Methemoglobinemia associated with loxoscelism

In twenty five patients who presented the cutaneous form of loxoscelism, serum haptoglobin and lactic dehydrogenase, erythrocyte glucose-6-phosphate dehydrogenase, glutathione reductase, glutathione peroxidase, methemoglobin, bilirubin and reticulocytes were investigated after bite. No hemolysis was detected but an increase in methemoglobin was found in 54% of the cases; in 7% it was between 1.1% and 2%, in 27% it ranged from 2.1% to 4%, and in 20% from 4.1% to 8%. Blood samples of a normal, blood group 0 individual and of a patient who exhibited methemoglobinemia after Loxosceles bite were incubated separately with antisera against Loxosceles gaucho, Crotalus terrificus, Bothrops jararaca, with Loxosceles gaucho venom and 0.3% phenol. No methemoglobin was found after 1, 4,8 and 15 days in both sets of samples. At the 25th day all the samples, including the controls, exhibited similar methemoglobin reductase decrease. The data suggest that the methemoglobinemia which occurs in 50% of the patients probably arises from in vivo venom metabolism, inasmuch as the crude venom does not induce methemoglobinemia.

Comparative studies of Yersinia pestis outer membrane isolation techniques and their potential use in plague epidemiology

In the present study three techniques for obtaining outer membrane enriched fractions from Yersinia pestis were evaluated. The techniques analysed were: differential solubilization of the cytoplasmic membrane with Sarkosyl or Triton X-100, and centrifugation in sucrose density gradients. The sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of outer membrane isolated by the different methods resulted in similar protein patterns. The measurement of NADH-dehydrogenase and succinate dehydrogenase (inner membrane enzymes) indicated that the outer membrane preparations obtained by the three methods were pure enough for analytical studies. In addition, preliminary evidences on the potential use of outer membrane proteins for the identification of geographic variants of Y. pestis wild isolates are presented.

Human cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis in Santiago del Estero, Argentina: identification of parasites by monoclonal antibodies and isoenzymes

Diagnostic and parasite characterization and identification studies were carried out in human patients with cutaneous leishmaniasis lesions in Santiago del Estero, Northern Province of Argentina. Diagnostic procedures were biopsies of lesions for smears and inoculations in hamster, needle aspirations of material from ulcers for "in vitro" cultures. Immunodiagnostic techniques applied were IFAT-IgG and Montenegro skin test. Primary isolation of eight stocks of leishmanial parasites was achieved from patients with active lesions. All stocks were biologically characterized by their behaviour in hamster, measurements of amastigote and promastigotes and growth "in vitro". Eight stocks were characterized and identified at species level by their reactivity to a cross-panel of sub-genus and specie-specific Monoclonal Antibodies through an Indirect Immunofluorescence technique and a Dot-ELISA. We conclude from the serodeme analysis of Argentina stocks that: stocks MHOM/AR/92/SE-1; SE-2; SE-4; SE-8; SE-8-I; SE-30; SE-34 and SE-36 are Leishmania (Viannia) braziliensis. Three Leishmania stocks (SE-1; SE-2 and SE-30) did not react with one highly specie-specific Monoclonal Antibody (Clone: B-18, Leishmania (Viannia) braziliensis marker) disclosing two serodeme group patterns. Five out of eight soluble extracts of leishmanial promastigotes were electrophoresed on thin-layer starch gels and examined for the enzyme MPI, Mannose Phosphate Isomerase; MDH, Malate Dehydrogenase; 6PGD, 6 Phosphogluconate Dehydrogenase; NH, Nucleoside Hydrolase, 2-deoxyinosinc as substrate; SOD, Superoxide Dismutase; GPI, Glucose Phosphate Isomerase and ES, Esterase. From the isoenzyme studies we concluded that stocks: MHOM/AR/92/SE-1; SE-2; SE-4; SE-8 and SE-8-I are isoenzymatically Leishmania (Viannia) braziliensis. We need to analyze more enzymes before assigning them to a braziliensis zymodeme.

