Utility of barium studies for patients with recurrent weight gain after Roux-en-Y gastric bypass.

AIM: To determine the utility of barium studies for detecting abnormalities responsible for recurrent weight gain after gastric bypass surgery. METHODS: A computerized search identified 42 patients who had undergone barium studies for recurrent weight gain after gastric bypass and 42 controls. The images were reviewed to determine the frequency of staple-line breakdown and measure the length/width of the pouch and gastrojejunal anastomosis. A large pouch exceeded 6 cm in length or 5 cm in width and a wide anastomosis exceeded 2 cm. Records were reviewed for the amount of recurrent weight gain and subsequent weight loss after additional treatment. RESULTS: Staple-line breakdown was present in 6/42 patients (14%) with recurrent weight gain. When measurements were obtained, 13/35 patients (37%) with recurrent weight gain had a large pouch, three (9%) had a wide anastomosis, and four (11%) had both, whereas 22/42 controls (52%) had a large pouch, one (2%) had a wide anastomosis, and two (5%) had both. Ten patients (24%) with recurrent weight gain underwent staple-line repair (n = 3) or pouch/anastomosis revision (n = 7). These 10 patients had a mean weight loss of 38.1 lbs versus a mean loss of 8.6 lbs in 19 patients managed medically. CONCLUSION: Only 14% of patients with recurrent weight gain after gastric bypass had staple-line breakdown, whereas 57% had a large pouch, wide anastomosis, or both. Not all patients with abnormal anatomy had recurrent weight gain, but those who did were more likely to benefit from surgical intervention than from medical management.

Successful ex vivo gene therapy directed to liver in a patient with familial hypercholesterolaemia.

An ex vivo approach to gene therapy for familial hypercholesterolaemia (FH) has been developed in which the recipient is transplanted with autologous hepatocytes that are genetically corrected with recombinant retroviruses carrying the LDL receptor. We describe the treatment of a 29 year old woman with homozygous FH by ex vivo gene therapy directed to liver. She tolerated the procedures well and in situ hybridization of liver tissue four months after therapy revealed evidence for engraftment of transgene expressing cells. The patient's LDL/HDL ratio declined from 10-13 before gene therapy to 5-8 following gene therapy, improvements which have remained stable for the duration of the treatment (18 months). This represents the first report of human gene therapy in which stable correction of a therapeutic endpoint has been achieved.

Comparison of infrared thermography and laser speckle contrast imaging for the dynamic assessment of digital microvascular function.

OBJECTIVES: Laser speckle contrast imaging (LSCI) is a novel non-invasive microvascular imaging modality. The present study evaluates the validity and reliability of LSCI by comparison with infrared thermography (IRT) for the dynamic assessment of digital microvascular function in healthy volunteers. METHODS: Subjects attended on 3 occasions. Simultaneous assessment of cutaneous perfusion at 3 distinct regions of interest (ROI) within the hands was undertaken using LSCI and infrared thermography (IRT) at baseline, and at 13s intervals over 15 min following a standardised local cold challenge. Endpoints for evaluation included absolute measurements at baseline and following cold stress, in addition to the characteristics of the re-warming curves (maximum % recovery and maximum gradient). Visits 1 and 2 were undertaken in identical conditions (ambient temperature 23°C) to assess reproducibility, whereas visit 3 was undertaken at a lower ambient room temperature of 18°C to evaluate responsiveness to reduction in ambient room temperature. RESULTS: Fourteen healthy participants completed the study. There was greater variability in the data generated using LSCI compared with the highly damped IRT, reflecting greater sensitivity of LSCI to physiological variation and movement artefact. LSCI and IRT correlated well at baseline and following cold challenge for all endpoints (r(s) for pooled data between 0.5 and 0.65, p<0.00005). Reproducibility of both IRT and LSCI was excellent (ICCs>0.75) for absolute assessments but lower for re-warming curve characteristics. LSCI provides greater spatial resolution than IRT identifying variation in cutaneous perfusion within the hands most likely associated with the presence of arteriovenous anastamoses. Both techniques were responsive to reduction in ambient room temperature. Effect sizes were greatest for IRT than LSCI (e.g. -1.17 vs. -0.85 at ROI 1 at baseline) although this may represent heat transfer rather than altered vascular perfusion. DISCUSSION: In the dynamic assessment of digital vascular perfusion, LSCI correlates well with IRT, is reproducible and responsive to reduction in ambient room temperature. Absolute measurements appear preferable to parameters derived from re-warming curve characteristics when assessing digital perfusion following cold challenge. The greater temporal and spatial resolution of LSCI compared with IRT may facilitate the development of novel assessment tools of autonomic function and digital cutaneous perfusion.

