Oropouche virus entry into HeLa cells involves clathrin and requires endosomal acidification

Oropouche virus (ORO), family Bunyaviridae, is the second most frequent cause of arboviral febrile illness in Brazil. Studies were conducted to understand ORO entry in HeLa cells. Chlorpromazine inhibited early steps of ORO replication cycle, consistent with entry/uncoating. The data indicate that ORO enters HeLa cells by clathrin-coated vesicles, by a mechanism susceptible to endosomal acidification inhibitors. Transmission electron microscopy and immunofluorescence indicated that ORO associates with clathrin-coated pits and can be found in association with late endosomes in a time shorter than 1 h. (C) 2008 Elsevier B.V. All rights reserved.

Oropouche virus experimental infection in the golden hamster (Mesocrisetus auratus)

Oropouche virus (OROV), of the family Bunyaviridae, is the second most frequent arbovirus causing febrile disease in Brazil. In spite of this, little is known about pathogenesis of OROV infection. This report describes an experimental model of OROV in golden hamster (Mesocricetus auratus). Following subcutaneous inoculation of OROV, over 50% of the animals developed disease characterized by lethargy, ruffled fur, shivering, paralysis, and approximately one third died. Animals were sacrificed on days 1, 3, 5, 8 and 11 post-inoculation to collect tissue samples from brain, heart, liver, lung, spleen, muscle and blood for virus titration, histology and OROV immunohistochemistry. OROV was detected in high titers in blood, liver and brain, but not in the other organs. Histopathology revealed meningoencephalitis and hepatitis, with abundant OROV antigen detected in liver and brain. Diffuse galectin-3 immunostaining in brain and liver supports microglial and Kupfer cells activation. This is the first description of an experimental model for OROV infection and should be helpful to study pathogenesis and possibly to test antiviral interventions such as drugs and vaccine candidates. (C) 2010 Elsevier B.V. All rights reserved.

Quercetin does not alter lipopolysaccharide-induced fever in rats

Fever is considered an important component of the acute phase response of the body in defence against invading organisms such as bacteria. Quercetin, an important representative of the flavonoid class, has been extensively studied as an anti-inflammatory agent. In the present study, we investigated the effect of quercetin, administered orally (5, 25 and 50 mg kg(-1)) or intraperitoneally (50 mg kg(-1)), on the febrile response induced by either intraperitoneally (50 mu g kg(-1)) or intravenously (5 mu g kg(-1)) injected lipopolysaccharide (LPS from Escherichia coli) in rats. In contrast with the well known anti-inflammatory activity of quercetin, the results demonstrate that quercetin, at the doses used, did not alter the fever induced by LPS, regardless of the route of administration.

Functional characterization of the hypothalamic-pituitary-adrenal axis of the Wistar Audiogenic Rat (WAR) strain

The Wistar Audiogenic Rat (WAR) strain is a genetic model of sound-induced reflex epilepsy which was selected starting from audiogenic seizures susceptible Wistar rats. Wistar resistant rats were used as WAR`s control in this study. In the acute situation, audiogenic seizures (AS) in WARs mimic tonic-clonic seizures and, in the chronic protocol, mimic temporal lobe epilepsy. AS have been shown to evoke neuroendocrine responses; however, the hypothalamic-pituitary-adrenal activity in the WAR has not been established. The aim of this study was to evaluate the hypothalamic-pituitary-adrenal axis (HPA) responses to exogenous ACTH stimulation (8 ng/rat), fifteen minute restraint stress and circadian variation (8 am and 8 pm) under rest conditions in these animals through plasma measurements of ACTH and corticosterone concentrations. We also measured the body weight from birth to the 9th week of life and determined adrenal gland weight. We found that WARs are smaller than Wistar and presented a higher adrenal gland weight with a higher level of corticosterone release after intravenous ACTH injection. They also showed altered HPA axis circadian rhythms and responses to restraint stress. Our data indicate that, despite the lower body weight, WARs have increased adrenal gland weight associated with enhanced pituitary and adrenal responsiveness after HPA axis stimulation. Thus, we propose WARs as a model to study stress-epilepsy interactions and epilepsy-neuropsychiatry comorbidities. (C) 2011 Elsevier B.V. All rights reserved.

Inflammatory mediators involved in the nociceptive and oedematogenic responses induced by Tityus serrulatus scorpion venom injected into rat paws.

