Primate Liver Research Articles

Similarity of Bisphenol A Pharmacokinetics in Rhesus Monkeys and Mice: Relevance for Human Exposure

ObjectiveDaily adult human exposure to bisphenol A (BPA) has been estimated at < 1 μg/kg, with virtually complete first-pass conjugation in the liver in primates but not in mice. We measured unconjugated and conjugated BPA levels in serum from adult female rhesus monkeys and adult female mice after oral administration of BPA and compared findings in mice and monkeys with prior published data in women.MethodsEleven adult female rhesus macaques were fed 400 μg/kg deuterated BPA (dBPA) daily for 7 days. Levels of serum dBPA were analyzed by isotope-dilution liquid chromatography–mass spectrometry (0.2 ng/mL limit of quantitation) over 24 hr on day 1 and on day 7. The same dose of BPA was fed to adult female CD-1 mice; other female mice were administered 3H-BPA at doses ranging from 2 to 100,000 μg/kg.ResultsIn monkeys, the maximum unconjugated serum dBPA concentration of 4 ng/mL was reached 1 hr after feeding and declined to low levels by 24 hr, with no significant bioaccumulation after seven daily doses. Mice and monkeys cleared unconjugated serum BPA at virtually identical rates. We observed a linear (proportional) relationship between administered dose and serum BPA in mice.ConclusionsBPA pharmacokinetics in women, female monkeys, and mice is very similar. By comparison with approximately 2 ng/mL unconjugated serum BPA reported in multiple human studies, the average 24-hr unconjugated serum BPA concentration of 0.5 ng/mL in both monkeys and mice after a 400 μg/kg oral dose suggests that total daily human exposure is via multiple routes and is much higher than previously assumed.

Progressive mitochondrial compromise in brains and livers of primates exposed in utero to nucleoside reverse transcriptase inhibitors (NRTIs).

Mitochondrial compromise has been documented in infants born to women infected with the human immunodeficiency virus (HIV-1) who received nucleoside reverse transcriptase inhibitor (NRTI) therapy during pregnancy. To model these human exposures, we examined mitochondrial integrity at birth and 1 year in brain cortex and liver from offspring of retroviral-free Erythrocebus patas dams-administered human-equivalent NRTI doses for the last half (10 weeks) of gestation. Additional infants, followed for 1 year, were given the same drugs as their mothers for the first 6 weeks of life. Exposures included: no drug, Zidovudine (AZT), Lamivudine (3TC), AZT/3TC, AZT/Didanosine (ddI), and Stavudine (d4T)/3TC. In brain and liver, oxidative phosphorylation (OXPHOS) enzyme activities (complexes I, II, and IV) showed minimal differences between unexposed and NRTI-exposed offspring at both times. Brain and liver mitochondria from most NRTI-exposed patas, both at birth and 1 year of age, contained significant (p < 0.05) morphological damage observed by electron microscopy (EM), based on scoring of coded photomicrographs. Brain and liver mitochondrial DNA (mtDNA) levels in NRTI-exposed patas were depleted significantly in the 3TC and d4T/3TC groups at birth and were depleted significantly (p < 0.05) at 1 year in all NRTI-exposed groups. In 1-year-old infants exposed in utero to NRTIs, mtDNA depletion was 28.8-51.8% in brain and 37.4-56.5% in liver. These investigations suggest that some NRTI-exposed human infants may sustain similar mitochondrial compromise in brain and liver and should be followed long term for cognitive integrity and liver function.

Therapeutic Liver Reconstitution With Murine Cells Isolated Long After Death

Due to the shortage of donor organs, many patients needing liver transplantation cannot receive one. For some liver diseases, hepatocyte transplantation could be a viable alternative, but donor cells currently are procured from the same sources as whole organs, and thus the supply is severely limited. Here, we investigated the possibility of isolating viable hepatocytes for liver cell therapy from the plentiful source of morgue cadavers. To determine the utility of this approach, cells were isolated from the livers of non-heart-beating cadaveric mice long after death and transplanted into fumarylacetoacetate hydrolase-deficient mice, a model for the human metabolic liver disease hereditary tyrosinemia type I and a stringent in vivo model for hepatic cell transplantation. Surprisingly, complete and therapeutic liver repopulation could be achieved with hepatocytes derived up to 27 hours post mortem. Competitive repopulation experiments showed that cadaveric liver cells had a repopulation capacity similar to freshly isolated hepatocytes. Importantly, viable hepatocytes also could be isolated from cadaveric primate liver (monkey and human) efficiently. These data provide evidence that non-heart-beating donors could be a suitable source of hepatocytes for much longer time periods than previously thought possible.

