Expression and characterization of a novel single-chain anti-vascular endothelial growth factor antibody in the goat milk.

Vascular endothelial growth factor (VEGF) has essential functions in angiogenesis, endothelial cell proliferation, migration, and tumor invasion. Different approaches have been developed to suppress tumor angiogenesis, which is considered a hallmark of cancer. Anti-VEGF monoclonal antibodies constitute an important strategy for cancer immunotherapy, which has been produced on several platforms. In this study, a novel single-chain anti-VEGF monoclonal antibody (scVEGFmAb) was produced in the goat mammary gland by adenoviral transduction. scVEGFmAb was purified by affinity chromatography. N-glycans were analyzed by exoglycosidase digestion and hydrophilic interaction ultra-performance liquid chromatography coupled to electrospray ionization mass spectrometry. The biological activity of scVEGFmAb was assessed by scratch and mouse aortic ring assays. scVEGFmAb was produced at 0.61 g/L in the goat milk, and its purification rendered 95 % purity. N-glycans attached to scVEGFmAb backbone were mainly neutral biantennary core fucosylated with Galß1,4GlcNAc motif, and charged structures were capped with Neu5Ac and Neu5Gc. The chimeric molecule significantly prevented cell migration and suppressed microvessel sprouting. These results demonstrated for the first time the feasibility of producing an anti-VEGF therapeutic antibody in the milk of non-transgenic goats with the potential to counteract tumor angiogenesis.

In-vitro production and pre-validation of lyophilized canine platelet-rich plasma for therapeutic use / Produção e validação de plasma rico em plaquetas canino liofilizado para fins de utilização terapêutica

Platelet-rich plasma (PRP) has been considered a promising therapeutic alternative, since platelets are rich in growth factors that are used in the Regenerative Medicine field. However, fresh PRP cannot be stored for long periods. This study aimed to develop a protocol for obtaining lyophilized canine PRP capable of maintaining viability after its reconstitution. For that purpose, canine PRP extraction and lyophilization protocols were initially tested. Subsequently, assays were carried out to quantify the growth factors VEGF and TGF-β, before and after the lyophilization process, gelation test and the three-dimensional gel structure analysis of the reconstituted lyophilized PRP by electron microscopy, as well as in vitro cell proliferation test in lyophilized PRP gel. Additionally, the immunogenicity test was performed, using allogeneic samples of lyophilized PRP. The results showed that the lyophilized PRP had adequate therapeutic concentrations of growth factors VEGF and TGF-β (9.1pg/mL and 6161.6pg/mL, respectively). The reconstituted PRP gel after lyophilization showed an in vitro durability of 10 days. Its electron microscopy structure was similar to that of fresh PRP. In the cell proliferation test, an intense division process was verified in mesenchymal stem cells (MSCs) through the three-dimensional mesh structure of the lyophilized PRP gel. The immunogenicity test showed no evidence of an immune reaction. The findings were promising, suggesting the possibility of having a lyophilized canine PRP that can be marketed. New in vivo and in vitro studies must be carried out for therapeutic confirmation.(AU)

O plasma rico em plaquetas (PRP) é uma alternativa terapêutica promissora, pois as plaquetas são ricas em fatores de crescimento com ação na regeneração de tecidos. No entanto, o PRP fresco não pode ser armazenado por longos períodos. Esse trabalho teve como objetivo desenvolver um protocolo de obtenção de PRP liofilizado canino capaz de manter a viabilidade pós reconstituição. Portanto, foram testados diversos protocolos de extração e liofilização. Para validação do PRP canino liofilizado foi analisada a concentração dos fatores de crescimento VEGF e TGF-β antes e após o processo de liofilização, a estrutura tridimensional do PRP liofilizado reconstituído em forma de gel por microscopia eletrônica e seu efeito in vitro na proliferação de células-tronco mesenquimais. Os resultados demonstraram que o PRP liofilizado apresentou concentrações terapêuticas adequadas dos fatores de crescimento VEGF e TGF- β (9,1pg/ml e 6161,6pg/ml, respectivamente). O gel de PRP reconstituído após liofilização apresentou uma durabilidade in vitro de 10 dias, sua estrutura tridimensional mostrou-se semelhante ao PRP fresco e proporcionou intensa proliferação de células-tronco mesenquimais durante o cultivo. O teste de imunogenicidade não demonstrou evidências de reação imune. Os achados foram promissores, sugerindo a possibilidade de uso de PRP canino liofilizado para o mercado. Novos estudos in vivo e in vitro deverão ser conduzidos para comprovação terapêutica.(AU)

