Blood cell attachment to root surfaces treated with EDTA gel.

Root debridement generates a smear layer which contains microorganisms and toxins that could interfere in periodontal healing. For this reason, different substances have been used to remove it and to expose collagen fibers at the tooth surface. Blood element adhesion to demineralized roots and clot stabilization by collagen fibers are extremely important for the success of periodontal surgery. The aim of this study was to evaluate the different patterns of blood element adsorption and adhesion to root surfaces only irrigated with distilled water and after application of a manipulated or an industrialized EDTA gel. Thirty samples were planed, equally divided into three groups and treated with distilled water (control), a manipulated EDTA gel or an industrialized one. Immediately after, samples were exposed to fresh blood and prepared for scanning electron microscopy. Untreated planed dentin presented the best results with blood cells entrapped in a thick web of fibrin. In the manipulated EDTA group, the web of fibrin was thick with sparse blood elements. The worst result was seen with the industrialized EDTA group, in which no blood elements could be seen. Statistical difference was obtained between control and industrialized EDTA groups. Surfaces only irrigated presented the most organized fibrin network and cell entrapment.

Immunomodulatory activities associated with β-glucan derived from Saccharomyces cerevisiae

In this study we investigated the effect of β-glucan derived from Saccharomyces cerevisiae on fungicidal activity, cytokine production and natural killer activity. Spleen and peritoneal cells from female C57BL/6 mice, previously injected (24 or 48 h) with 20 or 100 μg of glucan by i.p. route, were assayed. In vivo β-glucan administration primed spleen cells for a higher production of IL-12 and TNF-α when S. aureus was used as a stimulus. In addition, β-glucan increased NK spleen cells activity against YAC target cells. Some immunomodulatory activities not yet described for β-glucan were observed in this work. © 2005 Institute of Physiology, Academy of Sciences of the Czech Republic.

Clinical and cardiovascular alterations produced by scorpion envenomation in dogs

Scorpionism is a common problem that occurs in tropical and subtropical countries and assumes great medical-sanitary importance due to its fatal effect on sensitive individuals, being able to lead children and aged people to death. The envenomation lethal potential is responsible for the serious cardiopulmonary alterations the scorpion toxin produces in its victims. The present research evaluated the effects of Tityus serrulatus venom on dogs, using two distinct doses: a dose that simulates natural envenomation (0.4 mg/total dose), and an experimental dose (0.25 mg/kg). General clinical signs were observed at different moments after envenomation, and specific data related to the cardiopulmonary system were evaluated by systemic arterial pressure measurement, CK-MB enzymatic activity dosage, and radiographic, electrocardiographic and echocardiographic examinations. Results demonstrated that the scorpion venom, in experimental doses, was able to cause acute and reversible cardiac injury in few days, and, in the dose that simulated natural accident, it produced clinical signs of light envenomation, such as local pain, hyperesthesia, sialorrhea, vomiting, diarrhea, sneeze and prostration.

Selection of environmental temperature by the yellow scorpion Tityus serrulatus Lutz & Mello, 1922 (Scorpiones, Buthidae)

The preferred temperature of the yellow scorpion Tityus serrulatus was investigated since its dispersion is a matter of concern. Adult T. serrulatus, weighing 1.24 ± 0.20 g (mean + sd) and with a standard length of 59.3 ± 2.5 mm, were used. A metallic corridor (120 cm long, 5 cm large and 10 cm high) with thermal gradient ranging from 0°C to 40°C was used. Tityus serrulatus chose and stayed in temperatures ranging from 14° C to 38°C when safe conditions were offered (dark and thigmotactic stimuli). The number of animals that remained in the 11°C-20°C, 21°C-30°C, and 31°C-40°C temperature zones were 8, 8, and 9, respectively. The chi-square test (degree of freedom = 2) showed that differences were not significant (p>0.05). Some animals moved to lower temperature areas (less than 8°C) when the corridor was completely illuminated and thigmotactic stimuli were absent, which led the animals to present a torpor state. It is concluded that T. serrulatus does not select a specific environmental temperature. Associated with the capacity of temporally surviving at low temperatures, this species seems to be highly adaptable to different thermal zones.

