, i.e., 3.78% (5 of 132) with only adult males affected in contrast with Iraquara, which had an incidence of 34.5%, but a low prevalence of only one of 22 among children up to 14 years of age. Santo Inácio maintained a low (25.8%) seroprevalence for toxoplasmosis. Housewives presented a higher incidence of toxoplasmosis during both periods, probably due to related risk factors. Cats were found less frequently in Santo Inácio than in Iraquara, which showed an incidence of 65.5% seropositivity for Toxoplasma gondii. These results suggest that the environmental conditions of Santo Inácio were preserved after 20 years, with a low incidence of these selected protozoan diseases.

Differential biodistribution of native and 2 kGy 60Co irradiated crotoxin in tissues of CBA/J mice.

Crotalus durissus envenomation is treated using antivenins produced in horses. During production, animals have problems, sometimes followed by death, due to the high toxicity of the main toxin, crotoxin. Several methods tested to detoxify this toxin often resulted in decreased immunogenicity. Gamma irradiation has proved to be a successful method for crotoxin detoxification without loss of immunogenicity. We have studied the biodistribution of 2 kGy 60Co irradiated crotoxin (iCTX) in mouse tissues. We used both 125I-labeled iCTX or its detection by a specific immunohistochemistry assay (IHA). Both approaches showed similar early excretion of toxins by the kidneys. Higher iCTX uptake was seen in spleen and liver, which are rich in immune responder cells. In contrast to previous reports concerning native crotoxin (nCTX), we failed to detect iCTX in the neuromuscular junction, but both toxins were found on the kidney tubular cell surface, with rapid excretion that was more intense for iCTX. Kupffer cells and splenocyte macrophages presented IHA staining, as shown by the increased uptake of 125I toxin by these organs. No staining was observed in the brain, lung or heart, which also showed very low 125I counts. Allied to reduced toxicity, irradiation induced early endocytosis of crotoxin by phagocytic cells, improving antigen processing.

Prostaglandin and nitric oxide regulate TNF-alpha production during Trypanosoma cruzi infection.

The mechanisms that control TNF-alpha production by macrophages during Trypanosoma cruzi infection are still unknown. Destruction of intracellular forms by cytokine activated macrophages is considered to be a major mechanism of parasite elimination. Although in vitro TNF-alpha contributes to enhanced parasite destruction by macrophages, previous work in vivo has shown that as the parasite burden increases, serum TNF-alpha levels decline. In this report we show that TNF-alpha production by peritoneal adherent cells is elevated at the initial phase of T. cruzi infection. As infection progresses TNF-alpha production decreases. The observed reduction is partly due to inhibition, largely exerted by endogenous PG and secondarily by NO. Inhibition of their synthesis partially restored the ability to produce high levels of TNF-alpha to macrophages upon stimulation by LPS. Neither endogenous IL-10 nor TGF-beta seem to be involved in the negative regulation of TNF-alpha production.

Irradiation of Crotalus durissus terrificus crotoxin with 60Co gamma-rays induces its uptake by macrophages through scavenger receptors.

PURPOSE: To investigate the action of 2 kGy 60Co gamma-rays on crotoxin and its favoured uptake through scavenger receptor (ScvR) mouse peritoneal macrophages. MATERIALS AND METHODS: Native or irradiated crotoxin (iCTX) (50 microg/ml) dosed with 2 kGy 60Co gamma-rays (dose-rate 540 Gy/h) were offered to mouse peritoneal macrophages; their uptake was evaluated by immunohistochemistry and quantitative in situ ELISA. Receptors recognizing irradiated crotoxin were evaluated with specific ScvR blockers (Probucol and dextran sulphate) or with non-specific blocking using foetal calf serum (FCS). RESULTS: Immunohistochemical assays revealed more deeply staining intracytoplasmic vacuoles in macrophages incubated with iCTX. Using in situ ELISA with ScvR specific blockers, it was shown that the increased uptake of iCTX was blocked by Probucol or dextran sulphate, but not by FCS. On the other hand, the uptake of native crotoxin was decreased by FCS, but not affected by ScvR blockers. The morphology and viability of macrophages were preserved during the experiments. CONCLUSIONS: It is concluded that 60Co gamma-rays probably induced oxidative changes in crotoxin, driving this toxin towards ScvR mouse peritoneal macrophages. This suggests a different in vivo route of iCTX away from toxic neural sites by a preferential and rapid internalization and processing by macrophages, leading to the induction of a better immune response.

