RESUMO

TNFα is a pro-inflammatory cytokine that is a therapeutic target for inflammatory autoimmune disorders. Thus, TNFα antagonists are successfully used for the treatment of these disorders. Here, new association patterns of rhTNFα and its antagonists Adalimumab and Etanercept are disclosed. Active rhTNFα was purified by IMAC from the soluble fraction of transformed Escherichia coli. Protein detection was assessed by SDS-PAGE and Western blot. The KD values for rhTNFα interactions with their antagonists were obtained by non-competitive ELISA and by microscale thermophoresis (MST). Molecular sizes of the complexes were evaluated by size-exclusion chromatography-high performance liquid chromatography (SEC-HPLC). Surprisingly, both antagonists recognized the monomeric form of rhTNFα under reducing and non-reducing conditions, indicating unexpected bindings of the antagonists to linear epitopes and to rhTNFα monomers. For the first time, the interactions of rhTNFα with Adalimumab and Etanercept were assessed by MST, which allows evaluating molecular interactions in solution with a wide range of concentrations. Biphasic binding curves with low and high KD values (<10-9 M and >10-8 M) were observed during thermophoresis experiments, suggesting the generation of complexes with different stoichiometry, which were confirmed by SEC-HPLC. Our results demonstrated the binding of TNFα-antagonists with rhTNFα monomers and linear epitopes. Also, complexes of high molecular mass were observed. This pioneer investigation constitutes valuable data for future approaches into the study of the interaction mechanism of TNFα and its antagonists.

Assuntos

RESUMO

Erythropoietin (Epo) is a fundamental hormone in the regulation of hematopoiesis, and other secondary roles mediated by the binding of the hormone to its specific receptor (EpoR), which leads to an activation of key signaling pathways that induce an increase in cell differentiation, apoptosis control and neuroprotection. It has been suggested that their function depends on final conformation of glycosylations, related with affinity to the receptor and its half-life. The presence of EpoR has been reported in different tissues including central nervous system, where it has been demonstrated to exert a neuroprotective function against oxidative stress conditions, such as ischemic injury and neurodegenerative diseases. There is also evidence of an increase in EpoR expression in brain cell lysates of Alzheimer's patients with respect to healthy patients. These results are related with extensive in vitro experimental data of neuroprotection obtained from cell lines, primary cell cultures and hippocampal slices. Additionally, this data is correlated with in vivo experiments (water maze test) in mouse models of Alzheimer's disease where Epo treatment improved cognitive function. These studies support the idea that receptor activation induces a neuroprotective effect in neurodegenerative disorders including dementias, and especially Alzheimer's disease. Taken together, available evidence suggests that Epo appears to be a central element for EpoR activation and neuroprotective properties in the central nervous system. In this review, we will describe the mechanisms associated with neuroprotection and its relation with the activation of EpoR in order with identify new targets to develop pharmacological strategies.

RESUMO

Gaucher disease (GD) is an orphan disease characterized by the lack or incapacity of glucocerebrosidase (hGCase) to properly process glucosylceramide, resulting in its accumulation in vital structures of the human body. Enzyme replacement therapy supplies hGCase to GD patients with a high-cost recombinant enzyme produced in vitro in mammalian or plant cell culture. In this study, we produced hGCase through the direct injection of recombinant adenovirus in the mammary gland of a non-transgenic goat. The enzyme was secreted in the milk during six days at a level up to 111.1 ± 8.1 mg/L, as identified by mass spectrometry, showing high in vitro activity. The milk-produced hGCase presented a mass correspondent to the intermediary high-mannose glycosylated protein, which could facilitate its delivery to macrophages through the macrophage mannose receptor. Further studies are underway to determine the in vivo delivery capacity of milk-hGCase, but results from this study paves the way toward the generation of transgenic goats constitutively expressing hGCase in the milk.