Immune reconstitution in HIV-1 infected patients treated for two years with highly active antiretroviral therapy

The aim of this paper was to evaluate the immune reconstitution of HIV-1 patients subjected to highly active antiretroviral therapy (HAART) for two years or more according to CD 45RA and CD 45RO cell count; determination of IL-2, IFN-γ, IL-4, IL-10 and TNF-α serum levels; CD 4 + T and CD 8 + T lymphocyte count; and plasma viral load (VL) determination. For this purpose, a cross sectional study was carried out in the Tropical Diseases Area, Botucatu School of Medicine, São Paulo State University, UNESP, Botucatu, São Paulo, Brazil. Between June 2001 and April 2002, 37 HIV-1 infected patients were evaluated, 13 with treatment indication but untreated (G1), 9 subjected to HAART for 5-7 months (G2), and 15 treated for two years or more (G3); both treated groups used medication regularly and without failure. Forty-nine normal individuals were studied as controls (GC-1 and GC-2). There was a tendency (p<0.10) for the predominance of two nucleoside reverse transcriptase inhibitors (NRTI) associated with one non-nucleoside reverse transcriptase inhibitor (NNRTI) regimen in G2; and two NRTI associated with a protease inhibitor (PI) in G3. Statistical differences between groups were seen for CD 45RA (G1<[G3=GC-2]; p<0.05) and CD 45RO (G1[G2=G3]; p<0.001), TNF-α serum determination ([G1>G3; G2=intermediate]>GC-1; p<0.001), IL-2 (G1<[G2=G3=GC-1]; p<0.01), IFN-γ ([G1=GC-1]>[GC-2=G3]; p<0.001), IL-4 and IL-10 ([G1=G2=G3]>GC-1; p<0.001), serum cytokine profiles, with a higher proportion of subtype 2 in G1 and mature subtype 0 in G2 and G3 (p<0.005). There was no statistical difference for CD 8 + T lymphocyte counts (G1=G2=G3; p<0.50). Consistency was seen between positive correlations of profile 1 definer cytokines (IL-2 and IFN-γ), CD 45RA and CD 45RO cells, and CD 4 + T lymphocyte counts and between positive correlations of profile 2 definer cytokines (IL-4 and IL-10) with TNF-α, and VL. The negative correlations were also consistent as they expressed the inverse of the positives. The variables with the highest number of correlations were IL-2, IFN-γ, and VL, followed by CD 45RA and CD 45RO cells, and IL-10. The variables with the lowest number of correlations were CD 4 + T and CD 8 + T lymphocytes. The results express the partial but important immune reconstitution in HIV-1 infected individuals with the interference of HAART and the importance of cytokines especially IL-2 and IFN-γ, and CD 45RA and CD 45RO cells as surrogate markers of this reconstitution.

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.

Serological profile of anti-Toxoplasma gondii antibodies in apparently healthy dogs of the city of Botucatu, São Paulo state, Brazil

Toxoplasmosis is a zoonosis caused by Toxoplasma gondii, a coccidian protozoan of worldwide distribution. The seroprevalence in canine population can be an alternative for measuring T. gondii urban spreading. A total of 780 blood samples from dogs were collected, during the yearly anti-rabies campaign, carried out by the Department of Veterinary Hygiene and Public Health, School of Veterinary Medicine and Animal Husbandry (FMVZ), São Paulo State University, UNESP, together with the county health authorities, in August 1999. Using Indirect Fluorescent Antibody Test (IFAT) for detecting antibodies anti-T. gondii in the sera samples, we observed that 258 dogs (33.1%) were positive. The associations between the serological results and the epidemiological variables were studied. Statistically significant differences were not found regarding sex (32.2% male and 34.3% female reactors). Dogs without a defined breed showed seropositivity statistically higher than the pedigreed group. The occurrence of infection was considered higher with age.