Recombinant adeno-associated virus for muscle directed gene therapy.

Although gene transfer with adeno-associated virus (AAV) vectors has typically been low, transduction can be enhanced in the presence of adenovirus gene products through the formation of double stranded, non-integrated AAV genomes. We describe the unexpected finding of high level and stable transgene expression in mice following intramuscular injection of purified recombinant AAV (rAAV). The rAAV genome is efficiently incorporated into nuclei of differentiated muscle fibers where it persists as head-to-tail concatamers. Fluorescent in situ hybridization of muscle tissue suggests single integration sites. Neutralizing antibody against AAV capsid proteins does not prevent readministration of vector. Remarkably, no humoral or cellular immune responses are elicited to the neoantigenic transgene product E. coli beta-galactosidase. The favorable biology of rAAV in muscle-directed gene therapy described in this study expands the potential of this vector for the treatment of inherited and acquired diseases.

A pilot study of ex vivo gene therapy for homozygous familial hypercholesterolaemia.

The outcome of the first pilot study of liver-directed gene therapy is reported here. Five patients with homozygous familial hypercholesterolaemia (FH) ranging in age from 7 to 41 years were enrolled; each patient tolerated the procedure well without significant complications. Transgene expression was detected in a limited number of hepatocytes of liver tissue harvested four months after gene transfer from all five patients. Significant and prolonged reductions in low density lipoprotein (LDL) cholesterol were demonstrated in three of five patients; in vivo LDL catabolism was increased 53% following gene therapy in a receptor negative patient, who realized a reduction in serum LDL equal to approximately 150 mg dl-1. This study demonstrates the feasibility of engrafting limited numbers of retrovirus-transduced hepatocytes without morbidity and achieving persistent gene expression lasting at least four months after gene therapy. The variable metabolic responses observed following low-level genetic reconstitution in the five patients studied precludes a broader application of liver-directed gene therapy without modifications that consistently effect substantially greater gene transfer.

Temperature increase of 21st century mitigation scenarios.

Estimates of 21st Century global-mean surface temperature increase have generally been based on scenarios that do not include climate policies. Newly developed multigas mitigation scenarios, based on a wide range of modeling approaches and socioeconomic assumptions, now allow the assessment of possible impacts of climate policies on projected warming ranges. This article assesses the atmospheric CO(2) concentrations, radiative forcing, and temperature increase for these new scenarios using two reduced-complexity climate models. These scenarios result in temperature increase of 0.5-4.4 degrees C over 1990 levels or 0.3-3.4 degrees C less than the no-policy cases. The range results from differences in the assumed stringency of climate policy and uncertainty in our understanding of the climate system. Notably, an average minimum warming of approximately 1.4 degrees C (with a full range of 0.5-2.8 degrees C) remains for even the most stringent stabilization scenarios analyzed here. This value is substantially above previously estimated committed warming based on climate system inertia alone. The results show that, although ambitious mitigation efforts can significantly reduce global warming, adaptation measures will be needed in addition to mitigation to reduce the impact of the residual warming.

Transport of epidermal growth factor by rat liver: evidence for a nonlysosomal pathway.

