Carboxytherapy: Controls the inflammation and enhances the production of fibronectin on wound healing under venous insufficiency.

To examine the influence of carboxytherapy on wound healing under venous insufficiency, full-thickness excisional wounds were created on Wistar rats. We used three groups with 32 rats each: Group (I): daily cleaning with 0.9% saline solution; Group Sulfadiazine (II): 1% silver sulfadiazine; and Carboxytherapy (III): subcutaneous application of 0.3 mL of carbon dioxide. The predetermined periods of analysis were the 3rd, 7th, 14th, and 30th day. The slides were stained with haematoxylin and eosin and Picrosirius red and submitted for immunohistochemistry. Groups II and III presented a statistically significant decrease in relation to the presence of neutrophilic and lymphocytic infiltrates. The presence of collagen significant increased in groups II and III. However, group III presented better organisation. Only group I maintained the neovascularisation until the 30th day. The new epithelium statistically significantly increased in groups II and III. On immunohistochemistry, regarding fibronectin expression, only group III demonstrated a statistically significant increase since the beginning of the healing process. Thus, the use of carboxytherapy promotes the formation of a tissue better structured and that may be an important resource for the treatment of wounds under venous insufficiency, especially those of recurrent re-openings.

Lentivirus-meditated frataxin gene delivery reverses genome instability in Friedreich ataxia patient and mouse model fibroblasts.

Friedreich ataxia (FRDA) is a progressive neurodegenerative disease caused by deficiency of frataxin protein, with the primary sites of pathology being the large sensory neurons of the dorsal root ganglia and the cerebellum. FRDA is also often accompanied by severe cardiomyopathy and diabetes mellitus. Frataxin is important in mitochondrial iron-sulfur cluster (ISC) biogenesis and low-frataxin expression is due to a GAA repeat expansion in intron 1 of the FXN gene. FRDA cells are genomically unstable, with increased levels of reactive oxygen species and sensitivity to oxidative stress. Here we report the identification of elevated levels of DNA double strand breaks (DSBs) in FRDA patient and YG8sR FRDA mouse model fibroblasts compared to normal fibroblasts. Using lentivirus FXN gene delivery to FRDA patient and YG8sR cells, we obtained long-term overexpression of FXN mRNA and frataxin protein levels with reduced DSB levels towards normal. Furthermore, γ-irradiation of FRDA patient and YG8sR cells revealed impaired DSB repair that was recovered on FXN gene transfer. This suggests that frataxin may be involved in DSB repair, either directly by an unknown mechanism, or indirectly via ISC biogenesis for DNA repair enzymes, which may be essential for the prevention of neurodegeneration.

Intra-amniotic delivery of CFTR-expressing adenovirus does not reverse cystic fibrosis phenotype in inbred CFTR-knockout mice.

Due to its early onset and severe prognosis, cystic fibrosis (CF) has been suggested as a candidate disease for in utero gene therapy. In 1997, a study was published claiming that to how transient prenatal expression of CF transmembrane conductance regulator (CFTR) from an in utero-injected adenovirus vector could achieve permanent reversal of the CF intestinal pathology in adult CF knockout mice, despite the loss of CFTR transgene expression by birth. This would imply that the underlying cause of CF is a prenatal defect for which lifelong cure can be achieved by transient prenatal expression of CFTR. Despite criticism at the time of publication, no independent verification of this contentious finding has been published so far. This is vital for the development of future therapeutic strategies as it may determine whether CF gene therapy should be performed prenatally or postnatally. We therefore reinvestigated this finding with an identical adenoviral vector and a knockout CF mouse line (Cftr(tmlCam)) with a completely inbred genetic background to eliminate any effects due to genetic variation. After delivery of the CFTR-expressing adenovirus to the fetal mouse, both vector DNA and transgenic CFTR expression were detected in treated animals postpartum but statistically no significant difference in survival was observed between the Cftr(-/-) mice treated with the CFTR-adenovirus and those treated with the control vector.

Community Awareness on Rabies Prevention and Control in Bicol, Philippines: Pre- and Post-Project Implementation.