Hepatic AAV Gene Transfer and the Immune System: Friends or Foes?

Two ongoing clinical trials utilize different adeno-associated viral (AAV) vectors for liver-directed factor IX (F.IX) gene transfer with the goal of sustained therapy in patients with severe hemophilia B. Although preclinical studies have documented immune tolerance and long-term expression of F.IX in animals, the single prior clinical trial of this approach achieved only transient therapeutic gene expression and exposed preexisting immunity to the AAV vector as a major obstacle for therapy.1 Although accumulating preclinical data continue to fuel a debate over the potential impact of immune responses on hepatic AAV gene transfer, these data do not allow us to predict whether the human immune system will reject or tolerate therapy. This commentary dissects the relevant human and animal data, some of which are contradictory or allow us to draw only limited conclusions. More clinical trial data are critically needed and should ultimately help us develop better protocols.

365. The Rous Sarcoma Virus Promoter Carried by a rAAV Vector Is Resistant to DNA Methylation in Primate Muscle and Liver

365. The Rous Sarcoma Virus Promoter Carried by a rAAV Vector Is Resistant to DNA Methylation in Primate Muscle and Liver

Correlation of antinuclear antibody immunofluorescence patterns with immune profile using line immunoassay in the Indian scenario

Immunity status, individual response to disease and types of antibodies produced are well known to vary from person to person, place to place and probably from population to population. A broad spectrum of specific auto antibodies that have so far been associated with specific rheumatic diseases, as noted in Western literature, has been well taken as a reference standard all over the world. There is neither research work nor any data correlating the auto antibodies and their antinuclear antibody (ANA) patterns with the immunoprofile in the Indian population to date. To understand a definite association between ANA patterns and specific antibodies in the serum in the Indian study population and to document similarities / differences with the West. This prospective and retrospective double blind study was undertaken on the South Indian population referred for ANA testing by Indirect Immunofluorescence method and by immunoline methods. Serum samples of patients from a random South Indian population who sought medical help for rheumatic disease were subjected for ANA testing by indirect immunofluorescence (IIF) method and line immunoassay during the study period of 27 months. Serum samples were processed in dilution of 1:100 using HEp - 2010 / liver biochip (Monkey) (EUROIMMUN AG). The serum samples which were further processed for line immunoassay were treated in 1:100 dilution on nylon strips coated with recombinant and purified antigens as discrete lines with plastic backing (EUROIMMUN AG) coated with antigens nRNP / Sm, Sm, SSA, Ro-52, SSB, Scl-70, PM-Scl, PCNA, Jo-1, CENP-B, dsDNA, nucleosomes, histones, ribosomal protein-P, anti-mitochondrial antibodies (AMA-M2) along with a control band. The analysis was done by comparing the intensity of the reaction with positive control line by image analysis. The antinuclear antibody indirect immunofluorescence (ANA - IIF) patterns obtained were projectable to visualize a certain spectrum of specific antibodies such as homogenous (45.5%) with dsDNA, nucleosomes, histones, SSA / Ro-52, RIB and RNP / Sm, speckled pattern (35.6%) with Sm, RNP, SSA/Ro-52, SSB, Sm and RIB; nucleolar pattern with Scl-70, Sm, RNP and centromere pattern with CENP-B. The methodology indicated that, cytoplasmic pattern noted in ANA also needs to be correlated with primate liver in a biochip, which should prompt further decision for a request for line immunoassay and it is preferable for two pathologists to report independently and sign out a consensus ANA report for better predictive value. As a definite correlation between the ANA patterns and the group of antibodies was detected by line immunoassay, one could predict presence of certain specific auto antibodies for a particular ANA pattern identified. This may restrict one from requesting for line immunoassay, which is expensive and economizes on the cost of laboratory investigations in a developing country like India. Thus, screening of sera by ANA-IIF method alone may suffice and probably reduce the expense of detailed immunological work-up with minimal loss in diagnostic accuracy. This study, the first of its kind in India, provides database and reference for the Indian subpopulation.

Adeno-Associated Virus-Mediated Gene Transfer to Nonhuman Primate Liver Can Elicit Destructive Transgene-Specific T Cell Responses

Gene transfer to murine liver with vectors based on novel adeno-associated virus (AAV) serotypes is efficient, stable, and safe even in the setting of antigenic transgene products. We undertook a study in cynomolgus macaques to evaluate the relevance of these findings to primates. The vectors were based on AAV serotype 7 and expressed green fluorescence protein (GFP) from the cytomegalovirus enhanced beta-actin promoter in both single-stranded and self-complementary genomes. Transduction efficiencies from the single-stranded vectors were similar to those observed in mice, although there was no advantage in primates with the self-complementary vectors. Primates elicited vibrant cytotoxic T cell responses to GFP that correlated with hepatitis and loss of transgene expression. There was no evidence of T cell activation in response to the AAV capsid. These studies indicate that under some conditions primates may activate more robust T cell responses to transgene products than is observed in mice.