Immunogenic properties of the M. leprae recombinant 18-kDa antigen purified from Saccharomyces cerevisiae; enhancement of delayed-type hypersensitivity after gamma-irradiation.

In this paper we report the purification and study of the immunogenic properties of the Mycobacterium leprae 18-kDa protein antigen produced and secreted by the yeast Saccharomyces cerevisiae, using an expression system we recently described [Biotech. Lett. 16 (1994) 1241-1246]. The 18-kDa protein was purified from the yeast culture media by precipitation, ion exchange chromatography (MonoQ) and exclusion size chromatography (Sephacryl S-100). The biological properties of the recombinant protein, previously irradiated with gamma rays, were assayed by immunization of mice. Humoral and cellular responses, monitored by antibody production and delayed-type hypersensitivity, respectively, were obtained. Furthermore, gamma-irradiation of the recombinant protein prior to the administration was shown to significantly potentiate the T-cell response. The data suggest that this irradiated recombinant antigen could be used in a more sensitive standardized skin test to monitor M. leprae infection.

High resolution of Trypanosoma cruzi amastigote antigen in serodiagnosis of different clinical forms of Chagas' disease.

The serodiagnosis of Chagas' disease, a highly prevalent disorder in South American countries, is usually made by the detection of antibodies to Trypanosoma cruzi epimastigote antigen. In this study, we assess the diagnostic performance of the immunofluorescence test with T. cruzi (Y strain) amastigote antigen from an LLC-MK2-infected cell supernatant in comparison with a test with the conventional epimastigote antigen. A total of 238 serum samples from patients in the acute and chronic phases of the disease, with the chronic indeterminate, cardiac, and digestive forms, and from nonchagasic individuals were tested for the presence of immunoglobulin G (IgG), IgM, and IgA antibodies. The reactivity of the amastigote antigen in terms of geometric mean titers was 2 to 4 times higher than that of the epimastigote antigen. Clear-cut results were obtained with the amastigote antigen, with no overlapping of true and false positives. IgG antibodies to amastigotes were found in all patients with Chagas' disease, whereas all sera from nonchagasic patients were negative, except for those from patients with visceral leishmaniasis, in which 63% cross-reactivity was observed. IgM antibodies to amastigotes were detected in 100% of sera from patients with acute Chagas' disease and in 7.5% of sera from patients with chronic Chagas' disease, whereas IgA antibodies were found in 60% of sera from patients in the acute phase and in 33% of sera from patients in the chronic phase. Despite the cross-reactivity observed with sera from visceral leishmaniasis patients, the IgG immunofluorescence test with the amastigote antigen had the highest sensitivity, specificity, and efficiency. No relationship was observed between the class-specific antibodies or their titers and the clinical forms of patients in the chronic phase. Amastigotes from the cell culture supernatant proved to be useful as an alternative antigen to epimastigotes because of their high resolution in the serodiagnosis of Chagas' disease.

Treated-cured indeterminate leprosy: a search for predictive histopathological and immunohistochemical parameters in skin biopsies taken from patients at admission and at clinical discharge.

In a previous study an index (sigma 3) resulting from the summation of three parameters, i.e., presence of bacilli, even in small numbers, in various dermal structures, multiple positive antigen sites as detected by anti-BCG antiserum and dermal nerve involvement, identified 72.22% of cases of indeterminate leprosy which progressed to multibacillary leprosy. The present study was undertaken to investigate possible parameters which might be indicative of indeterminate leprosy which would persist unchanged or be cured (treated cured patients). Thirty treated cured indeterminate leprosy patients were selected from the files of the São Paulo Health Institute and studied by histopathological, immunohistochemical and statistical methods similar to those employed in the previous study. The sigma 3 index was 4.10 +/- 0.60, a finding that places this group of patients in a position close to that of patients changing to paucibacillary leprosy but statistically different from that of patients progressing to multibacillary leprosy. Moreover, it was found that patients belonging to this group have heterogeneous single parameters, some of them suggestive of multibacillary and others of paucibacillary leprosy. Immunologically based techniques mainly employing rabbit anti-BCG serum as the primary antibody have proved to be valuable to detect antigen sites in biopsies from indeterminate leprosy patients and should be used together with the bacillary index during the follow up and clinical discharge control of such patients. In the present study, we show that clinical discharge of these patients did not mean a complete clearance of bacillary antigens.