Snakebites by Crotalus durissus ssp in children in Campinas, São Paulo, Brazil

From January, 1984 to March, 1999, 31 children under 15 y old (ages 1-14 y, median 8 y) were admitted after being bitten by rattlesnakes (Crotalus durissus ssp). One patient was classified as "dry-bite", 3 as mild envenoming, 9 as moderate envenoming and 18 as severe envenoming. Most patients had neuromuscular manifestations, such as palpebral ptosis (27/31), myalgia (23/31) and weakness (20/31). Laboratory tests suggesting rhabdomyolysis included an increase in total blood creatine kinase (CK, 28/29) and lactate dehydrogenase (LDH, 25/25) levels and myoglobinuria (14/15). The main local signs and symptoms were slight edema (20/31) and erythema (19/31). Before antivenom (AV) administration, blood coagulation disorders were observed in 20/25 children that received AV only at our hospital (incoagulable blood in 17/25). AV early reactions were observed in 20 of these 25 cases (9/9 patients not pretreated and 11/16 patients pretreated with hydrocortisone and histamine H1 and H2 antagonists). There were no significant differences in the frequency of patients with AV early reactions between the groups that were and were not pretreated (Fisher's exact test, p = 0.12). Patients admitted less than and more than 6 h after the bite showed the same risk of developing severe envenoming (Fisher's exact test, p = 1). No children of the first group (< 6 h) showed severe complications whereas 3/6 children admitted more than 6 h post-bite developed acute renal failure. Patients bitten in the legs had a higher risk of developing severe envenoming (Fisher's exact test, p = 0.04). There was a significant association between both total CK and LDH blood enzyme levels and severity (p < 0.001 for CK and p < 0.001 for LDH; Mann-Whitney U test). No deaths were recorded.

HYPOXIC STRESS, HEPATOCYTES AND CACO-2 VIABILITY AND SUSCEPTIBILITY TO Shigella flexneri INVASION

SUMMARY Inflammation due to Shigella flexneri can cause damage to the colonic mucosa and cell death by necrosis and apoptosis. This bacteria can reach the bloodstream in this way, and the liver through portal veins. Hypoxia is a condition present in many human diseases, and it may induce bacterial translocation from intestinal lumen. We studied the ability of S. flexneri to invade rat hepatocytes and Caco-2 cells both in normoxic and hypoxic microenvironments, as well as morphological and physiological alterations in these cells after infection under hypoxia. We used the primary culture of rat hepatocytes as a model of study. We analyzed the following parameters in normoxic and hypoxic conditions: morphology, cell viability, bacterial recovery and lactate dehydrogenase (LDH) released. The results showed that there were fewer bacteria within the Caco-2 cells than in hepatocytes in normoxic and hypoxic conditions. We observed that the higher the multiplicity of infection (MOI) the greater the bacterial recovery in hepatocytes. The hypoxic condition decreased the bacterial recovery in hepatocytes. The cytotoxicity evaluated by LDH released by cells was significantly higher in cells submitted to hypoxia than normoxia. Caco-2 cells in normoxia released 63% more LDH than hepatocytes. LDH increased 164% when hepatocytes were submitted to hypoxia and just 21% when Caco-2 cells were in the same condition. The apoptosis evaluated by Tunel was significantly higher in cells submitted to hypoxia than normoxia. When comparing hypoxic cells, we obtained more apoptotic hepatocytes than apoptotic Caco-2 cells. Concluding our results contribute to a better knowledge of interactions between studied cells and Shigella flexneri. These data may be useful in the future to define strategies to combat this virulent pathogen.

