A novel oncogenic BTK isoform is overexpressed in colon cancers and required for RAS-mediated transformation.

Bruton's tyrosine kinase (BTK) is essential for B-cell proliferation/differentiation and it is generally believed that its expression and function are limited to bone marrow-derived cells. Here, we report the identification and characterization of p65BTK, a novel isoform abundantly expressed in colon carcinoma cell lines and tumour tissue samples. p65BTK protein is expressed, through heterogeneous nuclear ribonucleoprotein K (hnRNPK)-dependent and internal ribosome entry site-driven translation, from a transcript containing an alternative first exon in the 5'-untranslated region, and is post-transcriptionally regulated, via hnRNPK, by the mitogen-activated protein kinase (MAPK) pathway. p65BTK is endowed with strong transforming activity that depends on active signal-regulated protein kinases-1/2 (ERK1/2) and its inhibition abolishes RAS transforming activity. Accordingly, p65BTK overexpression in colon cancer tissues correlates with ERK1/2 activation. Moreover, p65BTK inhibition affects growth and survival of colon cancer cells. Our data reveal that BTK, via p65BTK expression, is a novel and powerful oncogene acting downstream of the RAS/MAPK pathway and suggest that its targeting may be a promising therapeutic approach.

Functional analysis of expression of human ecto-nucleoside triphosphate diphosphohydrolase-1 and/or ecto-5'-nucleotidase in pig endothelial cells.

Adenine nucleosides and nucleotides are important signaling molecules involved in control of key mechanisms of xenotransplant rejection. Extracellular pathway that converts ATP and ADP to AMP, and AMP to adenosine mainly mediated by ecto-nucleoside triphosphate diphosphohydrolase 1, (ENTPD1 or CD39) and ecto-5'-nucleotidase (E5NT or CD73) respectively, is considered as important target for xenograft protection. To clarify feasibility of combined expression of human ENTPD1 and E5NT and to study its functional effect we transfected pig endothelial cell line (PIEC) with both genes together. To do this we have produced a dicistronic construct bearing F2A sequence in frame between human E5NT and human ENTPD1 coding sequences. PIEC cells were mock-transfected as transfection control or transfected with plasmids encoding human ENTPD1 or human E5NT. PIEC cells were exposed to 50 µM ATP or 50 µM ADP or 50 µM AMP. Conversion of extracellular substrates into products (ATP/ADP/AMP/adenosine) was measured by HPLC in the media collected at specific time intervals. Following addition of AMP, production of adenosine in the medium of E5NT/ENTPD1- and E5NT- transfected cells increased to 14.2±1.1 and 24.5±3.4 µM respectively while it remained below 1 µM in controls and in ENTPD1-transfected cells. A marked increase of adenosine formation from ADP or ATP was observed only in E5NT/ENTPD1-transfected cells (11.7±0.1 and 5.7±2.2 µM respectively) but not in any other condition studied. This study indicates feasibility and functionality of combined expression of human E5NT and ENTPD1 in pig endothelial cells using F2A sequence bearing construct.

AAV-mediated photoreceptor transduction of the pig cone-enriched retina.

Recent success in clinical trials supports the use of adeno-associated viral (AAV) vectors for gene therapy of retinal diseases caused by defects in the retinal pigment epithelium (RPE). In contrast, evidence of the efficacy of AAV-mediated gene transfer to retinal photoreceptors, the major site of inherited retinal diseases, is less robust. In addition, although AAV-mediated RPE transduction appears efficient, independently of the serotype used and species treated, AAV-mediated photoreceptor gene transfer has not been systematically investigated thus so far in large animal models, which also may allow identifying relevant species-specific differences in AAV-mediated retinal transduction. In the present study, we used the porcine retina, which has a high cone/rod ratio. This feature allows to properly evaluate both cone and rod photoreceptors transduction and compare the transduction characteristics of AAV2/5 and 2/8, the two most efficient AAV vector serotypes for photoreceptor targeting. Here we show that AAV2/5 and 2/8 transduces both RPE and photoreceptors. AAV2/8 infects and transduces photoreceptor more efficiently than AAV2/5, similarly to what we have observed in the murine retina. The use of the photoreceptor-specific rhodopsin promoter restricts transgene expression to porcine rods and cones, and results in photoreceptor transduction levels similar to those obtained with the ubiquitous promoters tested. Finally, immunological, toxicological and biodistribution studies support the safety of AAV subretinal administration to the large porcine retina. The data presented here on AAV-mediated transduction of the cone-enriched porcine retina may affect the development of gene-based therapies for rare and common severe photoreceptor diseases.

