Strategies for transfection of bovine mesenchymal stem cells with pBC1-anti-CD3 vector.

Cells from different origins behave differently regarding the incorporation of exogenous DNA and formation of transgenic cells. Milk production of recombinant antibody may benefit from efficient transfection protocols to produce transgenic animals. In this context, the objective of this study was to verify the transfection potential of bovine mesenchymal stem cells from Wharton's jelly (MSC-WJ) and adipose tissue (MSC-AT), comparing co-transfection protocols with vectors pBC1-anti-CD3 and pEF-NEO-GFP, using transfection reagents Lipofectamine LTX with Plus Reagent or Xfect. Skin fibroblasts (FIB) were used as the control group. Forty-eight hours after transfection, neomycin was added and cells cultured for 2 weeks. Treated cells were submitted to fluorescence microscopy, flow cytometry, and PCR evaluations. Wharton's jelly cells were sensitive to treatments and started necrosis. In the flow cytometry assay, the median fluorescence was higher in adipocytes than fibroblasts, for both the Xfect (20.057 ± 1.620,7 and 10.601 ± 702,86, respectively, p < 0.05) and LTX (19.590 ± 113,84 and 10.518 ± 442,65, respectively, p < 0.05). These results, associated with evaluation of epifluorescence, demonstrated that adipocytes presented a better response to transfection than other cells, independent of the kit used. Performing PCR on co-transfected cells demonstrated the presence of anti-CD3, making this approach feasible for future experiments.

The glycosylation of anti-rhIFN-α2b recombinant antibodies influences the antigen-neutralizing activity.

OBJECTIVES: The influence of glycosylation on the antigen-neutralizing ability of two potential biotherapeutic anti-human IFN-α2b antibodies composed by murine and humanized single-chain Fv fused to human Fcγ1 (chimeric and humanized scFv-Fc, respectively) was studied. RESULTS: Chimeric antibodies produced in CHO-K1 and HEK293 mammalian cells showed no differences in the antigen-antibody affinity but demonstrated differences in the in vitro neutralization of IFN-α2b activity. On the other hand, the humanized antibodies produced in the same cell types showed differences in both the antigen-antibody affinity and the antigen-neutralizing ability. These differences are due to the scFv domain, as evidenced by its expression in CHO-K1 and HEK293 cells. In order to determine if the Fc glycosylation influences the antigen binding ability, both parameters were analyzed on chimeric and humanized deglycosylated scFv-Fc. Surprisingly, no differences in the antigen-antibody affinity were observed, but differences in the antigen-neutralizing ability of both chimeric and humanized antibodies, and their respectively deglycosylated glycoforms were found. CONCLUSIONS: Fc glycosylation influences the antigen neutralization ability of two anti-rhIFN-α2b recombinant antibodies. Although affinity is the widely accepted parameter to analyze antibody antigen binding, it does not appear to be sufficient to describe the behavior of recombinant antibodies in vitro. This work contributes with a high impact knowledge to develop therapeutic recombinant antibodies where glycosylation and producer cell lines must be taken into account for their influence on the antigen binding capacity and not only for their impact on the effector properties as it has been historically considered for antibodies.

Construção de um vetor de expressão para fragmentos de anticorpos anti-cd3 humanos com potencial imunorregulatório / Construction of an expression vector for fragments of human anti-cd3 antibodies with immunoregulatory potential

Construiu-se um novo vetor para expressão de fragmentos de anticorpos anti-CD3 humano (pMIRES FvFc R ­ Fc mutado) com potencial imunoregulatório. Foram utilizadas técnicas de clonagem no vetor pUC 57 e no vetor de expressão pMIRES FvFc R por meio de transformação bacteriana por choque térmico; seguida de preparação do DNA plasmidial em pequena e média escala; digestão dos mesmos com enzimas de restrição, e posterior análise e extração do DNA plasmidial por eletroforese. A clonagem foi confirmada pela técnica de sequenciamento de Sanger. Este trabalho resultou na construção de um novo cassete para expressão de fragmentos de anticorpos anti-CD3 humano contendo duas mutações na região variável e uma mutação na porção Fc, que se acredita que melhore a produção heteróloga do anticorpo. A construção deste novo vetor de expressão, com a adição da sequência de exportação de RNA e mutações na porção Fc, buscou contribuir para a caracterização e aprimoramento das características ligantes e efetoras dos anticorpos monoclonais anti-CD3.

Complete genome sequence of Mycobacterium massiliense.

Mycobacterium massiliense is a rapidly growing bacterium associated with opportunistic infections. The genome of a representative isolate (strain GO 06) recovered from wound samples from patients who underwent arthroscopic or laparoscopic surgery was sequenced. To the best of our knowledge, this is the first announcement of the complete genome sequence of an M. massiliense strain.

Comparative isolation protocols and characterization of stem cells from human primary and permanent teeth pulp

Aim: This study was developed to compare the morphological, proliferative and immunophenotypic profiles of pulp cells from permanent and primary teeth, obtained by two isolation methods. Methods: Normal human impacted third molars and exfoliated primary teeth were collected and cut around the cementoenamel junction. Pulp cells cultures were established by two approaches: enzyme digestion (3 mg/mL type I colagenase and 4 mg/mL dispase), or culture of the tissue explants in cell culture dishes. Morphological and proliferative analyses, as well as immunophenotype characterization with monoclonal antibodies against CD117, CD34 and CD45 surface receptors were performed. Results: For the permanent teeth, on the 4th day of culture, the cell number was significantly higher for the outgrowth method. By the end of the studied period (14th day), the enzymatic method was more efficient in promoting culture growth. On the other hand, for primary teeth, enzymatic digestion always promoted a higher cell proliferation. The immunophenotypic profiles were CD117+/ CD34-/ CD45- and CD117+/ CD34+/ CD45- for cells from permanent and primary teeth, respectively. Conclusions: The findings of this study indicate that both isolation methods can be efficiently used. The cell population displayed an immunophenotype compatible to the one of stem cells, with remarkable positive expression of CD117.