The present study investigated the role of kinins, prostaglandins (PGs) and nitric oxide (NO) in mechanical hypernociception, Spontaneous nociception and paw oedema after intraplantar (ipl) injection of Tityus serrulatus venom (Tsv) in male Wistar rats. Tsv was ipl-injected in doses of 0.01-10 mu g/paw. Pre-treatment (30 min prior) with DALBK (100 nmol/paw) and icatibant (10 nmol/paw), B1 and B2 selective kinin receptor antagonists, L-NAME (50 mg/kg, i.p., a non-selective nitric oxide synthase inhibitor) or celecoxib, selective COX-2 inhibitor, was given 1 h prior per os (5 mg/kg, p.o.), significantly reduced the hypernociceptive response (Von Frey method), the spontaneous nociception (determined by counting the number of flinches) and paw oedema (plethysmometer method) induced by Tsv at doses of 1.0 and 10 mu g/paw for both nociceptive and oedematogenic responses, respectively. Nevertheless, indomethacin (5 mg/kg, i.p.. 30 min prior) was ineffective in altering all of these events. The results of the present study show that Tsv, injected ipl into the rat paw, causes a dose-dependent paw oedema, mechanical hypernociception and flinches (a characteristic biphasic response) in which kinins and NO are substantially involved. Although celecoxib was effective against the oedema and pain caused by Tsv, COX-2 does not seem to be involved in the inflammatory response caused by Tsv. (C) 2008 Elsevier Ltd. All rights reserved.

Evaluation of Leptospiral Recombinant Antigens MPL17 and MPL21 for Serological Diagnosis of Leptospirosis by Enzyme-Linked Immunosorbent Assays

Leptospirosis is a zoonosis of multisystem involvement caused by pathogenic strains of the genus Leptospira. In the last few years, intensive studies aimed at the development of a vaccine have provided important knowledge about the nature of the immunological mechanisms of the host. The purpose of this study was to analyze the immune responses to two recombinant proteins, MPL17 and MPL21 (encoded by the genes LIC10765 and LIC13131, respectively) of Leptospira interrogans serovar Copenhageni in individuals during infection. The recombinant proteins were expressed in Escherichia coli as six-His tag fusion proteins and were purified from the soluble bacterial fraction by affinity chromatography with Ni2+ -charged resin. The recombinant proteins were used to evaluate their ability to bind to immunoglobulin G (IgG) (and IgG subclass) or IgM antibodies in serum samples from patients in the early and convalescent phases of leptospirosis (n = 52) by enzyme-linked immunosorbent assays. The prevalences of total IgG antibodies against MPL17 and MPL21 were 38.5% and 21.2%, respectively. The titers achieved with MPL17 were statistically significantly higher than those obtained by the reference microscopic agglutination test. The specificity of the assay was estimated to be 95.5% for MPL17 and 80.6% for MPL21 when serum samples from individuals with unrelated febrile diseases and control healthy donors were tested. The proteins are conserved among Leptospira strains that cause human and animal diseases. MPL17 and MPL21 are most likely new surface proteins of leptospires, as revealed by liquid-phase immunofluorescence assays with living organisms. Our results demonstrate that these recombinant proteins are highly immunogenic and, when they are used together, might be useful as a means of diagnosing leptospirosis.

Reduced stress fever is accompanied by increased glucocorticoids and reduced PGE(2) in adult rats exposed to endotoxin as neonates

Immune challenges during neonatal period may permanently program immune responses later in life, including endotoxin fever. We tested the hypothesis that neonatal endotoxin exposure affects stress fever in adult rats. In control rats (treated with saline as neonates; nSal) body temperature peaked similar to 1.5 degrees C during open-field stress, whereas in rats exposed to endotoxin (lipopolysaccharide, LPS) as neonates (nLPS) stress fever was significantly attenuated. Following stress, plasma corticosterone levels significantly increased from 74.29 +/- 7.05 ng ml(-1) to 226.29 +/- 9.87 ng ml(-1) in nSal rats, and from 83.43 +/- 10.31 ng ml(-1) to 324.7 +/- 36.87 ng ml(-1) in nLPS rats. Animals treated with LPS as neonates and adrenalectomized one week before experimentation no longer displayed the attenuated febrile response to stress. This attenuated stress fever caused by an increased corticosterone secretion is likely to be linked to an inhibitory effect of glucocorticoids on cyclooxygenase activity/PGE(2) production in preoptic/anteroventral third ventricular region (AV3V) since stress failed to cause a significant increase in PGE(2) in nLPS rats, and this effect was reverted by adrenalectomy. Altogether, the present results indicate that endogenous glucocorticoids are key modulators of the attenuated stress fever in adult rats treated with LPS as neonates, and they act downregulating PGE(2) production in AV3V. Moreover, our findings also support the notion that neonatal immune stimulus affects programming of stress responses during adulthood, despite the fact that inflammation and stress are two distinct processes mediated largely by different neurobiological mechanisms. (C) 2010 Elsevier B.V. All rights reserved.