In vitro production of multigene transgenic blastocysts via sperm-mediated gene transfer allows rapid screening of constructs to be used in xenotransplantation experiments.

Multigene transgenic pigs would be of benefit for large animal models and in particular for xenotransplantation, where extensive genetic manipulation of donor pigs is required to make them suitable for organ grafting to humans. We have previously produced multitransgenic pigs via sperm-mediated gene transfer (SMGT) using integrative constructs expressing 3 different reporter genes. The aim of the present work was to evaluate the efficacy and safety of using 3 integrative constructs carrying 3 different human genes involved in the modulation of inflammatory responses. We developed an in vitro fertilization system to demonstrate that SMGT can be used to efficiently produce multigene transgenic embryos through a 1-step genetic modification using multiple integrative constructs each carrying a different human gene involved in the modulation of inflammatory processes (hHO1, hCD39, and hCD73). The results suggest that this system allowed an effective preliminary test of transgenesis optimization, greatly reducing the number of animals used in the experiments and fulfilling important ethical issues. We performed 5 in vitro fertilization experiments using sperm cells preincubated with all 3 integrative constructs. A total of 1,498 oocytes were fertilized to obtain 775 embryos, among which 340 further developed into blastocysts. We did not observe any toxicity related to the transgenesis procedure that affected normal embryo development. We observed 68.5% transgenesis efficiency. Blastocysts were 48% single, 31% double, and 21% triple transgenic.

Carotid artery disease: novel pathophysiological mechanisms identified by gene-expression profiling of peripheral blood.

OBJECT: The pathogenesis of carotid artery stenosis (CAS) as well as the mechanisms underlying the different localisation of the atherosclerotic lesions remains poorly understood. We used microarray technology to identify novel systemic mediators that could contribute to CAS pathogenesis. Moreover, we compared gene-expression profile of CAS with that of patients affected by abdominal aortic aneurysm (AAA), previously published by our group. METHODS AND RESULTS: By global gene-expression profiling in a pool of 10 CAS patients and 10 matched controls, we found 82 genes differentially expressed. Validation study in pools used for profiling and replication study in larger numbers of CAS patients (n = 40) and controls (n = 40) of 14 genes by real-time polymerase chain reaction (RT-PCR) confirmed microarray results. Fourteen out of 82 genes were similarly expressed in AAA patients. Gene ontology analysis identified a statistically significant enrichment in CAS of differentially expressed transcripts involved in immune response and oxygen transport. Whereas alteration of oxygen transport is a common tract of the two localisations, alteration of immune response in CAS and of lipid metabolic process in AAA represents distinctive tracts of the two atherosclerotic diseases. CONCLUSIONS: We describe the systemic gene-expression profile of CAS, which provides an extensive list of potential molecular markers.

Sperm-mediated gene transfer-treated spermatozoa maintain good quality parameters and in vitro fertilization ability in swine.

