Flávia Aleixo Vasconcellos

An Immunomagnetic Separation-PCR Method for Detection of Pathogenic Leptospira in Biological Fluids

Abstract Leptospirosis is a zoonotic disease that occurs worldwide and is caused by pathogenic bacteria of the genus Leptospira. Clinical manifestations of leptospirosis are similar to other febrile illnesses and this fact frequently retards the beginning of antibiotic therapy. Thus, early and accurate diagnosis is a prerequisite for proper treatment of leptospirosis. Antigen and DNA-based detection tests offer potential advantage over tests based on antibody detection for early diagnosis of leptospirosis since antibodies only reach detectable levels several days after the onset of the infection. This work describes a method for detection of pathogenic Leptospira that associates an immunoseparation step with a PCR assay and uses an internal amplification control (IAC) to ensure accuracy of the test. The immunoseparation was performed with protein A-magnetic beads in house coated with an MAb specific for LipL32, the major outer membrane protein of pathogenic Leptospira; PCR was performed using lipL32 specific primers. The IMS-PCR method enhanced detection of Leptospira in experimentally contaminated human sera and urine when compared to PCR performed alone. IMS-PCR was able to detect 10(2) Leptospira cells per mL of human sera and urine, corresponding to 25 genomic copies per PCR reaction.

Monoclonal antibodies against the leptospiral immunoglobulin-like proteins A and B conserved regions

Leptospirosis is an infectious disease caused by pathogenic spirochetes of the genus Leptospira that affects humans and a wide variety of animals. Recently the genomes of Leptospira interrogans, Leptospira borgpetersenii and Leptospira biflexa species were sequenced allowing the identification of new virulence factors involved in survival and pathogenesis of bacteria. LigA and LigB are surface-exposed bacterial adhesins whose expression is correlated with the virulence of Leptospira strains. In this study, we produced and characterized five monoclonal antibodies (MAbs) against a recombinant fragment of LigB (rLigBrep) with approximately 54kDa that comprise the portions of LigA and LigB (domains 2-7). The 5 MAbs obtained were of the IgG1 (2) and IgG2b (3) isotypes and their affinity constants for rLigBrep ranged from 7×10(7) M(-1) to 4×10(8) M(-1). The MAbs were able to react with the native antigen on the L. interrogans, L. borgpetersenii and Leptospira noguchii surfaces by indirect immunofluorescence, immunoblotting and immunoelectron microscopy. These results demonstrate that the MAbs anti-rLigBrep can be useful to complement genetic studies and to aid studies aiming understanding the role of Lig proteins in Leptospira pathogenesis and the development of Lig-based vaccines and improved diagnostic tests for leptospirosis.

Testing different antigen capture ELISA formats for detection of Leptospira spp. in human blood serum

Leptospirosis is an infectious disease caused by pathogenic spirochetes of the genus Leptospira. The illness is characterized by an acute bacteremic phase followed by an immune phase, in which specific antibodies are found in blood and leptospires are eliminated in urine. Novel diagnostic strategies for use in the acute phase of leptospirosis are needed since clinical manifestations in this phase mimic other feverish tropical diseases. In the present study, mAbs and polyclonal IgY were used in the standardization of three different antigen capture ELISA formats for direct detection of leptospires in human blood during the acute phase of the disease. Detection limit of leptospires in experimentally contaminated human sera ranged from 10(5) to 10(7) cells ml(-1) in the different formats. The ELISA format with the best performance was able to detect 10(5) leptospires ml(-1) in human sera using a mAb against LipL32, the major outer membrane protein of pathogenic leptospires, as capture antibody, and a biotinylated polyclonal IgY against a pathogenic serovar of L. interrogans Icterohamorrhagiae as detection antibody. By increasing the degree of IgY biotinylation this detection limit could be improved to make the assay clinically useful.

Highly Virulent Leptospira borgpetersenii Strain Characterized in the Hamster Model

Abstract. A recent study by our group reported the isolation and partial serological and molecular characterization of four Leptospira borgpetersenii serogroup Ballum strains. Here, we reproduced experimental leptospirosis in golden Syrian hamsters (Mesocricetus auratus) and carried out standardization of lethal dose 50% (LD50) of one of these strains (4E). Clinical disease features and histopathologic analyses of tissue lesions were also observed. As results, strain 4E induced lethality in the hamster model with inocula lower than 10 leptospires, and histopathological examination of animals showed typical lesions found in severe leptospirosis. Gross pathological findings were peculiar; animals that died early had more chance of presenting severe jaundice and less chance of presenting pulmonary hemorrhages (P < 0.01). L. borgpetersenii serogroup Ballum has had a considerable growth in human leptospirosis cases in recent years. This strain has now been thoroughly characterized and can be used in more studies, especially evaluations of vaccine candidates.

Generation and characterization of new HER2 monoclonal antibodies

Antibodies are among the most commonly used research and diagnostic tools. Antibody type and clonality are important in any assay as they can influence epitope detection. HER2 oncoprotein is overexpressed or undergoes gene amplification in approximately 30% of invasive breast carcinomas and 20% of gastric adenocarcinomas. Overexpression of HER2 is primarily detected by immunohistochemistry (IHC) on neoplastic tissue sections. We produced five murine hybridoma clones secreting monoclonal antibodies (MAbs) against HER2 protein. For hybridoma production, spleen cells from BALB/c mice immunized with a recombinant fragment of the extracellular portion of HER2 (rHER2) were fused to SP2/O-Ag14 cells, selected in HAT medium and screened by indirect ELISA. MAbs secreted were characterized according to isotypes, functional affinity constants, reaction with the native protein in MCF-7 cells by indirect immunofluorescence and in tissue sections from HER2 positive breast cancer specimens by IHC. Two MAbs were IgG2b and three were IgG1, and their affinity constants ranged from 6×10(7) to 1×10(9)M(-1). All MAbs reacted with the native protein and two stained strongly the membrane of neoplastic cells overexpressing HER2. These two MAbs could be useful in assaying HER2 overexpression in human tissues for research and possibly diagnostic purposes after a proper large-scale validation study.