In-vitro production and pre-validation of lyophilized canine platelet-rich plasma for therapeutic use

ABSTRACT: Platelet-rich plasma (PRP) has been considered a promising therapeutic alternative, since platelets are rich in growth factors that are used in the Regenerative Medicine field. However, fresh PRP cannot be stored for long periods. This study aimed to develop a protocol for obtaining lyophilized canine PRP capable of maintaining viability after its reconstitution. For that purpose, canine PRP extraction and lyophilization protocols were initially tested. Subsequently, assays were carried out to quantify the growth factors VEGF and TGF-, before and after the lyophilization process, gelation test and the three-dimensional gel structure analysis of the reconstituted lyophilized PRP by electron microscopy, as well as in vitro cell proliferation test in lyophilized PRP gel. Additionally, the immunogenicity test was performed, using allogeneic samples of lyophilized PRP. The results showed that the lyophilized PRP had adequate therapeutic concentrations of growth factors VEGF and TGF- (9.1pg/mL and 6161.6pg/mL, respectively). The reconstituted PRP gel after lyophilization showed an in vitro durability of 10 days. Its electron microscopy structure was similar to that of fresh PRP. In the cell proliferation test, an intense division process was verified in mesenchymal stem cells (MSCs) through the three-dimensional mesh structure of the lyophilized PRP gel. The immunogenicity test showed no evidence of an immune reaction. The findings were promising, suggesting the possibility of having a lyophilized canine PRP that can be marketed. New in vivo and in vitro studies must be carried out for therapeutic confirmation.

RESUMO: O plasma rico em plaquetas (PRP) é uma alternativa terapêutica promissora, pois as plaquetas são ricas em fatores de crescimento com ação na regeneração de tecidos. No entanto, o PRP fresco não pode ser armazenado por longos períodos. Esse trabalho teve como objetivo desenvolver um protocolo de obtenção de PRP liofilizado canino capaz de manter a viabilidade pós reconstituição. Portanto, foram testados diversos protocolos de extração e liofilização. Para validação do PRP canino liofilizado foi analisada a concentração dos fatores de crescimento VEGF e TGF- antes e após o processo de liofilização, a estrutura tridimensional do PRP liofilizado reconstituído em forma de gel por microscopia eletrônica e seu efeito in vitro na proliferação de células-tronco mesenquimais. Os resultados demonstraram que o PRP liofilizado apresentou concentrações terapêuticas adequadas dos fatores de crescimento VEGF e TGF- (9,1pg/ml e 6161,6pg/ml, respectivamente). O gel de PRP reconstituído após liofilização apresentou uma durabilidade in vitro de 10 dias, sua estrutura tridimensional mostrou-se semelhante ao PRP fresco e proporcionou intensa proliferação de células-tronco mesenquimais durante o cultivo. O teste de imunogenicidade não demonstrou evidências de reação imune. Os achados foram promissores, sugerindo a possibilidade de uso de PRP canino liofilizado para o mercado. Novos estudos in vivo e in vitro deverão ser conduzidos para comprovação terapêutica.

Biochemistry detection of acetylcholinesterase activity in Trypanosoma evansi and possible functional correlations.

Several chemical and immunohistochemical techniques can be used for the detection of acetylcholinesterase (AChE) activity. In this experiment we aimed to detect AChE activity in Trypanosoma evansi. For this, the parasites were isolated from the blood of experimentally infected rats using a DEA-cellulose column. Enzymatic activity was determined in trypomastigote forms at 0, 0.2, 0.4, 0.8 and 1.2 mg/mL of protein concentrations by a standard biochemical protocol. At all concentrations tested, the study showed that T. evansi expresses the enzyme AChE and its activity was proportional to the concentration of protein, ranging between 0.64 and 2.70 µmol of AcSCh/h. Therefore, we concluded that it is possible to biochemically detect AChE in T. evansi, an enzyme that may be associated with vital functions of the parasite and also can be related to chemotherapy treatments, as further discussed in this article.

