Fernando Henrique Lojudice

Bone Morphogenetic Proteins Facts, Challenges, and Future Perspectives

Bone morphogenetic proteins (BMPs) are members of the TGF-β superfamily, acting as potent regulators during embryogenesis and bone and cartilage formation and repair. Cell and molecular biology approaches have unveiled the great complexity of BMP action, later confirmed by transgenic animal studies. Genetic engineering allows for the production of large amounts of BMPs for clinical use, but they have systematically been associated with a delivery system, such as type I collagen and calcium phosphate ceramics, to ensure controlled release and to maximize their biological activity at the surgical site, avoiding systemic diffusion. Clinical orthopedic studies have shown the benefits of FDA-approved recombinant human BMPs (rhBMPs) 2 and 7, but side effects, such as swelling, seroma, and increased cancer risk, have been reported, probably due to high BMP dosage. Several studies have supported the use of BMPs in periodontal regeneration, sinus lift bone-grafting, and non-unions in oral surgery. However, the clinical use of BMPs is growing mainly in off-label applications, with robust evidence to ascertain rhBMPs’ safety and efficacy through well-designed, randomized, and double-blind clinical trials. Here we review and discuss the critical data on BMP structure, mechanisms of action, and possible clinical applications.

Beneficial effects of prolactin and laminin on human pancreatic islet-cell cultures.

The problem of pancreas donor shortage could be addressed through in vitro islet-cell proliferation prior to transplantation into diabetic patients. Therefore, we set out to evaluate the effects of prolactin (rhPRL) and laminin on primary cultures of human pancreatic islets. Our results showed that rhPRL induced an increase in islet-cell number and in cumulative insulin secretion (p<0.01). However, glucose-induced insulin secretion was enhanced only in the presence of both laminin and rhPRL. In addition, we describe, for the first time in human islets, the PRL-induced activation of JAK2, and signal transducer and activator of transcription (STAT) 1, 3 and 5. Our results demonstrate a significant beneficial effect of rhPRL and laminin on human islets and support widely held notion that the closer physiological stimuli and environment of beta cells are mimicked, the better are the results in cell proliferation and secretory function, both essential for successful islet transplantation.

Prolactin-induced changes in protein expression in human pancreatic islets.

Ex vivo islet cell culture prior to transplantation appears as an attractive alternative for treatment of type 1 diabetes. Previous results from our laboratory have demonstrated beneficial effects of human prolactin (rhPRL) treatment on human islet primary cultures. In order to probe into the molecular events involved in the intracellular action of rhPRL in these cells, we set out to identify proteins with altered expression levels upon rhPRL cell treatment, using two-dimensional (2D) gel electrophoresis and mass spectrometry (MS). An average of 300 different protein spots were detected, 14 of which were modified upon rhPRL treatment (p<0.01), of which 12 were successfully identified using MS and grouped according to their biological functions. In conclusion, our study provides, for the first time, information about proteins that could be critically involved in PRLs action on human pancreatic islets, and facilitate identification of new and specific targets involved in islet cell function and proliferation.

NUCEL (Cell and Molecular Therapy Center) : A Multidisciplinary Center for Translational Research in Brazil

Social and economical development is closely associated with technological innovation and a well-developed biotechnological industry. In the last few years, Brazil’s scientific production has been steadily increasing; however, the number of patents is lagging behind, with technological and translational research requiring governmental incentive and reinforcement. The Cell and Molecular Therapy Center (NUCEL) was created to develop activities in the translational research field, addressing concrete problems found in biomedical and veterinary areas and actively searching for solutions by employing a genetic engineering approach to generate cell lines over-expressing recombinant proteins to be transferred to local biotech companies, aiming at furthering the development of a national competence for local production of biopharmaceuticals of widespread use and of life-saving importance. To this end, mammalian cell engineering technologies were used to generate cell lines over-expressing several different recombinant proteins of biomedical and biotechnological interest, namely, recombinant human Amylin/IAPP for diabetes treatment, human FVIII and FIX clotting factors for hemophilia, human and bovine FSH for fertility and reproduction, and human bone repair proteins (BMPs). Expression of some of these proteins is also being sought with the baculovirus/insect cell system (BEVS) which, in many cases, is able to deliver high-yield production of recombinant proteins with biological activity comparable to that of mammalian systems, but in a much more cost-effective manner. Transfer of some of these recombinant products to local Biotech companies has been pursued by taking advantage of the São Paulo State Foundation (FAPESP) and Federal Government (FINEP, CNPq) incentives for joint Research Development and Innovation partnership projects.

