Sibele Borsuk

Chemical characterization, antioxidant and cytotoxic activities of Brazilian red propolis.

Propolis is known for a long time for its health benefits and biological activities. Here, the red variety from the northeast of Brazil was chemically analyzed and extracts were investigated regarding their antioxidant and antitumor activity. Hydroalcoholic extracts, obtained from the red propolis, revealed polyphenol content, 2,2-diphenyl-1-picrylhydrazyl scavenging potential and enzymatic activities for catalase-like and superoxide dismutase-like. Cytotoxic activity was evaluated for human laryngeal epidermoid carcinoma cell (Hep-2), human cervical adenocarcinoma (HeLa) and human normal epithelial embryonic kidney (Hek-293). Survival analysis for non-tumor cell line showed greater IC50 compared to tumor cell lines, suggesting an increased sensitivity that may correlate with the higher proliferative index of the tumor vs. normal cells. Our results indicate that the Brazilian red propolis is capable of inhibiting cancer cell growth and constitutes an excellent source of antioxidant and antitumor natural agent.

Recombinant Mycobacterium bovis BCG

The Bacillus Calmette-Guerin (BCG) is an attenuated strain of Mycobacterium bovis that has been broadly used as a vaccine against human tuberculosis. This live bacterial vaccine is able to establish a persistent infection and induces both cellular and humoral immune responses. The development of mycobacterial genetic systems to express foreign antigens and the adjuvanticity of BCG are the basis of the potential use of this attenuated mycobacterium as a recombinant vaccine. Over the years, a range of strategies has been developed to allow controlled and stable expression of viral, bacterial and parasite antigens in BCG. Herein, we review the strategies developed to express heterologous antigens in BCG and the immune response elicited by recombinant BCG constructs. In addition, the use of recombinant BCG as an immunomodulator and future perspectives of BCG as a recombinant vaccine vector are discussed.

Identification of proteins from tuberculin purified protein derivative (PPD) by LC-MS/MS.

The tuberculin purified protein derivative (PPD) is a widely used diagnostic antigen for tuberculosis, however it is poorly defined. Most mycobacterial proteins are extensively denatured by the procedure employed in its preparation, which explains previous difficulties in identifying constituents from PPD to characterize their behaviour in B- and T-cell reactions. We here described a proteomics-based characterization of PPD from several different sources by LC-MS/MS, which combines the solute separation power of HPLC, with the detection power of a mass spectrometer. The technique is able to identify proteins from complex mixtures of peptide fragments. A total of 171 different proteins were identified among the four PPD samples (two bovine PPD and two avium PPD) from Brazil and UK. The majority of the proteins were cytoplasmic (77.9%) and involved in intermediary metabolism and respiration (24.25%) but there was a preponderance of proteins involved in lipid metabolism. We identified a group of 21 proteins that are present in both bovine PPD but were not detected in avium PPD preparation. In addition, four proteins found in bovine PPD are absent in Mycobacterium bovis BCG vaccine strain. This study provides a better understanding of the tuberculin PPD components leading to the identification of additional antigens useful as reagents for specific diagnosis of tuberculosis.

Seroprevalence of Toxocara Infection in Children from Southern Brazil

Abstract: The seroprevalence of Toxocara canis antibodies in children aged from 1 to 12 yr old was evaluated in Pelotas City, Rio Grande do Sul, Brazil. Human toxocariasis or visceral larva migrans (VLM) was diagnosed with the use of an ELISA based on the T. canis excretory–secretory (TES) antigens; Western blotting was used to confirm the ELISA-positive results. From 427 samples, 50.6% were positive for the presence of anti-TES antibodies. A confirmatory test (Western blot) was carried out on a sample of the ELISA-positive sera (n = 70), and all were positive. The Western blots had specific banding pattern characteristics, where the 30-kDa fraction demonstrated the highest reactivity. This fraction could be important for the specific diagnosis of toxocariasis.

Brazilian Red Propolis Induces Apoptosis-Like Cell Death and Decreases Migration Potential in Bladder Cancer Cells

Natural products continue to be an invaluable resource of anticancer drug discovery in recent years. Propolis is known for its biological activities such as antimicrobial and antitumor effects. This study assessed the effects of Brazilian red propolis (BRP) on apoptosis and migration potential in human bladder cancer cells. The effect of BRP ethanolic extract (25, 50, and 100 μg/mL) on 5637 cells was determined by MTT, LIVE/DEAD, and migration (scratch assay) assays. Apoptosis induction was investigated through flow cytometry and gene expression profile was investigated by qRT-PCR. Results showed cytotoxicity on MTT and LIVE/DEAD assays, with IC50 values of 95 μg/mL in 24 h of treatment. Cellular migration of 5637 cells was significantly inhibited through lower doses of BRP ethanolic extract (25 and 50 μg/mL). Flow cytometry analyses showed that BRP induced cytotoxicity through apoptosis-like mechanisms in 5637 cells and qRT-PCR revealed increased levels of Bax/Bcl-2 ratio, p53, AIF, and antioxidant enzymes genes. Data suggest that BRP may be a potential source of drugs to bladder cancer treatment.

