Characterization of a Molecularly Engineered Banlec-Type Lectin (rBTL).

Lectins are proteins that reversibly bind to carbohydrates and are commonly found across many species. The Banana Lectin (BanLec) is a member of the Jacalin-related Lectins, heavily studied for its immunomodulatory, antiproliferative, and antiviral activity. In this study, a novel sequence was generated in silico considering the native BanLec amino acid sequence and 9 other lectins belonging to JRL. Based on multiple alignment of these proteins, 11 amino acids of the BanLec sequence were modified because of their potential for interference in active binding site properties resulting in a new lectin named recombinant BanLec-type Lectin (rBTL). rBTL was expressed in E. coli and was able to keep biological activity in hemagglutination assay (rat erythrocytes), maintaining similar structure with the native lectin. Antiproliferative activity was demonstrated on human melanoma lineage (A375), evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT). rBTL was able to inhibit cellular growth in a concentration-dependent manner, in an 8-h incubation, 12 µg/mL of rBTL led to a 28.94% of cell survival compared to cell control with 100%. Through a nonlinear fit out log-concentration versus biological response, an IC50% of 3.649 µg/mL of rBTL was determined. In conclusion, it is possible to state that the changes made to the rBTL sequence maintained the structure of the carbohydrate-binding site without changing specificity. The new lectin is biologically active, with an improved carbohydrate recognition spectrum compared to nBanLec, and can also be considered cytotoxic for A375 cells.

Biopolymer Xanthan: A New Adjuvant for DNA Vaccines

Abstract DNA vaccines have been evaluated as an option to prevent several diseases. In this study, the capacity of the xanthan biopolymer to improve the DNA vaccines immune response, administered intramuscularly, was evaluated. The experimental vaccines consisted of genes encoding fragments of the proteins LigA and LigB of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Copenhageni strain Fiocruz L1-130. The humoral immune response was evaluated by indirect ELISA. Cytokine expression levels were determined by RT-qPCR. Compared to the control group, the IgG antibody levels of animals immunized with pTARGET/ligAni and pTARGET/ligBrep plasmids associated with xanthan biopolymer were significantly higher than the control group. Additionally, there was a significant increase in IL-17 expression in animals vaccinated with pTARGET/ligBrep and xanthan.

A novel chimeric protein composed of recombinant Mycoplasma hyopneumoniae antigens as a vaccine candidate evaluated in mice.

Enzootic Pneumonia (EP) is caused by the Mycoplasma hyopneumoniae pathogenic bacteria, and it represents a significant respiratory disease that is responsible for major economic losses within the pig industry throughout the world. The bacterins that are currently commercially available have been proven to offer only partial protection against M. hyopneumoniae, and the development of more efficient vaccines is required. Several recombinant antigens have been evaluated via different immunization strategies and have been found to be highly immunogenic. This work describes the construction and immunological characterization of a multi-antigen chimera composed of four M. hyopneumoniae antigens: P97R1, P46, P95, and P42. Immunogenic regions of each antigen were selected and combined to encode a single polypeptide. The gene was cloned and expressed in Escherichia coli, and the chimeric protein was recognized by specific antibodies against each subunit, as well as by convalescent pig sera. The immunogenic properties of the chimera were then evaluated in a mice model through two recombinant vaccines that were formulated as follows: (1) purified chimeric protein plus adjuvant or (2) recombinant Escherichia coli bacterin. The immune response induced in BALB/c mice immunized with each formulation was characterized in terms of total IgG levels, IgG1, and IgG2a isotypes against each antigen present in the chimera. The results of the study indicated that novel chimeric protein is a potential candidate for the future development of a more effective vaccine against EP.

Stable expression of Mycobacterium bovis antigen 85B in auxotrophic M. bovis bacillus Calmette-Guérin

BACKGROUND Bovine tuberculosis (TB) is a zoonotic disease caused by Mycobacterium bovis, responsible for causing major losses in livestock. A cost effective alternative to control the disease could be herd vaccination. The bacillus Calmette-Guérin (BCG) vaccine has a limited efficacy against bovine TB, but can improved by over-expression of protective antigens. The M. bovis antigen 85B demonstrates ability to induce protective immune response against bovine TB in animal models. However, current systems for the construction of recombinant BCG expressing multiple copies of the gene result in strains of low genetic stability that rapidly lose the plasmid in vivo. Employing antibiotic resistance as selective markers, these systems also compromise vaccine safety. We previously reported the construction of a stable BCG expression system using auxotrophic complementation as a selectable marker. OBJECTIVES The fundamental aim of this study was to construct strains of M. bovis BCG Pasteur and the auxotrophic M. bovis BCG ΔleuD expressing Ag85B and determine their stability in vivo. METHODS Employing the auxotrophic system, we constructed rBCG strains that expressed M. bovis Ag85B and compared their stability with a conventional BCG strain in mice. Stability was measured in terms of bacterial growth on the selective medium and retention of antigen expression. FINDINGS The auxotrophic complementation system was highly stable after 18 weeks, even during in vivo growth, as the selective pressure and expression of antigen were maintained comparing to the conventional vector. MAIN CONCLUSION The Ag85B continuous expression within the host may generate a stronger and long-lasting immune response compared to conventional systems.