Effect of prostaglandins on the production of H2O2 and cytokines that modulate the fungicidal activity of human monocytes against Paracoccidioides brasiliensis

Human monocytes lack fungicidal activity against high virulent strains of Paracoccidioides brasiliensis, the etiological agent of paracoccidioidomycosis, even after IFN-γ activation. However, monocytes treated with indomethacin (INDO) or INDO plus IFN-γ effectively killed this fungus, suggesting an inhibitory role of prostaglandins in this process. Thus, the purpose of this work was to test if this regulatory effect of prostaglandin was associated with alterations on H2O2 production and/or on modulatory cytokines levels, such as TNF-α, IL-10, and IL-6. Peripheral blood monocytes obtained from 10 healthy donors were incubated for 18 hours in the presence or absence of IFN-γ, INDO, or IFN-γ plus INDO, and further challenged with a high virulent strain of P. brasiliensis (Pb18) for 4 hours. Then, the monocytes cultures were evaluated for H2O2 release and fungicidal activity calculated by counting the colony forming units after plating. Moreover, on supernatants of the same cultures, TNF-α, IL-10, IL-6, and PGE2 concentrations were evaluated by ELISA. Monocytes treated with INDO or INDO plus IFN-γ presented higher fungicidal activity associated with the release of higher levels of H2O2 and TNF-α, but lesser levels of PGE2, when compared to nontreated cells. However, the levels of IL-10 and IL-6 were similar between treated and nontreated cells. The results suggest that human monocytes when challenged with high virulent strains of P. brasiliensis produce prostaglandins that inhibit the fungicidal activity of these cells by reducing H2O2 and TNF-α levels.

Cytokines profile and clinical-metabolic alterations in HIV-1-infected individuals with and without lypodistrophy

Highly active antiretroviral therapy (HAART) has lead to a dramatic decrease in the morbidity of patients infected with HIV. However, metabolic side effects, including lipodystrophy and dyslipidemia, have been reported in patients treated with antiretroviral therapy (HAART). The aim of this study was to analyze the clinical and metabolic alterations and the cytokines TNF-α, IFN-γ, IL-2, IL-10 and TNF-II receptors profile in the serum of treated HIV-1-infected individuals with or without lipodystrophy. Eighty-four adult patients were analyzed, 42 females and 42 males, their mean age was 37 years old, and they received HAART for at least 15 months. These patients were ambulatory outpatients from the Infectious and Parasitary Disease Area of Botucatu School of Medicine, UNESP. Subsequently the individuals were distributed into 2 groups, G1: 42 HIV-infected individuals with lipodystrophy, and G2: 42 HIV-infected individuals without lipodystrophy. Among the antiretrovirals used, stavudine was more associated to the lipodystrophy group and zidovudine to the group without lipodystrophy. CD4, CD8, viral load, glucose, albumin, and the circulating lipid did not present any difference in the group comparison, except for triglyceride that was elevated in the lipodystrophy group and HDL which was present in low concentration in more patients of G1. The cytokines TNF-α, TNF-RII, and IL-10 profile presented high levels in the lipodystrophy group; also it was positively correlated with this group. On the other hand, IL-2 and IFN-γ presented low levels in this group. High levels of TNF-α and its receptor seem to be associated to the development of lipodystrophy in patients receiving HAART.

Use of skin graft punch graft type for the healing of leg ulcers in treated Hansen's disease patients