FATAL DISSEMINATED CRYPTOCOCCOSIS WITH RENAL INVOLVEMENT IN AN HIV-INFECTED PATIENT

SUMMARY Introduction: We present a fatal case of disseminated cryptococcosis in a young man whose diagnosis of HIV infection was made at the time of admission to the emergency room. Case report: The patient was a twenty-three-year-old man, with a history of daily fever during one month associated with diarrhea, weight loss, headache, vomiting and generalized seizures. He also had a history of diabetes mellitus, alcoholism and drug addiction. Upon physical examination the patient was pale, disoriented and had periods of agitation. White blood cells count was 3,440/mm3 (5% lymphocytes), hemoglobin was 10g/dL, platelets were 83,000/ mm3. Creatinine was 0.7 mg/dL; urea 19 mg/dL; Na, K, and liver enzymes were within normal limits. Lactic dehydrogenase was 494 IU/L. Cerebrospinal fluid (CSF) analysis revealed 10 white blood cells/mm3 (58% neutrophils, 31% lymphocytes, 11% monocytes) and 2 red blood cells/mm3. India ink test revealed six Cryptococcus yeasts/mm3. CSF glucose was 122 mg/dL and protein was 36 mg/ dL. VDRL test was negative and anti-HIV test was positive. Intravenous hydration, insulin, phenytoin, fluconazole, pyrimethamine, sulfadiazine, folinic acid, and amphotericin B were started. The patient did not improve and became obtunded and hypotensive. He was intubated and put on mechanical respiration. He received vasoactive drugs and died less than 24 hours after admission. A postmortem examination was performed and revealed disseminated cryptococcosis, with severe involvement of the kidneys. Conclusion: Cryptococcosis, as a rule, is a systemic disease that affects mostly immunocompromised individuals, especially patients with AIDS. When diagnosed late in its course it has a very high mortality.

Echinococcus granulosus sensu lato GENOTYPES IN DOMESTIC LIVESTOCK AND HUMANS IN GOLESTAN PROVINCE, IRAN

Cystic echinococcosis (CE) is a globally parasitic zoonosis caused by larval stages of Echinococcus granulosus. This study investigated E. granulosus genotypes isolated from livestock and humans in the Golestan province, northern Iran, southeast of the Caspian sea, using partial sequencing data of the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase 1 (nad1) mitochondrial genes. Seventy E. granulosus isolates were collected from animals in slaughterhouses: 18 isolates from sheep, 40 from cattle, nine from camels, two from buffaloes and one from a goat, along with four human isolates (formalin-fixed, paraffin-embedded tissues) from CE patients of provincial hospitals. All isolates were successfully analysed by PCR amplification and sequencing. The sequence analysis found four E. granulosus genotypes among the 74 CE isolates: G1 (78.3%), G2 (2.7%), G3 (15%) and G6 (4%). The G1-G3 complex genotype was found in all of the sheep, goat, cattle and buffalo isolates. Among the nine camel isolates, the frequency of G1-G3 and G6 genotypes were 66.7% and 33.3%, respectively. All four human CE isolates belonged to E. granulosus sensu stricto. This study reports the first occurrence of the G2 genotype in cattle from Iran and confirms the previously reported G3 genotype in camels in the same country.

Africanized honeybee stings: how to treat them

INTRODUCTION: In 1956, Africanized honeybees (AHB) migrated from Brazil to other regions of the Western Hemisphere, including South, Central, and North America, except for Canada. Despite being productive, they are highly aggressive and cause fatal accidents. This study aimed to evaluate patients at the Clinical Hospital of Botucatu Medical School (HC-FMB) and to propose treatment guidelines. METHODS: From 2005 to 2006, the clinical and laboratorial aspects of 11 patients (7 male and 4 female) and the anatomopathological aspects of one patient who had died in 2003 were analyzed. RESULTS: The age of the surviving patients varied from 5 to 87 years, with a mean of 42.5 years. The majority of accidents occurred in the afternoon, and the number of stings ranged from 20 to 500. The principal signs and symptoms were pain and local inflammatory signs, nausea, tachycardia, and vomiting. Biochemical findings presented increased levels of creatine phosphokinase, lactate dehydrogenase, and aspartate/alanine aminotransferase. An 11-year-old male patient died upon entering the attic of a two-storey building where he was attacked by a swarm, receiving more than 1,000 stings. He was sent to HC-FMB where he was treated, but he died 24h later. Observed at the autopsy were erythematous-purpuric skin lesions besides necrosis at the sting locations, rhabdomyolysis, focal myocardial necrosis, tubular hydropic degeneration and focal tubular acute necrosis of the kidneys, myoglobinuria, and centrolobular necrosis in the liver. CONCLUSIONS: Accidents caused by multiple AHB stings always constitute a medical emergency. As there is no specific antivenom, we have developed guidelines, including first aid, drugs, and the proper removal of stingers.