Propyretic role of the locus coeruleus nitric oxide pathway

Nitric oxide has been reported to modulate fever in the brain. However, the sites where NO exerts this modulation remain somewhat unclear. Locus coeruleus (LC) neurons express not only nitric oxide synthase (NOS) but also soluble guanylyl cyclase (sGC). In the present study, we evaluated in vivo and ex vivo the putative role of the LC NO-cGMP pathway in fever. To this end, deep body temperature was measured before and after pharmacological modulations of the pathway. Moreover, nitrite/nitrate (NOx) and cGMP levels in the LC were assessed. Conscious rats were microinjected within the LC with a non-selective NOS inhibitor (NG-monomethyl-l-arginine acetate), a NO donor (NOC12), a sGC inhibitor (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) or a cGMP analogue (8-bromo-cGMP) and injected intraperitoneally with endotoxin. Inhibition of NOS or sGC before endotoxin injection significantly increased the latency to the onset of fever. During the course of fever, inhibition of NOS or sGC attenuated the febrile response, whereas microinjection of NOC12 or 8-bromo-cGMP increased the response. These findings indicate that the LC NO-cGMP pathway plays a propyretic role. Furthermore, we observed a significant increase in NOx and cGMP levels, indicating that the febrile response to endotoxin is accompanied by stimulation of the NO-cGMP pathway in the LC.

High circulating levels of the dengue virus nonstructural protein NS1 early in dengue illness correlate with the development of dengue hemorrhagic fever

Infection with any 1 of 4 dengue viruses produces a spectrum of clinical illness ranging from a mild undifferentiated febrile illness to dengue fever (DF) to dengue hemorrhagic fever (DHF), a potentially life-threatening disease. The morbidity and mortality of DHF can be reduced by early hospitalization and careful supportive care. To determine its usefulness as a predictor of DHF, plasma levels of the secreted dengue virus nonstructural protein NS1 (sNS1) were measured daily in 32 children with dengue-2 virus infections participating in a prospective, hospital-based study. Free sNS1 levels in plasma correlated with viremia levels and were higher in patients with DHF than in those with DF. An elevated free sNS1 level (greater than or equal to600 ng/mL) within 72 h of illness onset identified patients at risk for developing DHF.

Urine D-arabinitol/L-arabinitol ratio in diagnosing Candida infection in patients with haematological malignancy and HIV infection

Adult patients with hematologic malignancies along with HIV infected patients were prospectively studied to determine the performance of urine D-arabinitol/L-arabinitol (DA/LA) ratio in diagnosing invasive candidiasis. Ten evaluable febrile neutropenic patients had proven invasive candidiasis and elevated DA/LA ratios were found in 5. Invasive candidiasis with normal DA/LA ratios was most frequently due to Candida krusei infection. This Candida species is a non-producer of arabinitol. Only 4 of 81 febrile neutropenic patients given either antifungal prophylaxis or empiric antifungal treatment had elevated DA/LA ratios. Only 1 of 15 HIV positive patients with either oropharyngeal or esophageal candidiasis had elevated DA/LA ratios. Widespread use of fluconazole prophylaxis in bone marrow transplantation patients at the study hospital has led to an increased prevalence of C. krusei infection. This is the likely reason for the low sensitivity of the test in proven and suspected invasive Candida infections reported here. (C) 2002 Elsevier Science Inc. All rights reserved.

Evaluation of dengue fever reports during an epidemic, Colombia

OBJECTIVE To assess the validity of dengue fever reports and how they relate to the definition of case and severity. METHODS Diagnostic test assessment was conducted using cross-sectional sampling from a universe of 13,873 patients treated during the fifth epidemiological period in health institutions from 11 Colombian departments in 2013. The test under analyses was the reporting to the National Public Health Surveillance System, and the reference standard was the review of histories identified by active institutional search. We reviewed all histories of patients diagnosed with dengue fever, as well as a random sample of patients with febrile syndromes. The specificity and sensitivity of reports were estimated for this purpose, considering the inverse of the probability of being selected for weighting. The concordance between reporting and the findings of the active institutional search was calculated using Kappa statistics. RESULTS We included 4,359 febrile patients, and 31.7% were classified as compatible with dengue fever (17 with severe dengue fever; 461 with dengue fever and warning signs; 904 with dengue fever and no warning signs). The global sensitivity of reports was 13.2% (95%CI 10.9;15.4) and specificity was 98.4% (95%CI 97.9;98.9). Sensitivity varied according to severity: 12.1% (95%CI 9.3;14.8) for patients presenting dengue fever with no warning signs; 14.5% (95%CI 10.6;18.4) for those presenting dengue fever with warning signs, and 40.0% (95%CI 9.6;70.4) for those with severe dengue fever. Concordance between reporting and the findings of the active institutional search resulted in a Kappa of 10.1%. CONCLUSIONS Low concordance was observed between reporting and the review of clinical histories, which was associated with the low reporting of dengue fever compatible cases, especially milder cases.