Trypanosoma evansi: effects of zinc and copper in experimentally infected rats.

The aim of this study was to evaluate the effects of a treatment using injectable zinc and copper in rats infected with Trypanosoma evansi. 48 rats were divided into eight groups of six animals each. Group A was composed of uninfected animals. Animals from groups B-H were inoculated at the 5th day of experiment with 1.2×10(6) trypanosomes. Group B was used as a positive control. The infected groups received prophylactic (C, D and E) and therapeutic (F, G and H) treatments with the zinc and copper, both at a dose of 5 mg kg(-1). The effectiveness of treatment was confirmed by negative blood smears and Polymerase Chain Reaction (PCR) at the end of study. All treated animals had their prepatent period and survival prolonged when compared with control group (group B). Treatment efficacy was 17% (C: zinc), 33% (D: copper), 50% (E: zinc+copper), 0% (F: zinc), 50% (G: copper) and 50% (H: zinc+copper). Thus, we can conclude that treatment with zinc and copper are capable of controlling and/or curing T. evansi infection in rats, delaying the parasitemia and prolonging their survival.

Biochemical detection of adenosine deaminase in Trypanosoma evansi.

Biochemical and molecular research on parasites has increased considerably in trypanosomes in the recent years. Many of them have the purpose of identify areas, proteins and structures of the parasite which are vulnerable and could be used in therapy against the protozoan. Based on this hypothesis this study aimed to detect biochemically the enzyme adenosine deaminase (ADA) in Trypanosoma evansi, and to adapt an assay to the measurement of its activity in trypomastigotes. Firstly, the parasites were separated from the blood of mice experimentally infected with a DEAE-cellulose column. The ADA activity in trypomastigotes was evaluated at concentrations of 0.1, 0.2, 0.5, 0.6 and 0.8mg of protein by spectrophotometry. ADA activity was observed in the parasites at all concentrations tested and its activity was proportional to the concentration of protein, ranging between 0.64 and 2.24U/L in the lowest and highest concentration of protein, respectively. Therefore, it is possible to detect biochemically ADA in T. evansi, an enzyme that may be associated with vital functions of the parasite, similar to what occurs in mammals. This knowledge may be useful in the association of the chemotherapic treatment with specific inhibitors of the enzyme, in future studies.

Susceptibility of mice to Trypanosoma evansi treated with human plasma containing different concentrations of apolipoprotein L-1.

The aim of this study was to test the susceptibility of mice to Trypanosoma evansi treated with human plasma containing different concentrations of apolipoprotein L-1 (APOL1). For this experiment, a strain of T. evansi and human plasma (plasmas 1, 2, and 3) from 3 adult males clinically healthy were used. In vivo test used 50 mice divided in 5 groups (A to E) with 10 animals in each group. Animals of groups B to E were infected, and then treated with 0.2 ml of human plasma in the following outline: negative control (A), positive control (B), treatment with plasma 1 (C), treatment with plasma 2 (D), and treatment with plasma 3 (E). Mice treated with human plasma showed an increase in longevity of 40.9 ± 0.3 (C), 20 ± 9.0 (D) and 35.6 ± 9.3 (E) days compared to the control group (B) which was 4.3 ± 0.5 days. The number of surviving mice and free of the parasite (blood smear and PCR negative) at the end of the experiment was 90%, 0%, and 60% for groups C, D, and E, respectively. The quantification of APOL1 was performed due to the large difference in the treatments that differed in the source plasma. In plasmas 1, 2, and 3 was detected the concentration of 194, 99, and 115 mg/dl of APOL1, respectively. However, we believe that this difference in the treatment efficiency is related to the level of APOL1 in plasmas.

SECTAB: a new device for tabanid storage in field collections / SECTAB: um novo instrumento para armazenamento de tabanídeos em coletas de campo

O artigo descreve a construção de um novo aparato simples, para o armazenamento e transporte de tabanídeos vivos durante as coletas realizadas em campo, evitando a perda de espécimes.

The article describes the construction of a simple new device, for the storage of live tabanids during field collections and their transportation to the laboratory, avoiding the loss of specimens.

SECTAB: a new device for tabanid storage in field collections.

The article describes the construction of a simple new device, for the storage of live tabanids during field collections and their transportation to the laboratory, avoiding the loss of specimens.