Epidermal growth factor (EGF), circulating in the blood, is taken up by rat liver hepatocytes by means of specific and saturable receptor-mediated endocytosis. These experiments were undertaken to determine (a) the transport pathway(s) of EGF taken up by rat liver and (b) the effects of lysosomal inhibition on its transport. 125I-EGF was injected into rat portal veins, and bile samples were collected and analyzed for both total and immunoprecipitable radioactivity. In addition, the livers were examined by electron microscopic autoradiography. Some animals received injections of chloroquine before surgery, to disrupt lysosomal function. The results indicate that most of the EGF taken up by the hepatocytes is transported to lysosomes and degraded. However, a small but significant percentage of endocytosed EGF is transported by a pathway independent of the lysosomal system, resulting in secretion of intact EGF: (a) Both degraded and immunoprecipitable EGF are secreted into bile. (b) Immunoprecipitable radioactivity peaks at 20 min after EGF injection, whereas degradation-associated radioactivity does not peak until 40 min postinjection. (c) EGF isolated from bile is specifically taken up by isolated hepatocytes in monolayer culture, indicating that it is still recognizable by the EGF receptor. (d) When the lysosomal system is inhibited with chloroquine, secretion of degraded EGF is significantly inhibited, whereas the amount of intact EGF secreted into bile is unchanged. The utilization by liver of a dual transport process for EGF represents an unusual system of intracellular ligand processing, whose physiological significance has yet to be determined.

Interpretation of high projections for global-mean warming.

The Intergovernmental Panel on Climate Change (IPCC) has recently released its Third Assessment Report (TAR), in which new projections are given for global-mean warming in the absence of policies to limit climate change. The full warming range over 1990 to 2100, 1.4 degrees to 5.8 degrees C, is substantially higher than the range given previously in the IPCC Second Assessment Report. Here we interpret the new warming range in probabilistic terms, accounting for uncertainties in emissions, the climate sensitivity, the carbon cycle, ocean mixing, and aerosol forcing. We show that the probabilities of warming values at both the high and low ends of the TAR range are very low. In the absence of climate-mitigation policies, the 90% probability interval for 1990 to 2100 warming is 1.7 degrees to 4.9 degrees C.

Relative activities of thymidylate synthetase and thymidine kinase in rat tissues.

The activities of thymidylate synthetase and thymidine kinase were compared in tissues of normal (adult and developing), cortisol-injected, and tumor-bearing rats. The purpose of the study was to determine whether the activities of these two enzymes, which catalyze reactions leading to the same metabolic intermediate, changed proportionately, reciprocally, or independently under different physiological conditions. Both enzymes had high activities in fetal tissues. Thymidine kinase concentrations decreased shortly before or immediately after birth; in several tissues, transient postnatal peaks in thymidine kinase activities appeared within the first 3 weeks after birth. Thymidylate synthetase activities declined gradually after parturition and showed no significant postnatal rises. In sucklings given injections of cortisol, thymidine kinase activities were reduced substantially in eight tissues while thymidlyate synthetase decreased only in lung and thymus of 11-day-old rats. In tumor-bearing rats, thymidine kinase activity increased dramatically in spleen, whereas thymidylate synthetase activities only doubled. In host liver, rises in thymidine kinase activities were not always matched by increases in thymidlyate synthetase. In the tumors, both activities were higher than in most normal adult tissues. Despite the differential sensitivities of the two enzymes to cortisol and tumor bearing, thymidylate synthetase and thymidine kinase were closely correlated in tissues of untreated animals. The Spearman rank correlation coefficient for 112 tissues was 0.895, while the correlation coefficient between the standard scores of the activities was 0.839. The activities of the two enzymes did not appear to be reciprocal or compensatory during normal differentiation or during dedifferentiation associated with tumor bearing, but their potentials for activity were independent of each other.

Plasminogen activator inhibitor-1 is an immediate early response gene in regenerating rat liver.