A simple and efficient method for producing multitransgenic animals is required for medical and veterinary applications. Sperm-mediated gene transfer (SMGT) is an effective method for introducing multiple genes into pigs (Sus, Sus scrofa). The major benefits of this technique are the high efficiency, low cost, and ease of use compared with that of other methods: Sperm-mediated gene transfer does not require embryo handling or expensive equipment. The aim of this study was to investigate the influence of SMGT treatment and exogenous DNA uptake on sperm quality. Even after a coincubation with a 20-fold larger amount (100 microg/mL) of DNA than usual (5 microg/mL), sperm quality parameters were not significantly affected, confirming the hypothesis that the SMGT protocol itself or the amount of bound DNA do not compromise the possibility of an extended employment of SMGT. More importantly, we found that semen used for in vitro fertilization 24h after DNA uptake gave good cleavage (60% vs. 58%, treated vs. control) and developmental rates definitely positive (41% vs. 48%, treated vs. control). These good results are connected to a competitive efficiency of transformation (62%) due to the numerous improvements in SMGT technique. We demonstrate that SMGT-treated spermatozoa retain good quality and fertilization potential for at least 24h, expanding the possibility to apply transgenesis in field conditions in swine, where the greatest hurdles are fertilization timing and plain procedure.

Gene expression profiling of peripheral blood in patients with abdominal aortic aneurysm.

OBJECT: Abdominal aortic aneurysm (AAA) pathogenesis remains poorly understood. This study investigated the gene expression profile of peripheral blood from patients with AAA using microarray technology. METHODS AND RESULTS: We determined gene expression profiles in pooled RNA from 10 AAA patients and 10 matched controls with arrays representing 14,000 transcripts. Microarray data for selected genes were confirmed by real-time PCR in two different AAA (n=36) and control (n=36) populations and integrated with biochemical data. We identified 91 genes which were differentially expressed in AAA patients. Gene Ontology analysis indicated a significant alteration of oxygen transport (increased hemoglobin gene expression) and lipid metabolism [including monoglyceride lipase and low density lipoprotein receptor-related protein 5 (LRP5) gene]. LRP5 expression was associated inversely with serum lipoprotein(a) [Lp(a)] concentration. CONCLUSIONS: Increased expression of hemoglobin chain genes as well as of genes involved in erythrocyte mechanical stability were observed in the AAA RNA pools. The association between low levels of LRP5 gene expression and increased levels of Lp(a) in AAA patients suggests a potential role of LRP5 in Lp(a) catabolism. Our data underline the power of microarrays in identifying further molecular perturbations associated with AAA.

Differences in activities of the enzymes of nucleotide metabolism and its implications for cardiac xenotransplantation.

Xenotransplantation is one be possible solution for a severe shortage of human organs available for transplantation. However, only a few studies addressed metabolic compatibility of transplanted animal organs. Our aim was to compare activities of adenosine metabolizing enzymes in the heart of different species that are relevant to clinical or experimental xenotransplantation. We noted fundamental differences: ecto-5' nucleotidease (E5' N) activity was 4-fold lower in pig and baboon hearts compared to the human hearts while mouse activity was compatible with human and rat activity was three times higher than human. There also were significant differences in AMP-deaminase (AMPD), adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) activities. We conclude that differences in nucleotide metabolism may contribute to organ dysfunction after xenotransplantation.

Carbon monoxide pretreatment prevents respiratory derangement and ameliorates hyperacute endotoxic shock in pigs.

Endotoxic shock, one of the most prominent causes of mortality in intensive care units, is characterized by pulmonary hypertension, systemic hypotension, heart failure, widespread endothelial activation/injury, and clotting culminating in disseminated intravascular coagulation and multi-organ system failure. In the last few years, studies in rodents have shown that administration of low concentrations of carbon monoxide (CO) exerts potent therapeutic effects in a variety of diseases/disorders. In this study, we have administered CO (one our pretreatment at 250 ppm) in a clinically relevant, well-characterized model of LPS-induced acute lung injury in pigs. Pretreatment only with inhaled CO significantly ameliorated several of the acute pathological changes induced by endotoxic shock. In terms of lung physiology, CO pretreatment corrected the LPS-induced changes in resistance and compliance and improved the derangement in pulmonary gas exchange. In terms of coagulation and inflammation, CO reduced the development of disseminated intravascular coagulation and completely suppressed serum levels of the proinflammatory IL-1beta in response to LPS, while augmenting the anti-inflammatory cytokine IL-10. Moreover, the effects of CO blunted the deterioration of kidney and liver function, suggesting a beneficial effect in terms of end organ damage associated with endotoxic shock. Lastly, CO pretreatment prevents LPS-induced ICAM expression on lung endothelium and inhibits leukocyte marginalization on lung parenchyma.

