C Di Stefano

Human decay accelerating factor transgenic pigs for xenotransplantation obtained by sperm-mediated gene transfer

UMAN organs for transplantation are insufficient in quantity, and for every organ transplant undertaken there is a need for an additional five to ten organs. Waiting lists are constantly growing and many patients die before an organ can be found. Researchers continue to experiment with alternatives. One of these is xenotransplantation, which uses animals as donors and which presents an entirely new set of challenges. The pig is presently considered the most likely source of organs for human xenotransplantation because it is easy to breed, has compatibly sized organs, and offers the possibility of genetic manipulation. However, organ transplantation between distantly related species, such as pigs and humans, results in hyperacute rejection (HAR), involving the complement system. It may be possible to avoid rejection reactions by genetically engineering donor animals, so that the recipient’s immune system does not act on the graft. Thus, a number of research teams, including our group, have embarked on programs to produce pigs transgenic for the human regulators of complement activation (RCAs) genes, in the attempt to produce pigs whose organs may be suitable for transplantation into humans. 1‐3 The present study reports on the production of pigs transgenic for the human regulator of complement activation human decay accelerating factor (hDAF) by spermmediated gene transfer (SMGT), a highly efficient and reproducible alternative to microinjection, presently the most widely used system for generating transgenic animals.

A new clinical approach: Use of blood-derived stem cells (BDSCs) for superficial digital flexor tendon injuries in horses

AIMS In this study, we present an innovative therapy using stem cells that were obtained from the peripheral blood of racehorses affected by uninduced superficial digital flexor tendon (SDFT) injuries. MAIN METHODS Blood-derived stem cells (BDSCs) were generated from the blood samples of three horses in the presence of macrophage colony-stimulating factor (M-CSF). The racehorses received a single autologous BDSC treatment, which resulted in the successful repair of the tendons injuries. KEY FINDINGS The results demonstrated that the BDSCs injection into the damaged tendon stimulated the regeneration of normal tissue. Furthermore, a relationship may exist between the speed and the quality of new tissue formation and the welfare and management of the treated animals. SIGNIFICANCE This study demonstrates that stem cell technology offers new tools for tissue repair that in many cases is considered incurable, and provides additional evidence that BDScs injections increase the speed and quality of the regeneration process in different animal tissues.

Efficiency of transgenesis using sperm-mediated gene transfer: generation of hDAF transgenic pigs.

SINCE the beginning of this century, replacement of failing human organs with their animal counterparts has been an interesting topic of debate for writers and scientists. In the 1960s, prolonged survival after kidney transplantation from chimpanzee to human was obtained in the United States and Europe. Nevertheless, both the progressive improvement in surgical technique and in immunosuppressant therapy and the availability of cadaveric organs and living donation have reduced the interest in xenotransplantation. Because of the increasing requests for organs and the lack of donors to meet that need, xenotransplantation has become a reliable option again for temporary organ replacement (eg, of heart or liver) before definitive transplant. However, primates such as chimpanzees and baboons are expensive, can carry important zoonoses, and their use is burdened by ethical implications. A better choice for xenotransplantation might be offered by swine, which are closer to humans for anatomic and metabolic features. Discordant transplantation is associated with humoral hyperacute rejection, due to preformed antibodies and complement system activation (both classical and alternative pathway). This results in massive and irreversible vascular damage and cellular necrosis. Expression of the species-specific complement activation inhibitors could prevent this kind of rejection. Organs harvested from pigs transgenic for human decay accelerating factor (hDAF or CD55), membrane cofactor protein (MCP or CD46), and CD59 could be more efficiently grafted into human recipients. Therefore, a number of research teams, including our group, have generated pigs transgenic for human negative regulators of the complement cascade. This research has been aimed to generate hDAF transgenic swine with high efficiency and reproducibility. This goal has been reached through the innovative method of sperm-mediated gene transfer (SMGT), which was developed about 10 years ago by our group. Data presented in this paper show that hDAF-positive individuals can also be used as founders of stable lines of F1 generation transgenic pigs. MATERIALS AND METHODS hDAF Transgenic F1 Offspring Generation