Anthocyanins control neuroinflammation and consequent memory dysfunction in mice exposed to lipopolysaccharide.

Peripheral inflammatory stimuli may activate a brain neuroinflammatory processes with consequences in brain function. The present study investigated if anthocyanins (ANT) consumption was able to prevent the memory loss, the neuronal damage, and the neuroinflammatory processes triggered by the intraperitoneal lipopolysaccharide (LPS) administration. C57BL6 male mice were treated with ANT (30-100 mg/kg by gavage). With a single dose or during 10 days, before be challenged with LPS (250 µg/kg intraperitoneally single administration), a classical inductor of inflammation. The data obtained showed that ANT was able to confer protection against the memory impairment after 10 days of ANT treatment (100 mg/kg). This phytonutrient also prevented the hypothermia episode induced by LPS. Moreover, ANT prevented the increase in protein carbonyl, NOx, and MDA levels in the hippocampus and cerebral cortex (4 and 24 h) in animal challenged with LPS. ANT showed a protective effect on the increase in the pro-inflammatory cytokines content, especially Interleukin (IL)-1ß, tumoral necrosis factor-α and on the reduction of IL-10 induced by LPS. ANT 100 mg/kg prevented the infiltration of peripheral immune cells in the hippocampus at 24 h post-LPS administration. In parallel, LPS increased the activity of myeloperoxidase in cortex and hippocampus, and ANT prevented this effect, also reducing microglia (Iba-1) and astrocyte (GFAP) immunoreactivity. Thus, our data support that ANT are a promising therapeutic component against brain disorders associated with process of neuroinflammation. Graphical Abstract ᅟ.

Lectin from seeds of a Brazilian lima bean variety (Phaseolus lunatus L. var. cascavel) presents antioxidant, antitumour and gastroprotective activities.

Lectins are proteins able to interact specifically and reversibly with carbohydrates. They are present in all living beings, particularly in legume seeds, which have many biological functions. The aim of this study was to isolate, characterize and verify antioxidant, anti-hemolytic, antitumor and gastroprotective activities in a lectin present in seeds of Phaseolus lunatus L. var. cascavel (PLUN). The isolation of lectin was performed by size exclusion chromatography on Sephadex G-100, which was isolated from a protein capable of agglutinating only human erythrocytes type A, being this the only inhibited haemagglutination n-acetyl-d-galactosamine. Its weight was estimated by PAGE is 128kDa. The lectin is thermostable up to 80°C and is active between pH 2-11. As 8M urea was able to denature the lectin. PLUN is a glycoprotein consisting of 2% carbohydrate and has antioxidant action with ascorbic acid equivalent antioxidant capacity (µMAA/g) of 418.20, 326 and 82.9 for total antioxidant activity, ABTS radical capture and capture of DPPH radical, respectively. The lectin has antitumor activity against melanoma derived cells at doses of 100 and 50mg/ml, reducing up to 83% tumor cells, and gastroprotective action, reducing up to 63% damaged area of ​​the stomach induced by ethanol.

Diagnostic potential of anti-rNcp-43 polyclonal antibodies for the detection of Neospora caninum.

Neosporosis is a disease caused by the apicomplexan parasite Neospora caninum, which is closely related to Toxoplasma gondii. N. caninum infection represents an important cause of reproductive failure in sheep, goats, horses, and cattle worldwide. The diagnosis of neosporosis is based on the detection of pathogen-specific antibodies in animal sera or the presence of tissue cysts. However, morphological similarities and serological cross-reactivity between N. caninum and T. gondii can result in the misdiagnosis. In this study, the N. caninum tachyzoite surface protein Ncp-43 was expressed in a recombinant form to elicit polyclonal antibodies (pAb) response. The pAb was purified and conjugated to horseradish peroxidase (HRP) or fluorescein isothiocyanate (FITC) to detect the recombinant and native Ncp-43 proteins, respectively. The pAb and pAb/HRP were able to recognize rNcp-43 by dot blot and ELISA, and pAb/FITC immunolabeled the apical complex of tachyzoites. A blocking enzyme-linked immunosorbent assay (b-ELISA) was performed to evaluate pAb/HRP as a diagnostic tool. The mean percent inhibition for the positive and negative serum samples from cattle with neosporosis was significantly different (P < 0.0001). These results suggest that the pAb may bind to the same epitopes of Ncp-43 as anti-N. caninum antibodies in the positive samples tested. The b-ELISA using the pAb/HRP can facilitate diagnostic testing for neosporosis, since fewer steps are involved, and cross-reactivity with secondary antibodies is avoided. In summary, this report describes the production of antibodies against N. caninum, and evaluates the potential of these tools for the development of new diagnostic tests for neosporosis.

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.