Hansen's disease is an infectious illness caused by Mycobacterium leprae. It affects preferentially the skin and the peripheral nervous system leading to incapacities, such as leg ulcers, which happens due to the direct action of the bacillus on the organs or its indirect action on the peripheral nervous system. Leg ulcers can occur by two physiopathologic processes. There are many treatments for general leg ulcers, which include the ones caused by Hansen's disease sequels. Among them, surgical treatment shows to be effective when using skin graft, which can be performed by several techniques. Considering the low number of techniques known for treating leg ulcers in Hansen's disease sequels, the aims of this work were to standardize alternative techniques, to detect the main bacteria found in ulcer secretion cultures, to analyze the patients profile and the ulcers, to describe the histophatologies found, and to correlate these data with those of literature from all over the world. Skin graft punch type was carried out and analyzed; males had a mean age of 59.4 years old and females, 54.2 years old. Patients were 73.6% male and 26.3% female. Lepromatous type was present in 89.4% patients and tuberculoid type was seen in 10.5% of them. Associated systemic diseases were observed in 26.3% patients. Mean time of ulcers evolution was 11.6 years in male and 12.8 years in women. The average diameter of ulcers in the pre-treatment period was 8.5 X 9.5 cm in male and 10.2 X 6.8 cm in women. After the graft, their average diameters were 3.2 X 2.7 cm in male and 5.1 X 5.6 cm in women. Statistical analysis showed that there was no significant correlation between the ulcer diameter and its reduction or not in the post-surgery period (p=0.269732). The mean age of patients whose ulcers diameter did not change or reduced by only 20% was 63.5 years. Using the Spearman's coefficient, it was possible to observe that there was no significant correlation between the patients' age and the ulcers diameter reduction after the skin graft (p=0.222531). Evolution time of ulcers that did not present any satisfactory result in the post-surgery period was 12.1 years. The Spearman's coefficient showed that there was no significant correlation between the ulcers evolution time and the ulcers diameter reduction in the post-surgery period (p=0.191655). Cultures presented 50% of cases with Pseudomonas aeruginosa. Statistical analysis showed there is no correlation between the bacterial types found and the ulcer evolution in the post-surgery period (p=0.697531). The average of the ulcers diameter reduction was 42.4%, and in 26.3% of the patients the lesions disappeared after the skin graft.

Antibody and cytokine serum levels in patients subjected to anti-rabies prophylaxis with serum-vaccination

Rabies is considered a fatal disease once clinical symptoms have developed. The aim of this study was to evaluate epidemiological aspects and immune response in patients attacked by domestic and wild animals and subjected to post-exposure rabies treatment with equine serum and associated vaccine. Thirty-three patients were evaluated; they were between 13 and 65 years old, 75.8% were male and 24.2% female, and from the Botucatu neighborhood. Twenty healthy control individuals with the same age range were also studied. Specific antibodies to equine immunoglobulins and IFN-γ, IL-2, IL-4, and IL-10 production were evaluated by ELISA. IgM, IgE, IgG and subclasses, and rabies virus antibodies serum levels were determined by nephelometry and seroneutralization methods, respectively. No anaphylactic or serum sickness allergic reactions were observed in patients after treatment. Anti-equine IgG levels were significantly higher than those of IgM after 14 and 28 days of treatment. Protective antibodies to rabies virus > 0.5 UI/ml were detected in 84.6% and 75% of patients at days 14 and 28, respectively. IFN-γ, IL-2 and IL-10 levels in patients before and 48h after treatment were significantly higher than in controls suggesting that both Th1 and Th2 cells were activated in the patients. Serum IgM levels were higher at day 14, and IgG 2 and IgE levels were higher at day 28 of treatment. These results suggest that post-exposure rabies treatment in humans induces significant alterations in patient immune response characterized by increased levels of cytokines, serum levels of specific rabies virus antibodies, and the equine serum components employed in the treatment.

Interval between first dose and booster affected antibody production in cattle vaccinated against rabies

In this study, we compared the levels of neutralizing antibodies induced by inactivated rabies vaccine in cattle by using three alternative immunization procedures. Forty-five bovines (breed nelore) were then organized in three groups (A, B and C, with 15 animals/group). Group A received only one vaccine dose at day zero and Group B received the first dose at day zero and then another dose at day 30 (early booster). Group C was also immunized with two doses; however, the booster was postponed until day 180 after the first dose (delayed booster). Blood samples were withdrawn at days zero (before the first dose) and 30, 210, 390, and 540 after the beginning of immunization and the antibody titers were evaluated by mouse neutralization test. The protocol used to immunize Group C (booster at day 180) was clearly more efficient. In this group, antibody levels were higher and also remained higher for longer periods in comparison with the other two groups. These results show that booster timing significantly affected antibody levels. Therefore, programs addressed to control this disease in cattle should consider not only the use of a booster but also its administration time.