Interference of bovine herpesvirus 1 (BHV-1) in sorbitol-Induced apoptosis

In order to determine the ability of bovine herpesvirus type 1 (BHV‐1) to suppress apoptosis, we examined the effects of BHV‐1 infection on sorbitol‐induced apoptosis on Madin–Darby bovine kidney (MDBK) cells. BHV‐1 suppresses sorbitol‐induced apoptosis in a manner similar to that of herpes simplex virus type 1 (HSV‐1), indicating that BHV‐1 has one or more anti‐apoptotic genes. To elucidate the molecular mechanisms of apoptosis, expression of some genes encoding apoptosis‐inhibiting and ‐promoting factors were analyzed on BHV‐1 infected cells during the process of sorbitol‐induced apoptosis. Our results revealed that the expression of bcl‐2 and bcl‐xL decreased after 5 and 3 h p.i., respectively; while bax and procaspase‐3 expression increased with respect to control as a function of p.i. times and at 7 h p.i. they were not observed. We further show that the expression of p53 gene was also enhanced, suggesting that this apoptotic mechanism is p53 dependent. From these results, we propose that BHV‐1 has one or more genes encoding apoptosis‐inhibiting factors which interfere with the involvement of bcl‐2 gene family members and apoptotic pathway, depending upon caspase‐3, triggered by sorbitol. J. Cell. Biochem. 89: 373–380, 2003.

hDAF expression in hearts of transgenic pigs obtained by sperm-mediated gene transfer.

TRANSPLANTATON has been the choice option to treat successfully an increasing number of acute and chronic human pathologies with declining morbidity and mortality. However, availability of organs from human donors is limited and dramatically inadequate with respect to patient requests. Xenotransplantation from large-sized mammals has thus been reconsidered as a tool to overcome the present unbalance between organ offers and requests. Pigs have been chosen because they can be easily and cheaply bred; they do not raise ethical questions—their use as alimentary resources is generally admitted; and they possess organs largely human compatible for size, anatomical organization, and physiology. Nevertheless, this option is usually compromised by the occurrence of an irreversible hyperacute vascular rejection of the animal transplanted organ as soon as it is perfused with human blood, mediated by natural preformed antibodies and by the activation of the complement cascade. Downregulating the complement system by expressing on the surface of the swine organs one or more species-specific negative modulators of the complement complexes, such as human decay accelerating factor (hDAF), could prevent the rejection of the graft. Our group has obtained a number of transgenic pigs for hDAF using sperm-mediated gene transfer (SMGT), an innovative and highly efficient and reproducible method. In SMGT, spermatozoa are employed as natural vectors of foreign DNA and are bound, captured, and internalized into recipient egg cells to generate a genetically modified progeny. Revealing the expression of the protein on the surface of the organs is an important confirmation for successful transgenesis. The aim of this report is to show, by immunohistochemical detection, the expression of hDAF in transgenic hearts, organs maximally eligible for xenotransplantation.

Degenerate PCR method for identification of an antiapoptotic gene in BHV-1

To investigate on the hypothetical presence of an antiapoptotic gene, we utilized the CODEHOP (COnsensus‐DEgenerate Hybrid Oligonucleotide Primers) strategy amplifying unknown sequences from a background of genomic (bovine herpesvirus type‐1) BHV‐1 DNA. An alignment of carboxyl‐terminal domains belonging to three proteins encoded by γ34.5, MyD116 and GADD34 genes, was carried out to design degenerate PCR primers in highly conserved regions. This allowed the amplification of a 110 bp fragment. This fragment was subjected to automatic sequencing and DNA sequence analysis revealed that its position resided between the nt 14363 and the nt 14438 in bovine herpesvirus type‐1 (BHV‐1) Cooper strain sharing an identity of 86% (UL14). Transient transfections showed that UL14 protein is efficient in protecting MDBK and K562 cells from sorbitol induced apoptosis. The proteins anti‐apoptotic function may derive from its heat shock protein‐like properties. J. Cell. Biochem. 97: 813–823, 2006.

The Antiapoptotic Effect of BHV-1 in Sorbitol-induced Apoptosis in MDBK Cells

INTRODUCTION. Apoptosis is a mechanism of eukariotic cell suicide which plays a major role in many physiological and pathological processes. Many viruses have developed different pathways to induce or inhibit the apoptotic process sufficiently to allow the completion of viral replication. In this report, we show that bovine herpes virus type-1(BVH-1) is able to inhibit the sorbitol-induced apoptosis in Madin-Darby bovine kidney (MDBK) cells.