Plasminogen activator inhibitor-1 (PAI-1), a M(r) 50,000 serine protease inhibitor, is the major physiological inhibitor of plasminogen activation. Quiescent rat hepatocytes do not express the PAI-1 gene in vivo; however, PAI-1 is synthesized both by primary cultures of rat hepatocytes and by hepatoma cells in vitro. Furthermore, PAI-1 is expressed by fibroblastic cells in vitro, in response to mitogen stimulation, suggesting a possible connection between hepatocyte PAI-1 expression and cell proliferation. To determine whether PAI-1 is an early growth response gene in hepatocytes in vivo, we analyzed its expression in regenerating rat liver. Male rats underwent partial (70%) hepatectomy (PH) or sham operation (SO), and liver samples were analyzed by Northern blot analysis and in situ hybridization. PAI-1 mRNA was not present at time 0 h, nor at any other time in SO rats but was induced rapidly in regenerating livers, peaking at 2 h and declining to negligible levels by 8 h posthepatectomy. This induction was not inhibited by cycloheximide. In situ hybridization analysis localized PAI-1 transcripts to hepatocytes. Immunohistochemical analysis demonstrated PAI-1-specific staining in hepatocytes in the livers of both PH and SO rats, but the temporal and spatial distribution profiles differed between PH and SO rats. Our studies demonstrate that PAI-1 is an immediate early response gene, transiently expressed in regenerating liver, expression of which may be important in hepatocyte growth and proliferation in vivo.

An animal model for colon cancer metastasis: establishment and characterization of murine cell lines with enhanced liver-metastasizing ability.

A detailed understanding of the pathogenesis of colon cancer metastasis has been hindered by the lack of appropriate animal models which accurately reflect events in this complex process. An animal model for colon cancer metastasis is described in which spontaneously metastasizing colonic tumors are formed after injection of murine colon cancer cells into the cecal wall of BALB/c mice. Using this model, tumor cells with different liver-metastasizing potential were selected and shown to possess several properties known to be associated with other metastatic cell lines. The ability of tumor cells to invade a reconstituted basement membrane and to secrete type IV collagenase was directly proportional to their metastatic ability. In addition, liver-metastasizing cells preferentially migrated toward liver extracts in a Boyden chamber assay, as compared to extracts of brain or lung, and adhered rapidly to highly purified hepatic sinusoidal endothelial cells versus hepatic parenchymal cells in vitro. This model may thus be useful for studying many aspects of the pathogenesis of colon cancer metastasis.

Enzymes of ornithine metabolism in adult and developing rat intestine.

The levels of 11 enzymes, most of them involved in the metabolism of ornithine, were measured in whole upper intestine, or in duodenum, small intestine and colon of adult rats. The developmental formations in small intestine of arginase, ornithine aminotransferase, and ornithine transcarbamylase were compared with those in liver. Changes with age (late gestation of adult) of the intestinal activities of pyrroline-5-carboxylate reductase, proline oxidase and glutamyl transpeptidase are also described. The results suggest that the proximal part of the intestine is well endowed with enzymes involved in the conversion of ornithine to proline as well as to citrulline. Fetal intestine is rich in proline oxidase and pyrroline-5-carboxylate reductase. The peak levels of ornithine aminotransferase found in intestine in the first 3 postnatal weeks were higher than seen in any other rat tissue. Some of the properties of arginase, ornithine aminotransferase and pyrroline-5-carboxylate reductase in small intestine were compared with those in liver. Isozymes of arginase in small intestine differed from those in liver; the kinetic properties of ornithine aminotransferase were similar in the two tissues. In intestine of 14-day-old rats, the ornithine aminotransferase reaction was reversible, forming ornithine from pyrroline-5-carboxylate. The intestinal pyrroline-5-carboxylate reductase was cold-labile as was the hepatic enzyme in rat.

Transplantation of genetically modified autologous hepatocytes into nonhuman primates: feasibility and short-term toxicity.

Ex vivo gene therapy directed to the liver is being developed for the treatment of inherited metabolic diseases. Transplantation of hepatocytes that have been transduced with a low-density lipoprotein (LDL) receptor gene is a potential form of therapy for familial hypercholesterolemia (FH). We have demonstrated efficacy of ex vivo gene therapy for familial hypercholesterolemia in a rabbit animal model of this disease. In preparation for human trials, we describe in this report experiments in baboons for documentation of the feasibility and safety of autologous hepatocyte transplantation. Three baboons underwent a partial hepatectomy and their hepatocytes were isolated, cultured, and transduced with a retrovirus containing the human LDL receptor gene. The hepatocytes were harvested and infused into an indwelling catheter that had been placed into the inferior mesenteric vein at the time of liver resection. The baboons tolerated the procedures well and are being maintained and clinically evaluated for an indefinite time period. Follow-up evaluations have ranged from 3 to 8 months. Clinical evaluations have been unremarkable and blood chemistry and hematology determinations have stayed within normal limits.