Molecular identification and antifungal susceptibility profiles of Candida parapsilosis complex species isolated from culture collection of clinical samples

AbstractINTRODUCTION:Candida parapsilosis is a common yeast species found in cases of onychomycosis and candidemia associated with infected intravascular devices. In this study, we differentiated Candida parapsilosis sensu stricto, Candida orthopsilosis , and Candida metapsilosis from a culture collection containing blood and subungual scraping samples. Furthermore, we assessed the in vitro antifungal susceptibility of these species to fluconazole, itraconazole, voriconazole, posaconazole, amphotericin B, and caspofungin.METHODS:Differentiation of C. parapsilosis complex species was performed by amplification of the secondary alcohol dehydrogenase (SADH) gene and digestion by the restriction enzyme Ban I. All isolates were evaluated for the determination of minimal inhibitory concentrations using Etest, a method for antifungal susceptibility testing.RESULTS:Among the 87 isolates, 78 (89.7%) were identified as C. parapsilosis sensu stricto , five (5.7%) were identified as C. orthopsilosis , and four (4.6%) were identified as C. metapsilosis . Analysis of antifungal susceptibility showed that C. parapsilosis sensu strictoisolates were less susceptible to amphotericin B and itraconazole. One C. parapsilosis sensu stricto isolate was resistant to amphotericin B and itraconazole. Moreover, 10.2% of C. parapsilosis sensu stricto isolates were resistant to caspofungin. Two C. parapsilosis sensu strictoisolates and one C. metapsilosis isolate were susceptible to fluconazole in a dose-dependent manner.CONCLUSIONS:We reported the first molecular identification of C. parapsilosiscomplex species in State of Goiás, Brazil. Additionally, we showed that although the three species exhibited differences in antifungal susceptibility profiles, the primary susceptibility of this species was to caspofungin.

Indicators of inflammation and cellular damage in chronic asymptomatic or oligosymptomatic alcoholics: correlation with alteration of bilirubin and hepatic and pancreatic enzymes

Biochemical and hematimetric indicators of inflammation and cell damage were correlated with bilirubin and hepatic and pancreatic enzymes in 30 chronic male alcoholics admitted into psychiatric hospital for detoxification and treatment of alcoholism. Aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltransferase, alkaline phosphatase, and total bilirubin were altered, respectively, in 90%, 63%, 87%, 23% and 23% of the cases. None of the indicators of inflammation (lactic dehydrogenase, altered in 16% of the cases; alpha-1 globulin, 24%; alpha-2 globulin, 88%; leucocyte counts, 28%) was correlated with alterations of bilirubin or liver enzymes. Lactic dehydrogenase was poorly sensitive for detection of hepatocytic or muscular damage. Alterations of alpha-globulins seemed to have been due more to alcohol metabolism-induced increase of lipoproteins than to inflammation. Among indicators of cell damage, serum iron, increased in 40% of the cases, seemed to be related to liver damage while creatine phosphokinase, increased in 84% of the cases, related to muscle damage. Hyperamylasemia was found in 20% of the cases and significantly correlated with levels of bilirubin, alkaline phosphatase and gamma-glutamyltransferase. It was indicated that injuries of liver, pancreas, salivary glands, and muscle occurred in asymptomatic or oligosymptomatic chronic alcoholics.