Assuntos

RESUMO

BACKGROUND: Mesenchymal stem cells have a high capacity for trans-differentiation toward many adult cell types, including endothelial cells. Feto-placental tissue, such as Wharton's jelly is a potential source of mesenchymal stem cells with low immunogenic capacity; make them an excellent source of progenitor cells with a potential use for tissue repair. We evaluated whether administration of endothelial cells derived from mesenchymal stem cells isolated from Wharton's jelly (hWMSCs) can accelerate tissue repair in vivo. METHODS: Mesenchymal stem cells were isolated from human Wharton's jelly by digestion with collagenase type I. Endothelial trans-differentiation was induced for 14 (hWMSC-End14d) and 30 (hWMSC-End30d) days. Cell phenotyping was performed using mesenchymal (CD90, CD73, CD105) and endothelial (Tie-2, KDR, eNOS, ICAM-1) markers. Endothelial trans-differentiation was demonstrated by the expression of endothelial markers and their ability to synthesize nitric oxide (NO). RESULTS: hWMSCs can be differentiated into adipocytes, osteocytes, chondrocytes and endothelial cells. Moreover, these cells show high expression of CD73, CD90 and CD105 but low expression of endothelial markers prior to differentiation. hWMSCs-End express high levels of endothelial markers at 14 and 30 days of culture, and also they can synthesize NO. Injection of hWMSC-End30d in a mouse model of skin injury significantly accelerated wound healing compared with animals injected with undifferentiated hWMSC or injected with vehicle alone. These effects were also observed in animals that received conditioned media from hWMSC-End30d cultures. CONCLUSION: These results demonstrate that mesenchymal stem cells isolated from Wharton's jelly can be cultured in vitro and trans-differentiated into endothelial cells. Differentiated hWMSC-End may promote neovascularization and tissue repair in vivo through the secretion of soluble pro-angiogenic factors.

Assuntos

RESUMO

Background: With the advent of transgenic technology to farm animals, it became possible to express recombinant proteins of high complexity in the body compartments and fluids of these animals; the term "pharming" was coined. This was a tremendous achievement taking into consideration the high costs associated with conventional (cell-based) production methods and the incapacity of lower organisms to adequately process complex proteins. The mammary gland had been the organ of choice and milk the appropriate vector for successful expression of many recombinant drugs of high added value. Review: While theoretically the mammary gland is able to carry out all the complex post-translational changes related with glycosylation or others, in the practice, not all proteins can be actually processed in a way that closely remembers the wild protein, thus making difficult the production of some proteins in full biologically active form. This is especially true for complex (branched) forms of glycosylation as it is the case of human erythropoietin (hEPO), or gamma carboxylation of blood clotting factors, to mention a few examples. These cases are discussed in this review, with special emphasis in the glycosylation of hEPO. In spite of the imperfectness of the mammary gland to accurately add some sugar residues, it continues to be the most desirable organ to which target gene expression, due to its potent biosynthetic machinery and the possibilities to amend the said incapacity to glycosylate appropriately all kinds of proteins. In line with this, the European Medicines Agency first (in 2006) and the Food and Drug Administration later (2009) approved the first milk-derived recombinant protein for human use, (ATryn; human anti-thrombin-III) after more than two decades of thorough reviews and test, thus opening the way for future massive production of blockbuster drugs using the mammary gland as bioreactor. In this job we reviewed briefly the state of the art of mammary gland-based production of recombinant proteins with emphasis in two different systems to target it. In the first approach, a transgenic mammal carrying appropriate mammary specific gene promoter linked to a transgene is made, then grown, mated, its milk tested for the presence of the protein, if expression levels and biological activity of the proteins meet the requirements, then a production flock is created from the founder(s) and milk collected and processed. In this way, most of the recombinant proteins produced in the milk had been created, including the leading drug ATryn. We developed an alternative method for transient viral vectors-mediated transduction of the mammary gland, using constitutive viral promoters linked to the transgene, thus producing very quickly high amounts of the desired protein. The drawback of this method is its transient nature; the advantage is the fastness and easiness to produce grams of recombinant proteins in the milk of otherwise non-transgenic mammals. In this way several drugs had been produced. Notably one of them, the E2 antigen of classic swine fever (CSF) had been secreted in biologically active form at high levels in goats´ milk; a veterinary vaccine formulation was established and tested successfully in clinical trials that included viral challenging with CSF. It is foreseen that this vaccine could be in the market this year and became the first recombinant drug produced in the milk of non-transgenic animals to get regulatory approval. In this article, we also reviewed the state of the art of different body fluids as vectors for recombinant protein production Conclusions: At least for the next coming years, all the animal-based recombinant protein production ways will lead to the milk.

Assuntos