Characterization of the immune response after local delivery of recombinant adenovirus in murine pancreas and successful strategies for readministration.

The pancreas is an ideal organ for adenoviral gene therapy because of the high level of gene transfer that can be achieved and because of the many diseases that can potentially be treated using this technology. In this report, we characterize the immune response to direct pancreatic injection of adenovirus and we overcome some of the limitations it imposes by using immunosuppression. Direct injection of recombinant adenovirus into the pancreas leads to the production of neutralizing antibodies and to sensitized splenocytes which engage in increased cytotoxic, lymphoproliferative, and cytokine release activity when reexposed to adenovirus. Transgene expression is transient and the vector cannot be readministered. Deletion of CD4+ T helper cells improves expression over time (40% of pancreatic cells express transgene at day 28 vs. 5% in controls), and allows the vector to be readministered in the pancreas, albeit, inefficiently, when compared to naive animals. Similarly, blockade of CD40 ligand, which preserves the CD4+ T helper cell population, also improves expression over time (30% of pancreatic cells express transgene at day 28), and allows the vector to be readministered. With both approaches, neutralizing antibodies are decreased and the remaining splenocytes do not engage in activated immune responses. Thus, local delivery of the adenoviral vector induces a systemic response that prevents pancreatic readministration, even with direct injection. Blockade of CD40 ligand and T helper cell depletion are transient regimens that induce systemic immunosuppression. Until the development of newer strategies that selectively suppress adenoviral immune responses, these are viable alternatives for enhancement of pancreatic adenoviral delivery.

Functional consequences of adenovirus-mediated murine pancreatic gene transfer.

Pancreatic adenoviral gene transfer can be achieved with high efficiency; however, questions concerning tissue injury from this commonly used vector have not been addressed. In these experiments, the effects of adenoviral gene transfer on pancreatic exocrine function were evaluated. Direct pancreatic injection with an adenoviral vector containing the Escherichia coli beta-galactosidase (beta-Gal; lacZ) transgene (H5.010CBlacZ) resulted in a high level of transgene expression (64 +/- 6% of pancreatic cells expressed beta-Gal) at 3 days following infection. However, amylase levels in four of five different subcellular pancreatic fractions were significantly decreased at this time point. Direct pancreatic injection with either saline or psoralen/UV-inactivated adenovirus did not have this effect, whereas both transduction with an adenoviral vector containing a different transgene and transduction with a homologous transgene resulted in decreased pancreatic amylase. The decrease in subcellular amylase levels persisted at 7 days post-transduction, and then returned to baseline at 21 days post-transduction. There was associated histologic damage (increased edema, inflammation, cell destruction, and vacuolization) at 3 and 7 days post-transduction, which resolved by 21 days. In summary, adenoviral transduction of the pancreas results in increased viral transgene expression and a uniform decrease in host amylase production throughout the pancreas. The normalization of amylase levels and histology suggest that organ recovery occurs. Gene transfer technology as a novel strategy for pancreatic diseases such as diabetes, pancreatitis, and cystic fibrosis is feasible but will benefit from continued approaches to limit toxicity.

Evaluating the potential of germ line transmission after intravenous administration of recombinant adenovirus in the C3H mouse.