Purine metabolism in pigs and humans and its implications for xenotransplantation.

We compared concentrations of nucleotide substrates and activities of enzymes of nucleotide metabolism in pig and human blood, heart, and kidney. The most important difference was lower ecto-5-nucleotidase (ESN) activity in both pig hearts and kidney. Furthermore, higher hypoxanthine, inosine, adenine, and uracil, but lower uridine and uric acid concentrations were observed in pig blood as compared to human. A twofold increase in UTP concentration has been observed in pig hearts following 4 h perfusion with human blood. Purine metabolism is an important target for genetic and pharmacological manipulation during xenotransplantations.

Exposure to human blood inactivates swine endothelial ecto-5'-nucleotidase.

Ecto-5'-nucleotidase (E5'N) is an extracellular enzyme forming anti-inflammatory and immunosuppressive adenosine. We evaluated whether confrontation of pig heart and endothelial cells with human blood changes the activity of E5'N. Pig hearts were perfused ex vivo with fresh human blood for 4 h. Pig aortic endothelial cells (PAEC) were incubated in vitro with human plasma for 3 h. Ex vivo perfusion of pig heart with fresh human blood resulted in a decrease in E5'N activity to 62% and 61% of initial in wild-type and transgenic pig hearts, respectively. PAEC activity of E5'N decreased to 71% and 50% of initial after 3 h exposure to heat-inactivated and active complement human plasma, respectively, while it remained constant in controls. Pig heart activity of E5'N decreased following exposure to human blood, which may affect adenosine production and exacerbate hyperacute and vascular rejection.

Expression of human ecto 5' nucleotidase in pig endothelial cells and its implication for adenosine production and xenotransplantation.

Human endothelial activity of ecto-5'-nucleotidase (E5'N) is several times higher than in pig endothelial cells. This may have implication for xenotransplantation due to the role this enzyme plays in conversion of pro-inflammatory and pro-aggreggatory nucleotides into anti-inflammatory and antiaggregatory adenosine. We have shown in this study that human E5'N can be functionally expressed in pig endothelial cells leading to increased adenosine production from both extracellular AMP and ATP. We suggest that E5'N expression in transgenic pigs for xenotransplantation may help to prolong graft survival.

Laparoscopic insemination technique with low numbers of spermatozoa in superovulated prepuberal gilts for biotechnological application.

New biotechnologies, such as sperm-mediated gene transfer (SMGT), spermatozoa freezing and spermatozoa sorting have improved the possibilities to produce animals with desirable features. The main problem associated with these technologies is the scarce availability of spermatozoa for insemination. The objective of this study was to develop a laparoscopic insemination (LI) technique in gilt that allows the use of low semen doses resulting in high fertilization rates (FR) and minimal distress to the animal; the efficiency of this technique was compared to conventional artificial insemination (AI). Ten gilts were inseminated 36 h post hCG treatment near both utero-tubal junctions (UTJ) with 1.5 x 10(9)spermatozoa/5 mL per horn and 10 gilts (C) underwent conventional AI. Embryos were collected either at two to four cell stage (LI, n = 5; C, n = 5) for determination of fertilization rate or at day 6 for evaluation of developmental competence (LI, n = 5; C, n = 5). LI gilts showed a slightly higher FR than control animals. In a second trial, 24 gilts underwent LI with varying doses (1.5 x 10(8), 1.5 x 10(7), 1 x 10(7), 5 x 10(6) or 1 x 10(6)) of semen. Two to four stage embryos were collected and FR was evaluated in each tube. FR obtained with the lowest dose was significantly different from that with other dosages (P < 0.05). Embryos were cultured in vitro to blastocyst stages (percentage of blastocysts: 79.2 +/- 3.6%). In a third trial, five gilts were inseminated with semen processed by SMGT technique; both FR (86.1 +/- 9.9%) and transgene protein expression were satisfactory. In conclusion, this study shows that LI can be a useful tool for reducing doses of insemination, without affecting the efficiency of fertilization; this technique could have a wide range of biotechnological applications.