Auxotrophic recombinant Mycobacterium bovis BCG overexpressing Ag85B enhances cytotoxicity on superficial bladder cancer cells in vitro

BCG therapy remains at the forefront of immunotherapy for treating patients with superficial bladder cancer. The high incidence of local side effects and the presence of non-responder diseases have led to efforts to improve the therapy. Hence, we proposed that an auxotrophic recombinant BCG strain overexpressing Ag85B (BCG ∆leuD/Ag85B), could enhance the cytotoxicity to the human bladder carcinoma cell line 5637. The rBCG was generated using an expression plasmid encoding the mycobacterial antigen Ag85B to transform a BCG ∆leuD strain. The inhibitory effect of BCG ∆leuD/Ag85B on 5637 cells was determined by the MTT method, morphology observation and a LIVE/DEAD assay. Gene expression profiles for apoptotic, cell cycle-related and oxidative stress-related genes were investigated by qRT-PCR. Bax, bcl-2 and p53 induction by BCG ∆leuD/Ag85B treatment was evaluated by Western blotting. BCG ∆leuD/Ag85B revealed a superior cytotoxicity effect compared to the control strains used in this study. The results showed that the expression level of pro-apoptotic and cell cycle-related genes increased after BCG ∆leuD/Ag85B treatment, whereas the mRNA levels of anti-apoptotic genes decreased. Interestingly, BCG ∆leuD/Ag85B also increased the mRNA level of antioxidant enzymes in the bladder cancer cell line. Bax and p53 proteins levels increased following treatment. In conclusion, these results suggest that treatment with BCG ∆leuD/Ag85B enhances cytotoxicity for superficial bladder cancer cells in vitro. Therefore, rBCG therapy may have potential benefits in the treatment of bladder cancer.

Development of an indirect ELISA-NcSRS2 for detection of Neospora caninum antibodies in cattle

Neosporosis is of alarming economic concern in the cattle industry. The effectiveness of diagnostic tests for detecting specific antibodies against Neospora caninum is hampered by potential cross-reaction with other coccidia. Use of a single specific antigen might improve test specificity. An indirect enzyme-linked immunosorbent assay (ELISA) was developed using the truncated protein NcSRS2 expressed in Escherichia coli. The ELISA results were compared with those of the indirect fluorescence antibody test (IFAT). Receiver Operating Characteristic (ROC) and Tests in the Absence of a Gold Standard (TAGS) analysis revealed an assay having 96% specificity and 95% sensitivity when applied to 145 positive and 352 negative sera from two distinct cattle populations. Using OD ≤ 0.095 as the cut-off point, the assays negative and positive predictive values ranged from 98.8% to 50.8% and from 58.8% to 99.1%, respectively, depending on neosporosis prevalence in a given area. The novel ELISA-NcSRS2 format described in the present report constitutes a specific and sensitive method for detecting N. caninum in cattle.

Immunological characterization of Mycoplasma hyopneumoniae recombinant proteins

Mycoplasma hyopneumoniae, the primary pathogen of enzootic pneumonia, is highly prevalent worldwide and causes major economic losses to the pig industry. Commercial vaccines are widely used in the control of this disease, however, they provide only partial protection. The aim of this study was to evaluate 34 recombinant proteins of M. hyopneumoniae expressed in Escherichia coli. Antigenic and immunogenic properties of these proteins were analyzed. For this, the proteins were tested against hyperimmune and convalescent pig sera through ELISA and Western blot. Immunogenicity of the recombinant proteins was evaluated in BALB/c mice following intramuscular inoculation. Most antigens were able to induce a strong immune response and sera from inoculated mice were able to recognize native proteins by cell ELISA and Western blot. Several recombinant proteins were specifically recognized by convalescent pig sera, indicating they are expressed during infection. These data may help to develop more efficacious vaccines against M. hyopneumoniae.

Clonal diversity of M. tuberculosis isolated in a sea port city in Brazil

Genotyping tools have been widely used to study the occurrence of outbreaks and to identify the patterns of transmission of Mycobacterium tuberculosis strains. The clonal diversity of 65 clinical isolates of M. tuberculosis was determined by PCR methods. The Double Repeat Element method (DRE-PCR) and spoligotyping identified 45 and 26 distinct patterns respectively. Among these, LAM (38%) was the most frequent lineage, followed by Haarlem (31%) and T (20%). Five orphan patterns were not present in the SITVIT database. Mycobacterial interspersed repetitive units (MIRU) using 12 loci revealed 46 distinct patterns. MIRU loci 10, 23, 26 and 40 had the highest discriminatory power. The high genetic diversity found among M. tuberculosis isolates in this study suggests a high level of recent TB transmission, indicating an endemic mode of TB transmission and a putative importation of new TB genotypes. In addition, the high diversity among the isolates could indicate early detection of the infection in patients and an efficient rate of cure.

Expression of Neospora caninum NcSRS2 surface protein in Pichia pastoris and its application for serodiagnosis of Neospora infection

Abstract Neospora caninum is considerd a major cause of abortion in cattle worldwide. The antigenic domain of NcSRS2 in N. caninum is an important surface antigen present in the membrane of this parasite. In the present study, the Pichia pastoris expression system proved to be a useful tool for the production of recombinant protein. The truncated NcSRS2 gene (by removal of the N-terminal hydrophobic sequence), was cloned in the vector pPICZalphaB, and integrated on the genome of the methylotrophic yeast P. pastoris. Subsequently, the NcSRS2 protein was expressed, purified, and characterized using naturally infected cattle sera and Mab 6xhistag. The recombinant protein NcSRS2 was present in the supernatant of the culture, where later it was concentrated and purified using ammonium sulfate (∼100 mg/ml). An indirect immunoenzymatic assay (ELISA) was performed using cattle sera from endemic N. caninum area.