Enhanced Proteolytic Processing of Recombinant Human Coagulation Factor VIII B-Domain Variants by Recombinant Furins

Recombinant human factor VIII (rFVIII) is used in replacement therapy for hemophilia A. Current research efforts are focused on bioengineering rFVIII molecules to improve its secretion efficiency and stability, limiting factors for its efficient production. However, high expression yield in mammalian cells of these rFVIII variants is generally associated with limited proteolytic processing. Non-processed single-chain polypeptides constitute non-natural FVIII molecule configurations with unpredictable toxicity and/or antigenicity. Our main objective was to demonstrate the feasibility of promoting full-proteolytic processing of an rFVIII variant retaining a portion of the B-domain, converting it into the smallest natural activatable form of rFVIII, while keeping its main advantage, i.e., improved secretion efficiency. We generated and employed a CHO-DG44 cell clone producing an rFVIII variant retaining a portion of the B-domain and the FVIII native cleavage site between Arg1648 and Glu1649. By bioengineering CHO-DG44 cells to express stably the recombinant human endoproteases PACE, PACE-SOL, PCSK5, PCSK6, or PCKS7, we were able to achieve complete intra- or extracellular proteolytic processing of this rFVIII variant. Additionally, our quantitative data indicated that removal of the B-domain segment by intracellular proteolytic processing does not interfere with this rFVIII variant secretion efficiency. This work also provides the first direct evidence of (1) intracellular cleavage at the Arg1648 FVIII processing site promoted by wild-type PACE and PCSK7 and (2) proteolytic processing at the Arg1648 FVIII processing site by PCSK6.

VP22 herpes simplex virus protein can transduce proteins into stem cells

The type I herpes simplex virus VP22 tegument protein is abundant and well known for its ability to translocate proteins from one cell to the other. In spite of some reports questioning its ability to translocate proteins by attributing the results observed to fixation artifacts or simple attachment to the cell membrane, VP22 has been used to deliver several proteins into different cell types, triggering the expected cell response. However, the question of the ability of VP22 to enter stem cells has not been addressed. We investigated whether VP22 could be used as a tool to be applied in stem cell research and differentiation due to its capacity to internalize other proteins without altering the cell genome. We generated a VP22.eGFP construct to evaluate whether VP22 could be internalized and carry another protein with it into two different types of stem cells, namely adult human dental pulp stem cells and mouse embryonic stem cells. We generated a VP22.eGFP fusion protein and demonstrated that, in fact, it enters stem cells. Therefore, this system may be used as a tool to deliver various proteins into stem cells, allowing stem cell research, differentiation and the generation of induced pluripotent stem cells in the absence of genome alterations.

Pancreatic Stem Cells

During embryogenesis, the first pancreatic cells arise from a region located at the posterior foregut. Numerous signals are required to induce the formation of both the ventral and the dorsal buds of the pancreas, as well as for differentiation of the different types of pancreatic cells. Other factors are also important to maintain a proliferative pool of pancreatic progenitor cells within the embryonic ductal epithelium. After organogenesis, new beta cells may also originate by expansion or neogenesis. Different stem cell populations have been described in the pancreas, namely: ductal and acinar cells, nestin-positive cells, HGF receptor-positive cells, c-Kit-positive cells, and DCAMKL-1-positive cells. Transdifferentiation or dedifferentiation, followed by differentiation, is also described to be present in the pancreas. Therefore, other pancreatic cells, which do not belong to the endocrine lineage, are possible sources of new beta cells. Extrapancreatic tissue may also be modified to express insulin, including the hepatic tissue, bone marrow, and monocytes. In addition, stem cells from several sources have been subjected to differentiation protocols aiming to achieve insulin-producing cells in the presence and in the absence of genetic manipulation.