The goal of this study is to assess the likelihood that an adenoviral vector disseminated to gonads will be transmitted to offspring. This study is based on the observation that systemically administered vector can be detected in both ovaries and testes, using sensitive nested PCR techniques. Although the extent of vector dissemination to gonads is extremely small, as it is detectable only by nested PCR, it is unclear where it is located within these tissues and whether the DNA is capable of integration and transmission to offspring. A protocol was developed in C3H mice to address this question. Both male and female C3H mice were injected with a high dose of H5.001CBhOTC, an E1- and E4-deleted vector expressing human ornithine transcarbamylase. This dose of vector was sufficient to target 80% of hepatocytes (Gao et al., J. Virol. 1996; 70:8934-8943) and disseminate, at low levels, to both ovaries and testes in 94% of animals as determined by PCR. Vector-administered animals and controls were mated and 814 offspring were evaluated for germ line transmission of the adenoviral vector by DNA hybridization of total cellular DNA extracted from the fetus. Southern blot analysis showed no evidence of germ line transmission in 578 offspring of crosses in which either one or both parents received recombinant adenovirus.

Gene transfer into the liver of nonhuman primates with E1-deleted recombinant adenoviral vectors: safety of readministration.

Preclinical studies were designed to investigate the safety of recombinant adenoviruses infused into the portal vein of adult rhesus monkeys, as well as the safety and efficacy of readministration of these agents. The vectors used were recombinant adenoviruses, the E1 region of which was replaced with a marker gene expression cassette. Four 3- to 5-kg rhesus monkeys underwent portal vein cannulation, and infusion of escalating doses of recombinant first-generation vector. Serial sequential liver biopsies were performed, and necropsies were performed out to 14 months. X-Gal histochemical analysis of the liver showed evidence of dose-dependent increased gene transfer throughout the liver. Quantitative analysis of histopathology showed that portal inflammation was also present in transduced livers, and occurred in a dose-dependent manner. Severe toxicity, including mortality, was noted at the highest dose of vector. Readministration of a second vector was associated with the same degree of toxicity as the first vector, but prompted a much more vigorous neutralizing antibody response. The data suggest that intraportal administration and readministration of recombinant adenoviral E1-deleted vectors are feasible and safe. Vector administration at the highest dose (1 x 10(13) particles/kg) was associated with severe clinical and biochemical toxicity, and significant gene expression was associated with transaminitis. Readministration of vector is safe, but gene transfer is limited by the presence of neutralizing antibody.

Intracranial administration of adenovirus expressing HSV-TK in combination with ganciclovir produces a dose-dependent, self-limiting inflammatory response.

Replication-defective adenovirus expressing the herpes simplex thymidine kinase gene (H5.010RSVtk) may be useful in treating human gliomas. To determine the toxicity of this therapeutic strategy, we injected H5.010RSVtk stereotactically into the normal brain of Wistar rats, cotton rats, and rhesus monkeys in conjunction with systemic ganciclovir (GCV) at 10 mg/kg per day. In the Wistar rat, 5.7 x 10(9) pfu resulted in histopathologic injury consisting of localized necrosis, mild gliosis, marked malacia, and focal astrocytosis; however, 1.0 x 10(8) pfu resulted in only mild gliosis and trace meningitis and approximates a "no toxic effect" dose. A dose of 1.0 x 10(9) pfu in both adenoviral immune and adenoviral naive cotton rats resulted in similar findings. In the rhesus monkey, doses ranging from 1.4 x 10(8) pfu to 1.5 x 10(11) pfu resulted in localized gliosis, necrosis, perivascular cuffing, meningitis, and roughly correlated in severity with increasing dose. No histologic evidence of toxicity was found in non-central nervous system (CNS) tissues, and no virus could be cultured from cerebrospinal fluid (CSF), blood, urine, and stool samples. All animals survived to prescribed end points without signs of general toxicity or neurologic symptoms, except for 2 of the rhesus monkeys, one of which became febrile and the other of which developed a grand mal seizure (both subsequently resolved). These toxicology studies define the parameters for developing a phase I clinical trial.