889. Systemic Gene Therapy of Lysosomal Storage Disorders: Efforts To Improve AAV8-Mediated Expression from Non-Human Primate Liver

889. Systemic Gene Therapy of Lysosomal Storage Disorders: Efforts To Improve AAV8-Mediated Expression from Non-Human Primate Liver

Gene expression in primate liver during viral hemorrhagic fever

BackgroundRhesus macaques infected with lymphocytic choriomeningitis virus (LCMV) provide a model for human Lassa fever. Disease begins with flu-like symptoms and progresses rapidly with fatal consequences. Previously, we profiled the blood transcriptome of LCMV-infected monkeys (M. Djavani et al J. Virol. 2007) showing distinct pre-viremic and viremic stages that discriminated virulent from benign infections. In the present study, changes in liver gene expression from macaques infected with virulent LCMV-WE were compared to gene expression in uninfected monkeys as well as to monkeys that were infected but not diseased.ResultsBased on a functional pathway analysis of differentially expressed genes, virulent LCMV-WE had a broader effect on liver cell function than did infection with non-virulent LCMV-Armstrong. During the first few days after infection, LCMV altered expression of genes associated with energy production, including fatty acid and glucose metabolism. The transcriptome profile resembled that of an organism in starvation: mRNA for acetyl-CoA carboxylase, a key enzyme of fatty acid synthesis was reduced while genes for enzymes in gluconeogenesis were up-regulated. Expression was also altered for genes associated with complement and coagulation cascades, and with signaling pathways involving STAT1 and TGF-β.ConclusionMost of the 4500 differentially expressed transcripts represented a general response to both virulent and mild infections. However, approximately 250 of these transcripts had significantly different expression in virulent infections as compared to mild infections, with approximately 30 of these being differentially regulated during the pre-viremic stage of infection. The genes that are expressed early and differently in mild and virulent disease are potential biomarkers for prognosis and triage of acute viral disease.

First description of the surgical anatomy of the cynomolgus monkey liver

No detailed description of nonhuman primate liver anatomy has been reported and little is known about the similarity between such livers and human liver. The cynomolgus monkey (Macaca fascicularis) was used to establish a preclinical model of genetically modified hepatocytes auto transplantation. Here, we report information gleaned from careful observation and notes obtained from 59 female cynomolgus monkeys undergoing 44 anatomical hepatic resections, 12 main portal vein division dissections and selective branch ligations, and 46 portographies. Additionally, three anatomical liver dissections after total resection at autopsy were performed and served to confirm peroperative observations and for photography to provide illustrations. Our results indicate that the cynomolgus monkey liver has four lobes: the median (the largest), the right and left lateral, and the caudate lobes. In 60% (N=20) of individuals the portal bifurcates into right and left portal veins, in the remaining 40% (N=14) the portal vein trifurcates into right anterior, right posterior, and left portal veins. The anatomy and branching pattern of the hepatic artery and bile ducts closely follow those of the portal branches. Functionally, the cynomolgus monkey liver can be divided into eight independent segments. Thus, we report the first detailed description of the hepatic and portal surgical anatomy of the cynomolgus monkey. The cynomolgus monkey liver is more similar to the human liver than are livers of any small or large nonprimate mammals that have been described.

Efficient, Long-term Hepatic Gene Transfer Using Clinically Relevant HDAd Doses by Balloon Occlusion Catheter Delivery in Nonhuman Primates

Helper-dependent adenoviral vectors (HDAd) are devoid of all viral coding sequences and are thus an improvement over early generation Ad because they can provide long-term transgene expression in vivo without chronic toxicity. However, high vector doses are required to achieve efficient hepatic transduction by systemic intravenous injection, and this unfortunately results in dose-dependent acute toxicity. To overcome this important obstacle, we have developed a minimally invasive method to preferentially deliver HDAd into the liver of nonhuman primates. Briefly, a balloon occlusion catheter was percutaneously positioned in the inferior vena cava to occlude hepatic venous outflow. HDAd was injected directly into the occluded liver via a percutaneously placed hepatic artery catheter. Compared to systemic vector injection, this approach resulted in substantially higher hepatic transduction efficiency using clinically relevant low vector doses and was accompanied by mild-to-moderate acute but transient toxicities. Transgene expression was sustained for up to 964 days. These results suggest that our minimally invasive method of delivery can significantly improve the vector's therapeutic index and may be a first step toward clinical application of HDAd for liver-directed gene therapy.