Rabies is endemic in the Philippines. To support the rabies campaign in the Bicol region at the southeastern part of Luzon, the BAI-OIE Stop Transboundary Animal Diseases and Zoonoses (STANDZ) Rabies project was implemented in the pilot provinces of Camarines Norte, Camarines Sur, Albay, and Masbate. A community awareness survey was conducted with the residents of these provinces to determine their knowledge, attitude, and practices (KAP) on rabies during the start and end of the project. Qualitative, descriptive research was done with a structured KAP questionnaire. Pet owners in the pilot provinces were chosen as respondents. Results showed that respondents know that they can acquire rabies in animals through the bite of a rabid dog (pre-project implementation (PRI): 19.6%, post-project implementation (POI): 38.0%). Vaccination was the top rabies preventive measure (PRI: 61.8%, POI: 92.8%). Biting incidents were noted in some respondents, and observing the dog and killing it immediately were some of the actions taken by bite victims. If a supposed rabid dog was seen, respondents would either: immediately kill the dog (PRI: 20.3%, POI: 13.7%), report it to authorities (PRI: 26.3%, POI: 63.1%), and capture and observe the dog concerned (PRI: 13.5%, POI: 6.0%). Pet owners increased their KAP about rabies prevention and control as compared to the pre-implementation study. However, certain gaps in their KAP need to be given attention; thus continuous education of pet owners must be done.

Clinically applicable procedure for gene delivery to fetal gut by ultrasound-guided gastric injection: toward prenatal prevention of early-onset intestinal diseases.

Targeting gene therapy vectors to the fetal intestinal tract could provide a novel means toward prevention of the early postnatal intestinal pathology of cystic fibrosis and other conditions, such as congenital enteropathy, that cause intestinal failure. Among these conditions, cystic fibrosis is by far the most common lethal genetic disease. It is caused by a functional absence or deficiency of the cystic fibrosis transmembrane conductance regulator and manifests in the gut as meconium ileus. Prenatal treatment of genetic disease may avoid early-onset tissue damage and immune sensitization, and may target cells that are less accessible in the adult. We investigated gene transfer to the fetal gut, using a minimally invasive injection technique. First-generation replication-deficient adenoviral vectors encoding the beta-galactosidase gene and transduction-enhancing agents were injected into the stomach of early-gestation fetal sheep (n = 8, 60 days of gestation; term, 145 days) under ultrasound guidance. Reporter gene expression was observed 2 days after injection in the villi of the gastrointestinal epithelia after 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside staining and beta-galactosidase immunohistochemistry of fetal tissues. Expression of beta-galactosidase, as measured by enzyme-linked immunosorbent assay, was enhanced after pretreatment of the fetal gut with sodium caprate, which opens tight junctions, and after adenovirus complexation with DEAE-dextran, which confers a positive charge to the virus. Instillation of the fluorocarbon perflubron after virus delivery resulted in tissue transduction from the fetal stomach to the colon. Using a clinically relevant technique, we have demonstrated widespread gene transfer to the fetal gastrointestinal epithelia.

Preventive and therapeutic moderate aerobic exercise programs convert atherosclerotic plaques into a more stable phenotype.

UNLABELLED: The mechanisms by which exercise affects atherosclerotic plaque stability remain incompletely understood. We evaluated the effects of two training protocols on both atherosclerotic plaque structure and the signaling pathways involved in plaque rupture. METHODS: Male low-density lipoprotein (LDL) receptor knockout mice were fed a high-fat, high-cholesterol diet (HFD). One group was subjected to moderate exercise using a treadmill for 14weeks (preventive protocol). The other group started an exercise regimen after 16weeks of the HFD (therapeutic group). Atherosclerotic plaques within the aorta were evaluated for lipid and collagen contents, as well as for inflammatory markers. Plasma cholesterol and cytokine levels were also determined. RESULTS: The mice receiving a HFD developed hypercholesterolemia and atherosclerotic plaques within the aorta. The aortas from the animals in the preventive protocol exhibited smaller lipid cores and higher collagen content. These animals also exhibited lower CD40 expression within the plaques. The aortas of the mice in the therapeutic group exhibited higher collagen content, but no differences in either lipid core size or plaque size were noted. No differences in blood pressure, plasma cholesterol, cytokine levels, plaque size or metalloproteinase 9 expression were observed in the trained animals compared with the sedentary animals. CONCLUSION: Moderate aerobic exercise modified atherosclerotic plaque characteristics and converted the plaques into a more stable phenotype, increasing the collagen content in response to both exercise programs. Furthermore, moderate aerobic exercise reduced the animals' fat content and decreased the activity of the CD40-CD40L signaling pathway in the preventive group.