Antioxidant, antihyperglycemic, and antidyslipidemic effects of Brazilian-native fruit extracts in an animal model of insulin resistance

ABSTRACT Objective: Insulin resistance (IR) plays an important role in the development of many diseases, such as diabetes mellitus. Therefore, the aim of the present study was to evaluate the effects of the extracts from fruits native to Brazil on metabolic parameters and hepatic oxidative markers in an animal model of insulin resistance induced by dexamethasone (DEX). Methods: Wistar rats received water or extracts of Eugenia uniflora or Psidium cattleianum, once a day for 21 days. For the last 5 days, the rats received an intraperitoneal injection of saline or DEX. Results: DEX caused a reduction in body weight gain and relative pancreatic weight, as well as glucose intolerance, and an increase in serum glucose and triacylglycerol levels. The extracts were found to prevent hyperglycemia and hypertriglyceridemia. DEX caused an increase in the levels of thiobarbituric acid-reactive substances and reactive oxygen species production in the liver of rats, and both extracts prevented these changes. In addition, hepatic glutathione peroxidase activity was reduced by DEX. However, total thiol content and activities of catalase, superoxide dismutase, and delta-aminolevulinate dehydratase were not altered in any of the tested groups. Conclusion: Fruit extracts of E. uniflora and P. cattleianum exhibited considerable antihyperglycemic, antidyslipidemic, and antioxidant effects, and may be useful in the therapeutic management of alterations due to IR.

Synthesis and antioxidant activity of 3-(Pyridin-2-ylmethyl)-1,3-thiazinan(thiazolidin)-4-ones.

The antioxidant properties of two series of thiazolidinones and thiazinanones were reported. The novel six‐membered thiazinanones were synthesized from the efficient multicomponent reaction of 2‐picolylamine (2‐aminomethylpyridine), arenaldehydes, and the 3‐mercaptopropionic acid in moderate to excellent yields. These novel compounds were fully identified and characterized by NMR and GC‐MS techniques. In vitro antioxidant activities of all compounds were evaluated by 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) and 2,2′‐azinobis‐3‐ethylbenzothiazoline‐6‐sulfonic acid (ABTS) tests. The antioxidant assays of thiobarbituric acid reactive species and total thiol content levels in the cerebral cortex and liver of rats were also performed. Thiazinanone 5a showed the best radical scavenging activity in DPPH and ABTS tests, as well as reduced lipid peroxidation and increased total thiol group in biological systems. Altogether, the results may be considered a good starting point for the discovery of a new radical scavenger.

Eugenia uniflora fruit (red type) standardized extract: a potential pharmacological tool to diet-induced metabolic syndrome damage management

The aim of this study was to investigate the effect of Eugenia uniflora fruit (red type) extract on metabolic status, as well as on neurochemical and behavioral parameters in an animal model of metabolic syndrome induced by a highly palatable diet (HPD). Rats were treated for 150days and divided into 4 experimental groups: standard chow (SC) and water orally, SC and E. uniflora extract (200mg/kg daily, p.o), HPD and water orally, HPD and extract. Our data showed that HPD caused glucose intolerance, increased visceral fat, weight gain, as well as serum glucose, triacylglycerol, total cholesterol and LDL cholesterol; however, E. uniflora prevented these alterations. The extract decreased lipid peroxidation and prevented the reduction of superoxide dismutase and catalase activities in the prefrontal cortex, hippocampus and striatum of animals submitted to HPD. We observed a HPD-induced reduction of thiol content in these cerebral structures. The extract prevented increased acetylcholinesterase activity in the prefrontal cortex caused by HPD and the increase in immobility time observed in the forced swim test. Regarding chemical composition, LC/MS analysis showed the presence of nine anthocyanins as the major compounds. In conclusion, E. uniflora extract showed benefits against metabolic alterations caused by HPD, as well as exhibited antioxidant and antidepressant-like effects.

Evaluation of HER2 Protein Expression Using 2 New Monoclonal Antibodies.

This study describes the performance of 2 new mouse anti-HER2 monoclonal antibodies (Abs), clones 33F and 410G, in evaluating HER2 overexpression in a series of 123 invasive breast carcinoma cases. In-house immunohistochemistry (IHC) was performed and the results were compared with those for the SP3 and A0485 anti-HER2 Abs. Chromogenic in situ hybridization was used to detect ERBB2 amplification and its concordance with IHC was analyzed. Comparison of IHC results for 33F with SP3 and A0485 yielded concordance rates (K) of 0.81 and 0.75, respectively; the same concordance rates were found when comparing results for 410G with SP3 and A0485. Compared with SP3 and A0485, 33F and 410G specificities were 98.6% and 98.6%, and 100% and 100%, respectively, whereas the sensitivities were 80% and 74.1%, and 78% and 72.2%, respectively. The K values between 33F and 410G HER2+ expression and chromogenic in situ hybridization-positive amplification were 1 and 0.96, respectively. These concordance rates were reproduced in another production batch (K=0.96 and K=0.96). Together, these results show that the tested monoclonal Abs would be well suited for detecting HER2 protein overexpression by IHC.