Association study of the Ile349val polymorphism of the gene ADH1C and alcohol dependence

OBJECTIVE: The aim of this study was to investigate the polymorphism Ile349Val of the enzyme alcohol dehydrogenase ADH1C gene among individuals with alcohol dependence syndrome (ADS) attending Alcoholics Anonymous (AA) meetings. METHODS: A total of 120 subjects residing in Rio de Janeiro city participated in this study. Subjects were divided into two groups: a group consisting of 54 individuals from the ADS group and 66 individuals that declared not having any alcohol dependence (control group). DNA was extracted from mouth epithelial cells by phenol-chloroform method and further submitted to amplification by polymerase chain reaction (PCR). RESULTS: Our results did not show differences between the genotypes of control individuals and ADS subjects. Nevertheless, we found increased rates of alcoholism in families of ADS subjects as compared to controls. CONCLUSIONS: Our results did not show any genotype difference on the ADH1C gene when control and AA genotypes are compared.

Pentoxifylline Attenuates Cardiac Remodeling Induced by Tobacco Smoke Exposure

Abstract Background: Tobacco smoke exposure is an important risk factor for cardiac remodeling. Under this condition, inflammation, oxidative stress, energy metabolism abnormalities, apoptosis, and hypertrophy are present. Pentoxifylline has anti‑inflammatory, anti-apoptotic, anti-thrombotic and anti-proliferative properties. Objective: The present study tested the hypothesis that pentoxifylline would attenuate cardiac remodeling induced by smoking. Methods: Wistar rats were distributed in four groups: Control (C), Pentoxifylline (PX), Tobacco Smoke (TS), and PX-TS. After two months, echocardiography, invasive blood pressure measurement, biochemical, and histological studies were performed. The groups were compared by two-way ANOVA with a significance level of 5%. Results: TS increased left atrium diameter and area, which was attenuated by PX. In the isolated heart study, TS lowered the positive derivate (+dp/dt), and this was attenuated by PX. The antioxidants enzyme superoxide dismutase and glutathione peroxidase were decreased in the TS group; PX recovered these activities. TS increased lactate dehydrogenase (LDH) and decreased 3-hydroxyacyl Coenzyme A dehydrogenases (OH-DHA) and citrate synthase (CS). PX attenuated LDH, 3-OH-DHA and CS alterations in TS-PX group. TS increased IL-10, ICAM-1, and caspase-3. PX did not influence these variables. Conclusion: TS induced cardiac remodeling, associated with increased inflammation, oxidative stress, apoptosis, and changed energy metabolism. PX attenuated cardiac remodeling by reducing oxidative stress and improving cardiac bioenergetics, but did not act upon cardiac cytokines and apoptosis.

Distribuição da xantina oxidase no fígado e no sôro de rato

The localization of the xanthine oxidase (X.O.) and xanthine dehydrogenase (X.D.) activities in rat liver have been studied using separation of cytoplasmic particles into fractions by differential centrifugation. The results clearly demonstrate that practically all the enzymic activity is present in the supernatant fluid corresponding to the cell sap containing the soluble proteins of the cell. No activity could be detected for the nuclear, mitocondrial and microsomal fractions. The enzymatic activity of the mixture of the four factions was 102 per cent of that of the original homogenate. The distribution of the xanthine dehydrogenase in the protein fractions of the rat serum was accomplished in preliminary experiments by means of 50% ammonium sulphate precipitation and subsequent dialysis against water. All enzymatic activity was confined to the globulin fractions of the serum. Paper electrophoresis was performed and the protein and lipoprotein fractions determined. A method for the localization of the X.D. activity in the protein fractions separated by paper electrophoresis was developed. The results obtained suggest that xanthine dehydrogenase is localized in the globulin fractions possessing mobilities of [alpha 1], [beta] and [gamma] globulins and are probably bound to the lipoproteins.