Hepatic stem cells: from inside and outside the liver?

The liver is normally proliferatively quiescent, but hepatocyte loss through partial hepatectomy, uncomplicated by virus infection or inflammation, invokes a rapid regenerative response from all cell types in the liver to perfectly restore liver mass. Moreover, hepatocyte transplants in animals have shown that a certain proportion of hepatocytes in foetal and adult liver can clonally expand, suggesting that hepatoblasts/hepatocytes are themselves the functional stem cells of the liver. More severe liver injury can activate a potential stem cell compartment located within the intrahepatic biliary tree, giving rise to cords of bipotential transit amplifying cells (oval cells), that can ultimately differentiate into hepatocytes and biliary epithelial cells. A third population of stem cells with hepatic potential resides in the bone marrow; these haematopoietic stem cells may contribute to the albeit low renewal rate of hepatocytes, but can make a more significant contribution to regeneration under a very strong positive selection pressure. In such instances, cell fusion rather than transdifferentiation appears to be the underlying mechanism by which the haematopoietic genome becomes reprogrammed.

The hopes and fears of in utero gene therapy for genetic disease--a review.

Somatic gene delivery in utero is a novel approach to gene therapy for genetic disease. It is based on the concept that application of gene therapy vectors to the fetus in utero may prevent the development of early disease related tissue damage, may allow targeting of otherwise inaccessible organs, tissues and still expanding stem cell populations and may also provide postnatal tolerance against the therapeutic transgenic protein. This review outlines the hypothesis and scientific background of in utero gene therapy and addresses some of the frequently expressed concerns raised by this still experimental, potentially preventive gene therapy approach. We describe and discuss the choice of vectors, of animal models and routes of administration to the fetus. We address potential risk factors of prenatal gene therapy such as vector toxicity, inadvertent germ line modification, developmental aberration and oncogenesis as well as specific risks of this procedure for the fetus and mother and discuss their ethical implications.

Characterization of long-term survival of syngeneic hepatocytes in rat peritoneum.

Hepatocyte transplantation is a potential therapy for both acute and chronic hepatic insufficiency and also for treatment of inborn errors of metabolism affecting the liver. The peritoneum is one site for implantation and has several advantages: cells implanted there can be easily identified and observed, and it has a relatively large capacity. Long-term survival using "pure" hepatocytes in the peritoneum have been disappointing. We hypothesized that cotransplantation of hepatocytes with nonparenchymal cells would help maintain differentiated hepatocyte function. Rat liver cells transplanted intraperitoneally into August rats were sacrificed at 7 days, 1, 3, 6, 9, and 12 months and analyzed for presence, basal proliferation, and functionality of hepatocytes. To demonstrate that ectopic hepatocytes remained susceptible to exogenous growth factors affecting cell proliferation, rats 9 and 12 months after transplantation were stimulated with tri-iodothyronine and KGF. Hepatocytes were identified 7 days to >12 months, by H&E and immunohistochemically, as ectopic islands in the omental fat. Functionality was confirmed by glycogen deposition. Basal proliferation in 7-day rats was 28.0 +/- 10/1000 hepatocytes in ectopic islands (cf. 5.70 +/- 2.7/1000 in recipient liver). Proliferation in ectopic islands was greater than host liver. Growth factor-stimulated proliferation in ectopic islands induced a 70-fold increase in DNA synthesis. In conclusion, hepatocytes transplanted with nonparenchymal cells survive, proliferate, and function in the peritoneum of normal rats, and respond to exogenous growth stimuli. Their survival and proliferation in the presence of a normal functioning liver has implications for the potential use of the peritoneal site clinically for supplementation of liver function in metabolic disorders.