Telomeres and telomerase activity in pig tissues.

The current state of the art concerning telomeres and telomerase stems almost exclusively from the analysis of protozoa, yeast, and a small number of mammals. In the present study, we confirm that the pig telomeric sequence is indeed T(2)AG(3), as previously suggested. By making use of sequence analysis of pig telomeric DNA variant telomeric repeats in the medial region of the telomeres, interspersed with canonical T(2)AG(3) repeats, were identified. This telomere organization is similar to the one present in humans. Analysis of terminal restriction fragments showed that the majority of telomeres from different pig tissues are longer than in humans but shorter than in Mus musculus. Telomeres from spermatozoa were found to be longer, ranging in size between 13 and 44 kb. Most of the somatic pig tissues expressed significant levels of telomerase activity, a situation more similar to mouse and that contrasts with the one in humans and dog. Moreover, the analysis of sperm cells from different epididymal compartments of an adult animal showed that telomerase activity is absent in maturing spermatozoa, suggesting that sperm telomere elongation is restricted during spermatogenesis.

'Advanced' generation lentiviruses as efficient vectors for cardiomyocyte gene transduction in vitro and in vivo.

Efficient gene transduction in cardiomyocytes is a task that can be accomplished only by viral vectors. Up to now, the most commonly used vectors for this purpose have been adenoviral-derived ones. Recently, it has been demonstrated that lentiviral vectors can transduce growth-arrested cells, such as hematopoietic stem cells. Moreover, a modified form of lentiviral vector (the 'advanced' generation), containing an mRNA-stabilizer sequence and a nuclear import sequence, has been shown to significantly improve gene transduction in growth-arrested cells as compared to the third-generation vector. Therefore, we tested whether the 'advanced' generation lentivirus is capable of infecting and transducing cardiomyocytes both in vitro and in vivo, comparing efficacy in vitro against the third-generation of the same vector. Here we report that 'advanced' generation lentiviral vectors infected most (>80%) cardiomyocytes in culture, as demonstrated by immunofluorescence and FACS analyses: in contrast the percentage of cardiomyocytes infected by third-generation lentivirus was three- to four-fold lower. Moreover, 'advanced' generation lentivirus was also capable of infecting and inducing stable gene expression in adult myocardium in vivo. Thus, 'advanced' generation lentiviral vectors can be used for both in vitro and in vivo gene expression studies in the cardiomyocyte.

Transfected human dendritic cells to induce antitumor immunity.

Dendritic cells are professional antigen-presenting cells able to prime naive T lymphocytes and regulate steadily the delicate balance between tolerance and activation during the immune response. In past years several reports have shown that genetically engineered dendritic cells (DCs) can be a powerful tool for inducing an antigen-specific immune response. The use of such modified antigen-presenting cells is a real working hypothesis in preclinical studies and in clinical vaccination approaches for cancer treatment. The definition of optimal transfection conditions for preserving DC survival and functionality is necessary to design a correct immunotherapeutic protocol. Different lipid-based transfection compounds were studied for their effects on DC survival, phenotype and functional properties. All the transfection procedures were able to select DCs with a higher expression of activation and costimulatory molecules (ie MHCII-DR, CD83, CD86, CD25) than the untreated DCs. However, only two compounds (LipofectAMINE PLUS and FuGENE 6), preserved or even increased the immunopotency of DCs as antigen-presenting cells. These protocols were applied to modify DCs in order to express an epithelial tumor-associated antigen, MUC1, and such cells were able to induce in vitro a specific immune response in healthy donors.