Evaluation of the antimicrobial susceptibility of methicillin-resistant Staphylococcus aureus isolated from human infections

Multiresistant Staphylococcus aureus constitutes an important public health problem, especially in view of its possible spread in nosocomial environments. In the present work, we analyzed the susceptibility profile of 80 S. aureus stains from human infections resistant to at least 10 drugs. For this study, the techniques used were the disk method and minimum inhibitory concentration (MIC) of the following drugs: cefuroxime, ciprofloxacin, clindamycin, erythromycin, gentamycin, imipenem, oxacillin, rifampicin, tetracycline and vancomycin, according the criteria of the National Committee for Clinical Laboratory Standards (NCCLS). Methicillin was included in the antibiogram as a marker, which is usually used in drugs selection for the treatment of staphylococcal infections. Results indicated that the most effective drug was vancomycin. For the other 10 drugs, the percentage of resistant strains ranged from 85% to 93.75%. In relation to the MICs, it was observed that vancomycin (MIC 90% = 0.615ug/ml) was the most effective drug; followed by rifampicin (MIC 90% = 2.6ug/ml) and ciprofloxacin (MIC 90% = 26.6ug/ml). The drugs that showed the least effective activity were cefuroxime, clindamycin, erythromycin, gentamycin, and oxacillin. On the other hand, observation of β-lactamase production revealed that most of the methicillin-resistant strains produced β-lactamase (83.7%), potentiating the risks of nosocomial infections. In general, vancomycin still continues to be one of the most effective drugs for staphylococcal infections therapy.

Bacterial contamination of the catfish (Cathorops agassizii - Valenciennes, 1839 and Genidens genidens - Agassiz, 1829) sting

This work aimed to study the bacterial contamination in stings of the catfish Genidens genidens and Cathorops agassizii found in the São Vicente estuarine system (São Paulo State, Brazil). For bacteriological analyses, we used fish samples distributed into a group of 50 specimens (25 C. agassizii and 25 G. genidens) and a group of 14 specimens (7 C. agassizii and 7 G. genidens). Results showed contamination of 13 different bacterial species of Enterobacteriaceae, being Klebsiella pneumoniae the most frequent bacteria (26.80%) followed by Enterobacter sp and Escherichia coli (16.27%), and Serratia marcescens, Serratia sp. and Proteus mirabilis (1.16%). Gram-positive bacteria as well as fungi were not detected in the samples. According to the Gram-negative species characterized and with regard to the environmental conditions, it can also be considered that accidents with these catfish stings may develop significant acute secondary infections in humans.

Risk of peritonitis during peritoneal dialysis in carriers of Staphylococcus aureus and coagulase-negative staphylococci

The presence of Staphylococcus aureus in the nasal cavities and pericatheter skin of peritoneal dialysis patients put them at high risk of developing peritonitis. However, it is not clear whether the presence of coagulase-negative staphylococci (CNS) in the nasal passages and skin of patients is related to subsequent occurrence of peritoneal infection. The aim of the present study was to verify the relationship between endogenous sources of S. aureus and CNS and occurrence of peritonitis in patients undergoing peritoneal dialysis. Thirty-two patients on peritoneal hemodialysis were observed for 18 months. Staphylococcus species present in their nasal passage, pericatheter skin and peritoneal effluent were identified and compared based on drug susceptibility tests and dendrograms, which were drawn to better visualize the similarity among strains from extraperitoneal sites as well as their involvement in the causes of infection. Out of 288 Staphylococcus strains isolated, 155 (53.8%) were detected in the nasal cavity, 122 (42.4%) on the skin, and 11 (3.8%) in the peritoneal effluent of patients who developed peritonitis during the study. The most frequent Staphylococcus species were CNS (78.1%), compared with S. aureus (21.9%). Among CNS, S. epidermidis was predominant (64.4%), followed by S. warneri (15.1%), S. haemolyticus (10.7%), and other species (9.8%). Seven (64%) out of 11 cases of peritonitis analyzed presented similar strains. The same strain was isolated from different sites in two (66%) out of three S. aureus infection cases. In the six cases of S. epidermidis peritonitis, the species that caused infection was also found in the normal flora. From these, two cases (33%) presented highly similar strains and in three cases (50%), it was difficult to group strains as to similarity. Patients colonized with multidrug-resistant S. epidermidis strains were more predisposed to infection. Results demonstrated that an endogenous source of S. epidermidis could cause peritonitis in peritoneal dialysis patients, similarly to what has been observed with S. aureus.