Cell immortalization as a key, rate-limiting event in malignant transformation: approaches toward a molecular genetic analysis.

Recent advances using somatic cell genetic approaches have provided a convincing body of evidence that the senescence of mammalian cells in culture is controlled by a small group of genes, one or more of which are functionally deleted in the process of immortalization. Microcell-mediated mono-chromosomal transfer methods should permit precise mapping of these genes to specific chromosomal regions. Cloning of senescence genes, using either conventional 'positional cloning' techniques or retroviral insertion mutagenesis, is now a realistic possibility. The leap in our understanding of the molecular genetic events driving the alternative cellular states of limited proliferative capacity and immortality, which such advances should precipitate, will finally permit the question of the role of cell immortalization in cancer to be addressed, and may open the door to the design of new modes of cancer therapy. In addition, the precise mechanism underlying the wide difference in transformability between human and rodent cells, which should also emerge from these investigations, is likely to make a significant contribution towards resolving the key issue of the relevance of rodent tumour induction assays in assessing the potential carcinogenicity of environmental chemicals.

Urogenital anomalies in human male fetuses.

BACKGROUND: There are few studies in the literature performed in human fetuses evaluating the incidence of genitourinary anomalies. AIMS: Analyze the incidence of congenital urogenital malformations in human male fetuses. STUDY DESIGNS AND SUBJECTS: We analyzed 166 human male fetuses well preserved. The gestational age was determined in weeks post conception (WPC) according to the foot length criterion and ranged from 10 to 35 WPC. The fetuses were dissected with the aid of a stereoscopic lens with 2.5x magnification. We performed abdominal and pelvic incisions to expose the urogenital organs. We studied the incidence of renal, ureteral, vesicle, urethral, testicular, epididymal, vas deferens, prostate and penile anomalies. RESULTS: Of the 166 fetuses, 7 (4.2%) presented some kind of anomaly of the urogenital system. Renal anomalies were found in two fetuses (1.2%). Unilateral renal agenesis was found in a 25 WPC fetus. Horseshoe kidney was found in a 20 WPC fetus. In a 23 WPC fetus (0.6%) the two testes were absent. Epididymal disjunction anomalies were found in four fetuses (2.4%). CONCLUSIONS: The urogenital anomalies in human male fetuses are rare and have an incidence around 4%.

Ultrasound-guided injection and occlusion of the trachea in fetal sheep.

OBJECTIVES: To access the fetal sheep trachea by ultrasound-guided transthoracic injection in order to deliver gene therapy vectors or occlude the trachea with a detachable balloon. METHODS: Fetal sheep were operated on at a mean gestational age of 102 (range, 81-116) days (term = 145 days). Under ultrasound guidance, either a 20-G spinal (for vector delivery) or a 16-G Kellett (for placement of an occlusive balloon) needle was inserted via the fetal thorax into the fetal trachea. RESULTS: Using the 20-G spinal needle the trachea was accessed successfully in 33/36 fetuses, with 97% survival. Failure to inject was related to fetal position and gestational age. Blood vessel damage causing significant morbidity occurred in two fetuses (6%). Tracheal occlusion was achieved by puncturing the trachea with the 16-G needle and advancing an endoluminal balloon in three out of five attempts in a mean time of 17 (range, 16-19) min, with 100% survival. In one case, the balloon became sited within the accessory lobe bronchus and was not inflated. At postmortem examination 21 days later, all balloons remained inflated and occluded the trachea, and the lung-to-body weight ratio and airways morphometric indices were consistent with relative pulmonary hyperplasia in the obstructed lungs. CONCLUSIONS: Ultrasound-guided transthoracic tracheal puncture is a reliable technique in fetal sheep, with low morbidity and mortality. Using this technique, a detachable endotracheal balloon can be placed to provoke pulmonary growth. Advances in needle design and balloon size may improve the success rate.