Ex vivo gene therapy of familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is an autosomal dominant disorder caused by a deficiency in the receptor that clears low density lipoprotein (LDL) from the serum (reviewed in Ref. 1 and 2). Patients with one abnormal LDL receptor allele have moderate elevations in plasma LDL and suffer premature coronary artery disease (CAD). Approximately 5% of all patients under 45 who have had a myocardial infarction carry this trait. Patients with two abnormal LDL receptor genes (homozygous deficient patients) have severe hypercholesterolemia and life-threatening coronary artery disease in childhood. Strategies for treating patients with FH are directed at lowering the plasma level of LDL. In heterozygotes, this is accomplished through the administration of drugs that stimulate the expression of LDL receptor from the normal allele (2). This therapeutic approach is not effective in the treatment of homozygous deficient patients, especially those that retain less than 2% of residual LDL receptor activity. Partial amelioration of hyperlipidemia has been achieved in some homozygous deficient patients by diverting the portal circulation through a portacaval anastomosis (3) and by chronic plasmapheresis therapy (4). A more direct approach has been to correct the deficiency of hepatic LDL receptor by transplanting a liver that expresses normal levels of LDL receptor. Three patients that survived this procedure normalized their serum LDL-cholesterol (5-9). We have used an authentic animal model for FH, the Watanabe Heritable Hyperlipidemic rabbit (WHHL), to develop gene therapies for the homozygous form of FH (10-13). The WHHL rabbit has a mutation in its LDL receptor gene which renders the receptor completely dysfunctional (12) leading to severe hypercholesterolemia, diffuse atherosclerosis, and premature death. The potential efficacy of gene therapy for FH is supported by a series of studies we have performed in the WHHL rabbit in which we have achieved metabolic improvement (14-18). Liver tissue was removed from WHHL rabbits and used to isolate hepatocytes and establish primary cultures. A functional rabbit LDL receptor gene was transduced into a high proportion of hepatocytes using recombinant retroviruses, and the genetically corrected cells were transplanted into the animal from which they were derived. Transplantation of the genetically corrected, autologous hepatocytes was associated with a 30-40% decrease in serum cholesterol that persisted for the duration of the experiment (4 months, Ref. 18). Recombinant derived LDL receptor RNA was detected in liver for at least 6 months. There was no apparent immunological response to the recombinant derived LDL receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Selective gene transfer into the liver of non-human primates with E1-deleted, E2A-defective, or E1-E4 deleted recombinant adenoviruses.

Preclinical studies were designed to investigate the feasibility and safety of recombinant adenoviruses transduced into the hepatic artery of nonhuman primates. The vectors used are recombinant adenoviruses deleted in E1 and contain either a temperature-sensitive mutation in the E2a gene, which encodes a defective DNA-binding protein at nonpermissive temperatures, or a deletion of the E4 region, including open reading frame (ORF) 6. Six 8- to 10-kg baboons underwent femoral artery cannulation, and angiographic techniques were used to introduce vector selectively into either a portion of the right lobe of the liver via a branch of the right hepatic artery or the common hepatic artery. Necropsies were performed at 4, 29, or 61 days. Serial sequential liver biopsies were performed in the baboons that survived 29 or 61 days. In the 2 baboons with vector transduction into the right hepatic artery, X-Gal histochemical analysis of the liver showed evidence of quantitatively increased gene transfer in the targeted lobe; however, gene transfer was present throughout the liver. Quantitative analysis of histopathology showed that portal inflammation was present throughout both livers transduced with the highest dose of vector. No differences were seen in the level of portal inflammation in targeted and untargeted lobes despite the observed qualitative and quantitative differences in gene expression. Southern blot analysis of total cellular DNA isolated from targeted and nontargeted lobes showed similar levels of viral DNA throughout the liver. Polymerase chain reaction (PCR) analysis was able to detect viral DNA sequence in gonads and brain as well as many other tissues in baboons treated with high-dose vector. In baboons treated with lower doses of an E1-E4 deleted vector expressing the human ornithine transcarbamylase (OTC) gene, DNA was detectable by nested PCR in liver but not gonads at days 29 and 61. The data suggest that intraarterial administration of recombinant adenoviral E1-E4 deleted vector is feasible and safe. At high doses of vector, widespread dissemination of vector DNA is seen. At low doses, hepatic gene transfer is not associated with vector DNA dissemination to gonads.