Transcriptome analysis of the Amazonian viper Bothrops atrox venom gland using expressed sequence tags (ESTs).

Bothrops atrox is a highly dangerous pit viper in the Brazilian Amazon region. We produced a global catalogue of gene transcripts to identify the main toxin and other protein families present in the B. atrox venom gland. We prepared a directional cDNA library, from which a set of 610 high quality expressed sequence tags (ESTs) were generated by bioinformatics processing. Our data indicated a predominance of transcripts encoding mainly metalloproteinases (59% of the toxins). The expression pattern of the B. atrox venom was similar to Bothrops insularis, Bothrops jararaca and Bothrops jararacussu in terms of toxin type, although some differences were observed. B. atrox showed a higher amount of the PIII class of metalloproteinases which correlates well with the observed intense hemorrhagic action of its toxin. Also, the PLA2 content was the second highest in this sample compared to the other three Bothrops transcriptomes. To our knowledge, this work is the first transcriptome analysis of an Amazonian rain forest pit viper and it will contribute to the body of knowledge regarding the gene diversity of the venom gland of members of the Bothrops genus. Moreover, our results can be used for future studies with other snake species from the Amazon region to investigate differences in gene patterns or phylogenetic relationships.

Genetic variability and phylogeny of the high-risk HPV-31, -33, -35, -52, and -58 in central Brazil.

More than 100 HPV types have been described, 13 of which are classified as high-risk due to their association with the development of cervical cancer. The intratype genomic diversity of HPV-16 and -18 has been studied extensively, while little data have been generated for other less common high-risk types. The present study explores the nucleotide variability and phylogeny of the high-risk HPV-31, -33, -35, -52, and -58, in samples from Central Brazil. For this purpose, the LCR and the E6 and L1 genes were sequenced. Several variants of these HPV types were detected, some of which have been detected in other parts of the world. Furthermore, new variants of all types examined were characterized in a total of 13 new variants. Based on the E6 and L1 sequences, variants were described comprising conservative and non-conservative amino acid changes. For phylogenetic tree construction, samples characterized in this study were compared to others described and submitted to GenBank previously. The phylogenetic analysis of HPV-31, -33, -35, and -58 isolates did not reveal ethnic or geographical clustering as observed previously for HPV-16 and -18. HPV-35 analysis showed a dichotomic branching characteristic of viral subtypes. Interestingly, four clusters relative to the analysis of HPV-52 isolates were identified, two of which could be classified as Asian and European branches. The genomic characterization of HPV variants is crucial for understanding the intrinsic geographical relatedness and biological differences of these viruses and contributes further to studies on their infectivity and pathogenicity.

Transporters in the Paracoccidioides brasiliensis transcriptome: insights on drug resistance.

In the struggle for life, the capacity of microorganisms to synthesize and secrete toxic compounds (inhibiting competitors) plays an important role in successful survival of these species. This ability must come together with the capability of being unaffected by these same compounds. Several mechanisms are thought to avoid the toxic effects. One of them is toxin extrusion from the intracellular environment to the outside vicinity, using special transmembrane proteins, referred to as transporters. These proteins are also important for other reasons, since most of them are involved in nutrient uptake and cellular excretion. In cancer cells and in pathogens, and particularly in fungi, some of these proteins have been pointed out as responsible for an important phenotype known as multidrug resistance (MDR). In the present study, we tried to identify in the Paracoccidioides brasiliensis transcriptome, transporter-ortholog genes from the two major classes: ATP binding cassette and major facilitator superfamily transporter. We found 22 groups with good similarity with other fungal ATP binding cassette transporters, and four Paracoccidioides brasilienses assembled expressed sequence tags that probably code for major facilitator superfamily proteins. We also focused on fungicide resistance orthologs already characterized in other pathogenic fungi. We were able to find homologs to C. albicans CDR1, CDR2, and MDR1, Saccharomyces cerevisiae PDR5 and Aspergillus AtrF genes, all of them related to azole resistance. As current treatment for paracoccidioidomycosis mainly uses azole derivatives, the presence of these genes can be postulated to play a similar role in P. brasiliensis, warning us for the possibility of resistant isolate emergence.

Oxidative stress response in Paracoccidioides brasiliensis.

Survival of pathogenic fungi inside human hosts depends on evasion from the host immune system and adaptation to the host environment. Among different insults that Paracoccidioides brasiliensis has to handle are reactive oxygen and nitrogen species produced by the human host cells, and by its own metabolism. Knowing how the parasite deals with reactive species is important to understand how it establishes infection and survives within humans. The initiative to describe the P. brasiliensis transcriptome fostered new approaches to study oxidative stress response in this organism. By examining genes related to oxidative stress response, one can evaluate the parasite's ability to face this condition and infer about possible ways to overcome this ability. We report the results of a search of the P. brasiliensis assembled expressed sequence tag database for homologous sequences involved in oxidative stress response. We described several genes coding proteins involved in antioxidant defense, for example, catalase and superoxide dismutase isoenzymes, peroxiredoxin, cytochrome c peroxidase, glutathione synthesis enzymes, thioredoxin, and the transcription factors Yap1 and Skn7. The transcriptome analysis of P. brasiliensis reveals a pathogen that has many resources to combat reactive species. Besides characterizing the antioxidant defense system in P. brasiliensis, we also compared the ways in which different fungi respond to oxidative damage, and we identified the basic features of this response.