Permanent partial phenotypic correction and tolerance in a mouse model of hemophilia B by stem cell gene delivery of human factor IX.

Immune responses against an introduced transgenic protein are a potential risk in many gene replacement strategies to treat genetic disease. We have developed a gene delivery approach for hemophilia B based on lentiviral expression of human factor IX in purified hematopoietic stem cells. In both normal C57Bl/6J and hemophilic 129/Sv recipient mice, we observed the production of therapeutic levels of human factor IX, persisting for at least a year with tolerance to human factor IX antigen. Secondary and tertiary recipients also demonstrate long-term production of therapeutic levels of human factor IX and tolerance, even at very low levels of donor chimerism. Furthermore, in hemophilic mice, partial functional correction of treated mice and phenotypic rescue is achieved. These data show the potential of a stem cell approach to gene delivery to tolerize recipients to a secreted foreign transgenic protein and, with appropriate modification, may be of use in developing treatments for other genetic disorders.

Factors influencing adenovirus-mediated airway transduction in fetal mice.

Intra-amniotic injection of adenovirus allows transduction of the fetal airways following natural fetal breathing movements. This administration method is promising for use in gene therapy for cystic fibrosis and other diseases for which the main target for exogenous gene expression is the lung. Here we have investigated factors that may affect the efficacy of gene transfer to the murine fetal lung. We examined marker compound distribution and transgene expression (from a first-generation adenoviral vector) at different stages of development. This demonstrated that fetal breathing movements at 15-16 days of gestation are of sufficient intensity to carry marker/vector into the fetal lungs. These movements can be significantly stimulated by the combination of intra-amniotic theophylline administration and postoperative exposure of the dam to elevated CO(2) levels. However, the most important factor for efficient and consistent pulmonary transgene delivery is the dose of adenoviral vector used, as both the degree of transduction and the percentage of lungs transduced increases with escalating viral dose.

Gene therapy progress and prospects: fetal gene therapy--first proofs of concept--some adverse effects.

Somatic gene delivery in utero is a novel approach to gene therapy for genetic disease based on the hypothesis that prenatal intervention may avoid the development of severe manifestations of early-onset disease, allow targeting of otherwise inaccessible tissues including expanding stem cell populations, induce tolerance against the therapeutic transgenic protein and thereby provide permanent somatic gene correction. This approach is particularly relevant in relation to prenatal screening programmes for severe genetic diseases as it could offer prevention as a third option to families faced with the prenatal diagnosis of a genetically affected child. Most investigations towards in utero gene therapy have been performed on mice and sheep fetuses as model animals for human disease and for the application of clinically relevant intervention techniques such as vector delivery by minimally invasive ultrasound guidance. Other animals such as dogs may serve as particular disease models and primates have to be considered in immediate preparation for clinical trials. Proof of principle for the hypothesis of fetal gene therapy has been provided during the last 2 years in mouse models for Crigler Najjar Disease, Leber's congenital amaurosis, Pompe's disease and haemophilia B showing long-term postnatal therapeutic effects and tolerance of the transgenic protein after in utero gene delivery. However, recently we have also observed a high incidence of liver tumours after in utero application of an early form of third-generation equine infectious anaemia virus vectors with SIN configuration. These findings highlight the need for more investigations into the safety and the ethical aspects of in utero gene therapy as well as for science-based public information on risks and benefits of this preventive gene therapy approach before application in humans can be contemplated.

A novel human suspension culture packaging cell line for production of high-titre retroviral vectors.