Prevalência de anemia em crianças de 1 a 5 anos moradoras do bairro Passo, Vila Arneldo Matter - São Borja/RS e sua relação com estado nutricional e enteroparasitoses / Prevalence of anemia in children from 1 to 5 years old inhabithantes of the Passo district, Arneldo Matter neighbourhood - São Borja/RS and its relationship with nutritional condition and enteroparasitosis

Introdução: A anemia ferropriva está relacionada com a deficiência de ferro no organismo devido à dieta pobre em ferro ou a presença de enteroparasitoses. O objetivo deste trabalho foi determinar a prevalência de anemia em crianças de 1 a 5 anos moradora do bairro Passo, vila Arneldo Matter-São Borja/RS e sua relação com estado nutricional e enteroparasitoses. Materiais e Métodos: A amostra constou de 42 crianças carentes de 1 a 5 anos. Para avaliação hematológica analisou-se o hematócrito, hemoglobina, eritócritos e leucócitos totais, volume corpuscular médio, concentração de hemoglobina corpuscular média e a hemoglobina corpuscular média e avaliação microscópica do esfregaço. No diagnóstico parasitológico utilizou-se o método de centrífugo-sedimentação modificado. O estado nutricional das crianças foi avaliado por um questionário. Resultados: Foram encontrados índices de 5% de anemia e 38% enteroparasitoses, sendo os mais encontrados Giárdia lamblia (19%), Ascaris lumbricoides e hymenolepis nana (14%), Entamoeba coli (11%), Trichuris trichiura e Enterobius vermicularis (3%). Além disso, 73,8% das crianças consumiam diariamente carne, leguminosas e alimentos com vitamina C. Conclusão: O estudo demonstrou baixa incidência de anemia pelo estado nutricional satisfatório, mas uma alta prevalência de enteroparasitoses, demonstrando a necessidade da continuidade de trabalhos científicos

Triagem dos perfis lipídico e glicídico emcaminhoneiros que trafegam no centro unificado de fronteira, entre Brasil e Argentina (AU) / Screen of the profiles of lipid and glucose in truck drivers that transite in the unified center of

Introducao: Atualmente no Brasil, as doencas cardiovasculares (DCVs) sao as patologias de maior morbimortalidade. Os principais fatores de risco sao ma alimentacao, sedentarismo, tabagismo, etilismo, obesidade, dislipidemias, Diabetes mellitus e estresse. O objetivo do estudo foi avaliar os perfis lipidico e glicidico e identificar fatores de risco associados as doencas cardiovascularesem caminhoneiros. Materiais e Metodos: A amostra foi composta por 45 caminhoneiros que passaram pelo Centro Unificado de Fronteira, entre Brasil e Argentina, nos dias 23 e 24 de abril e 21 de maio de 2007. Foi avaliado o indice de massa corporal, a circunferencia abdominal, a pressao arterial, habitos alimentares, tabagismo e historia familiar de DCVs. O perfil lipidico foi definido pelas determinacoes do colesterol total, HDL e LDL colesterol, triglicerideos; e o perfil glicidico, atraves da glicemia de jejum. Resultados: Foram encontrados 33 casos de dislipidemias, classificados em hipercolesterolemia (33%), hiperlipidemia mista (15%) e hipertrigliceridemia (30%). Os fatores de risco presentes foram: obesidade (42%), obesidade visceral (78%), hipertensao (47%) e tabagismo (20%).Conclusao: Mesmo se referindo a um grupo especifico, o trabalho foi importante, pois identificou fatores de risco presentes na categoria profissional de motoristas de caminhao e com isso sua maior predisposicao ao desenvolvimento de DCVs.

Introduction: Nowadays in Brazil, the cardiovascular diseases (CVDs) are the major morbidities pathologies. The maincause of these pathologies is improper lifestyle that results in risk factors as: bad feeding, sedentary lifestyle, smoking, alcoholism, obesity, dyslipidemia, Diabetes mellitus and stress. This study had as objective the lipid and glucose profiles evaluation, and the identification of risk factors related to the truck drivers CVDs development. Materials and Methods: The sample was composed by 45 truck drivers that went by the Unified Center of Border, among Brazil and Argentina, at the days April 23, April 24 and May 21, 2007. Thestudy evaluated the CMI (Corporal mass index), the abdominal circumference and the arterial pressure and investigated the physical activity practice, alimentary behavor, tobacco consumption, coronary artery disease family history and Diabetes mellitus. The lipid profile was defined by the biochemical determinations of the total cholesterol, the HDL fraction, the LDL fraction, triglyceride, and the glucose profile, through the fast glycemia. Results: The study demonstrated that 33 of dislipidemias cases classified at hypercholesterolemia(33%), mingle hiperlipidemia (15%) and hypertriglyceridemia (30%). The risk factors showed were obesity (42%), visceral obesity (78%), hypertension (47%) and smoke (20%). Conclusion: Although the data presented in this study refers to only one placeand a specific group, the work was important, because it evaluated the risk factors exhibition extension that the truck drivers' professional category is submitted, and with that its largest predisposition to the development of CVDs.