Advances in pharmacological studies of silymarin

Silymarin is the flavonoids extracted from the seeds of Silybum marianum (L) Gearth as a mixture of three structural isomers: silybin, silydianin and silychristin, the former being the most active component. Silymarin protects liver cell membrane against hepatotoxic agents and improves liver function in experimental animals and humans. It is generally accepted that silymarin exerts a membrane-stabilizing action preventing or inhibiting membrane peroxidation. The experiments with soybean lipoxygenase showed that the three components of silymarin brought about a concentration-dependent non-competitive inhibition of the lipoxygenase. The experiments also showed an analogous interaction with animal lipoxygenase, thus showing that an inhibition of the peroxidation of the fatty acid in vivo was self-evident. Silybin almost completely suppressed the formation of PG at the highest concentration (0.3 mM) and proved to be an inhibitor of PG synthesis in vitro. In our experiments, silybin at lower dose (65 mg/Kg) decreased liver lipoperoxide content and microsomal lipoperoxidation to 84.5% and 68.55% of those of the scalded control rats respectively, and prevented the decrease of liver microsomal cytochrome p-450 content and p-nitroanisole-0-demethylase activity 24 h post-scalding. Effects of silymarin on cardiovascular systen have been studied in this university since 1980. O. O silymarin 800 mg/Kg/d or silybin 600 mg/Kg/d reduced plasma total cholesterol, LDL-C and VLDL-C. They however, enhanced HDL-C in hyperlipenic rats. Further studies showed that silymarin enhanced HDL-C in hyperlipemic rats. Further studies showed that silymarin enhanced HDL-C but didn't affect HDL-C, a property of this component which is beneficial to treatment of atherosclerosis. The results showed silymarin 80 mg or silybin 60 mg decreased in vitro platelet aggregation (porcentagem) in rats. The maximal platelet aggregation induced by ADP declined significantly, and time to reach maximal platelet aggregation and five-minute disaggregation didn't change. In our experiments, iv silybin 22,4 mg/kg lowered the amplitude and duration of diastolic blood pressure (DBP) more than those of systolic (SBP), but the descending aortic blood flow, cardiac contractility and ECG did not change significantly in anesthetized open-chest cats. The results indicated a reduction of peripheral resistance and dilatatory action on the resistant blood vessels. These effects are beneficial to coronary heart disease. We also observed the effects of silybin on morphological change, the release of glutamic oxaloacetate aminotrasferase (GOT) and lactate dehydrogenase (LDH) as well as the radioactivity of 3H-TdR incorporated into DNA in normal cardiac cells and cells infected by coxsackie B5, virus os newborn rats. The results showed that silynin did not affect the morphology of normal cell, and that the pathological change of cells infected by virus was delayed and reduced as compared to control. We have investigated the effect of silybin on synthesis and release of LTs in the cultured porcine cerebral basilar arteries (PCBA). Silybin 100 and 500 µmol/L declined the amounts of LTs released from the PCBA incubsated in the presence of A 23187, AA and indomenthacin. The result suggests that silybin can inhibit the activity of 5-lipoxygenase of cerebral blood vessel and may protect the brain from ischemia.

Pharmacological and toxicological evaluation of Alpinia speciosa

Alpinia speciosa Schum or A. nutans is a plant of the Zingiberanceae family, Known popularly as "colony" (colônia) and used as a diuretic and to control hypertensión. We have determinated the concentration of Na+ and K+ found in the alcoholic extract and in the tea concoction. They contained 51.0mEq Na+, and 132 mEq K+ in the extract, and 0,0 mEq of Na+ and 26 mEq K+ in the tea. Phytochemical analysis of the leaves demonstrated the presence of catecquic tanins, phenols and alkaloids, and also some essential oils. When injected intra-peritoneally the hydroalcoholic extract, in range of 100 a 1400 mg/Kg, (or 2500-18000 mg/Kg orally) produced in mice: writhing, psychomorot excitation, hypokinesis and pruritus. The LD50 by ip was 0.760 + or - 0.126 g/Kg and 10.0 + or - 2.5 g/Kg by oral administration for the hydroalcoholic extract. Subacute toxicity made injecting daily for 30 days the LD10 in rats caused an increase in transaminases and lactate dehydrogenase, whereas other parameters such as nlood glucose, urea and creatinine were normal. A histopathological analysis of liver, spleen, gut, lung and heart showed no alterations. The drug also produced a prolongation of the sleeping time. The hydroalcoholic extract induced int he rat and in the dog a dose-dependent fall in blood pressure in doses of 10 to 30 mg/Kg. In isolated atria the extract induced a reduction of the frequnecy and in the inotropic responses. Neither the extract nor the tea had an effect on the diuresis of the rat.