Retroviruses are currently the most widely used vectors in clinical trials for gene therapy. These vectors are, however, limited by low titres partly due to the restrictive nature of monolayer cell culture. We have developed a stable suspension producer cell line derived from human lymphoblastoid cells (WIL-2) by electroporating these cells with the necessary trans components required for production of defective retrovirus particles which encode a nuclear localising beta-galactosidase gene. We show that this anchorage-independent cell line generates viruses at a titre of 7 x 10(5) iu/ml on NIH3T3 indicator cells which remains constant after at least 2 months in culture. The producer cells can be cultured at a density of 6 x 10(6) cells/ml with consistent virus titre production. WIL-2 can also be grown as single cells by rotation culture while maintaining virus production. By treating the cells with the transcriptional activator sodium butyrate titres above 1 x 10(6) i.u./ml are achieved. Concentrating viral supernatants by ultrafiltration can further increase virus titre to 5 x 10(8) i.u./ml. Even at these high titres no replication-competent virus was detected. Virus titre fell only slightly when cells were placed in serum-free media before harvest. The generation of this novel cell line provides proof-of-principle that large-scale production of retroviral vectors in serum-free growth conditions can be safely generated for use in gene therapy.

Mutational effects of retrovirus insertion on the genome of V79 cells by an attenuated retrovirus vector: implications for gene therapy.

Attenuated retroviruses are currently the most widely used vectors in clinical gene therapy because of their potential to effect stable and permanent gene transfer. Since gene delivery is accompanied by random insertion of foreign genetic material into the recipient chromosomal DNA, the potential for insertional mutagenesis exists. In this study, we used a defective retrovirus vector containing a selectable marker, the hygromycin phosphotransferase gene, to investigate the mutagenic effects of vector integration on the mammalian genome. V79 Chinese hamster cells were infected with virus supernatants or by coculture with virus producer cells, and provirus insertion events occurred at low and high frequencies, respectively. The frequency of hprt mutagenesis was increased by a factor of 2.3 over the spontaneous hprt mutation frequency only following multiple provirus insertions/cell genome. Multiple provirus insertions (>3/genome) resulted in instability at the hprt locus in 63% of the virally induced hprt mutants, as indicated by rearrangements at the molecular level, whereas no rearrangements were found when the provirus copy number was 1-2/genome. To demonstrate direct proviral involvement in mutagenesis, the defective MLV vector was retrieved along with flanking genomic hprt sequences from one mutant, and localized within intron 5 of the hprt gene. These data suggest that provirus copy number is a key factor when considering the potential hazards of using retrovirus vectors for gene therapy.

Fetal somatic gene therapy.

Fetal somatic gene therapy is emerging as a new experimental approach, in particular to prevent irreversible perinatal disease manifestation for many inherited conditions. Early therapeutic gene application may also allow targeting of still expanding stem cell populations of organ or cell systems inaccessible later in life and help to avoid immune sensitization against the therapeutic vector system or transgene protein product. The progress in development of ultrasound scanning and embryofetoscopy over the last decade has made minimally invasive administration of therapeutic gene transfer vectors to the fetus in utero possible in principle. We review here the different considerations in choosing candidate diseases, the possible routes of administration and times in fetal development for application of a therapeutic gene and discuss the benefits and problems of present vector systems in this context. Given the many unknown aspects of fetal gene transfer, it is essential to extensively investigate this new approach to gene therapy in animal models for specific diseases, to improve on the technology of delivery and to assess efficacy of expression as well as the possible side effects before application to humans can be considered.

Novel use of a selectable fusion gene as an "in-out" marker for studying genetic loss in mammalian cells.

Recent demonstrations of loss of heterozygosity in a wide variety of human cancers suggest that large multilocus genetic deletions (presumably including tumor suppressor genes) constitute a major class of genetic alteration in human carcinogenesis. Here we show that a bifunctional fusion gene (Hytk), suitable for both positive and negative selection, is an effective marker for studying genetic loss in mammalian cells with minimal interference from point-mutational changes. Studies with a transgenic V79 cell line in which a single functional copy of Hytk was stably inserted into the genome in a retroviral vector showed that loss of the marker (and presumably flanking cellular genetic material) could be induced efficiently by ionizing radiation (gamma-rays and fast neutrons) but only weakly by the powerful point-mutagen benzo[a]pyrene diol epoxide. In a first application of the system, we provide evidence that radiation-induced loss can occur through an indirect mechanism after a high-frequency event. Collectively, our results suggest that the Hytk marker should be a valuable tool for studying genome position effects on the tolerance of genetic loss in cultured human cells that represent different stages in clonal evolution and tumor progression.