Survey on the clinical trial results achieved in Brazil comparing praziquantel and oxamniquine in the treatment of mansoni schistosomiasis

A random, double-blind, parallel group clinical trial program was carried out to compare praziquantel, a recently developed anti-helmintic drug, and oxamniquine, an already established agent for treating mansoni schistosomiasis. Both drugs were administered orally as a single dose, on the average, praziquantel 55 mg/kg and oxamniquine 16 mg/kg BWT. The diagnosis and the parasitological follow-up lasting for a minimum of six months, were based on stool examinations according to Kato/Katz technique. A patient was considered cured if all results were negative and if he had performed at least three post-treatment controls, each one comprising three stool examinations. The finding of a single S. mansoni egg in any stool examination indicated, a therapeutical failure. A total of 267, cases were treated with praziquantel and 272 with oxamniquine. The two groups were homogeneous in regard to patients, age, clinical form of the disease, risk of reinfection and worm burden, relevant factors in the therapeutical response. The incidence and severity of untoward, effects were similar in both groups but abdominal distress and diarrhoea were more frequently reported under praziquantel and dizzines under oxamniquine (p < 0.05). In the former group a marked urticariform reaction was observed whereas in the latter one patient presented convulsion. The laboratory work-up. failed to disclose any significant alteration although the AST, ALT and y-GT mean values revealed a tendence to increase on the 7th day after oxamniquine intake. The overall parasitological cure rates were 75.5% (139/ 184) with praziquantel and 69.8% (134/192) with oxamniquine (p > 0.05). Amongst the noncured aptients a reduction of 88.6% and 74.6% in the mean number of eggs/g of feces Was seen following the treatment with praziquantel and oxamniquine, respectively (p < 0.05). In conclusion, in spite of their different chemical, pharmacological and toxicological profiles as well as mechanisms-of-action, inclusively praziquantel already had proved to be 100% active against S. mansoni strains resistant to oxamniquine, both drugs showed comparable tolerance and therapeutical efficacy.

Possible oral transmission of acute Chagas' disease in Brazil

In October, 1986, 7 to 22 days after a meeting at a farm in Paraíba state, 26 individuals presented with a febrile illness associated with bilateral eyelid and lower limb edema, mild hepatosplenomegaly, lymphadenopathy and, occasionally a skin rash. A 11-year-old boy exhibited atrial premature complexes and a 74-year-old patient developed acute heart failure. In two patients hospitalized in São Paulo city, acute Chagas' disease was diagnosed by the demonstration of circulating Trypanosoma cruzi. At autopsy in a fatal case, acute Chagas' cardiomyopathy was demonstrated. Xenodiagnosis were positive in 9 out of 14 tested patients. A specific IgG immune response was found in all patients and specific IgM antibodies were identified in 20 out of 22 tested patients. A epidemiological survey showed the existence of Triatoma brasiliensis in the outbuildings of this farm, but none in the house where most of the guests stayed. A high rate of infection with Trypanosoma cruzi was found in opossums. These observations together with those related to the food consumed by the patients, lead the authors to suggest that the human infections resulted from oral contamination probably originating from naturally infected marsupials in the area or crushed infected bugs.

VISCERAL LARVA MIGRANS AND TROPICAL PYOMYOSITIS: A CASE REPORT

We report a case of tropical pyomyositis in a boy who presented with a severe febrile illness associated with diffuse erythema, and swelling in many areas of the body which revealed on operation extensive necrotic areas of various muscles that required repeated débridement. The patient gave a history of contact with dogs, and an ELISA test for Toxocara canis was positive. He also presented eosinophilia and high serum IgE levels. Staphylococcus aureus was the sole bacteria isolated from the muscles affected. We suggest that tropical pyomyositis may be caused by the presence of migrating larvae of this or other parasites in the muscles. The immunologic and structural alterations caused by the larvae, in the presence of concomitant bacteremia, would favour seeding of the bacteria and the development of pyomyositis.

HANTAVIRUS PULMONARY SYNDROME (HPS) IN GUARIBA, SP, BRAZIL: REPORT OF 2 CASES

Human infections caused by a hantavirus were reported in different regions of the State of São Paulo (SP), Brazil during the first six months of 1998. Two cases of fatal pulmonary syndrome occurred in May of 1998 in the City of Guariba, located in the Northeastern Region of SP. Both patients worked in a corn storage barn infested by rodents. These patients, after 2 or 3 days of non-specific febrile illness, developed a severe interstitial pneumonia spreading widely in both lungs, causing respiratory failure and death. At autopsy both patients showed lung interstitial edema with immunoblast-like mononuclear cell infiltrates, consistent with a viral etiology. Hantavirus infection was diagnosed by ELISA in both cases and by RT-PCR in one of the patients. Aspects of the clinical presentation, physiopathology and differential diagnosis of Hantavirus Pulmonary Syndrome are discussed.