Maternal high-fat diet triggers lipotoxicity in the fetal livers of nonhuman primates

Maternal obesity is thought to increase the offspring's risk of juvenile obesity and metabolic diseases; however, the mechanism(s) whereby excess maternal nutrition affects fetal development remain poorly understood. Here, we investigated in nonhuman primates the effect of chronic high-fat diet (HFD) on the development of fetal metabolic systems. We found that fetal offspring from both lean and obese mothers chronically consuming a HFD had a 3-fold increase in liver triglycerides (TGs). In addition, fetal offspring from HFD-fed mothers (O-HFD) showed increased evidence of hepatic oxidative stress early in the third trimester, consistent with the development of nonalcoholic fatty liver disease (NAFLD). O-HFD animals also exhibited elevated hepatic expression of gluconeogenic enzymes and transcription factors. Furthermore, fetal glycerol levels were 2-fold higher in O-HFD animals than in control fetal offspring and correlated with maternal levels. The increased fetal hepatic TG levels persisted at P180, concurrent with a 2-fold increase in percent body fat. Importantly, reversing the maternal HFD to a low-fat diet during a subsequent pregnancy improved fetal hepatic TG levels and partially normalized gluconeogenic enzyme expression, without changing maternal body weight. These results suggest that a developing fetus is highly vulnerable to excess lipids, independent of maternal diabetes and/or obesity, and that exposure to this may increase the risk of pediatric NAFLD.

Publication incorrectly characterizes dose of bisphenol A

In their report on exposures of green monkeys to bisphenol A (BPA) Leranth et al. (1) represent their study as the evaluation of an exposure at the Environmental Protection Agency (EPA) oral reference dose (RfD) of 50 μg per kilogram of body weight per day. The EPA oral RfD (which can be found at www.epa.gov/ncea/iris/subst/0356.htm) is intended to apply to chronic oral exposures, not subcutaneous continuous-release exposures as used in the study. It is incorrect to suggest that this exposure paradigm results in potential physiological levels of BPA comparable to those after ingestion exposures by primates. It is well known that ingested BPA undergoes rapid first-pass conjugation in the liver of humans and other primates (2). The result of these metabolic events is that actual tissue exposures to BPA at sites distal to the liver, after ingestion exposures, is only a fraction of the amount ingested. Thus, the exposure route used in the Leranth et al. study (1) does not result in potential target organ exposures that might result from ingestion of BPA at the EPA oral RfD. The intent to link the unconventional exposure route to human risk was stated explicitly in a report of the study by The Washington Post (3). The toxicological evaluation of potential human exposures to environmental chemicals is difficult under the best circumstances, so it is important that the relevance of exposure routes be understood and explicitly recognized in studies such as the one reported by Leranth et al. (1).

Percutaneous Transendocardial Delivery of Self-complementary Adeno-associated Virus 6 Achieves Global Cardiac Gene Transfer in Canines

Achieving efficient cardiac gene transfer in a large animal model has proven to be technically challenging. Previous strategies have used cardiopulmonary bypass or dual catheterization with the aid of vasodilators to deliver vectors, such as adenovirus, adeno-associated virus (AAV), or plasmid DNA. Although single-stranded AAV (ssAAV) vectors have shown the greatest promise, they suffer from delayed expression, which might be circumvented using self-complementary vectors. We sought to optimize cardiac gene transfer using a percutaneous transendocardial injection catheter to deliver adeno-associated viral vectors to the canine myocardium. Four vectors were evaluated--ssAAV9, self-complementary AAV9 (scAAV9), scAAV8, scAAV6--so that comparison could be made between single-stranded and self-complementary vectors as well as among serotypes 9, 8, and 6. We demonstrate that scAAV is superior to ssAAV and that AAV 6 is superior to the other serotypes evaluated. Biodistribution studies revealed that vector genome copies were 15-4,000 times more abundant in the heart than in any other organ for scAAV6. Percutaneous transendocardial injection of scAAV6 is a safe, effective method to achieve efficient cardiac gene transfer.

PET imaging of thymidine kinase gene expression in the liver of non-human primates following systemic delivery of an adenoviral vector

Non-invasive in vivo imaging of transgene expression is currently providing very important means to optimize gene therapy regimes. Results in non-human primates are considered the most predictive models for the outcome in patients. In this study, we have documented that tumour and primary cell lines from human and non-human primates are comparably gene-transduced in vitro by serotype 5 adenovirus expressing HSV1-thymidine kinase. Transgene expression can be quantified in human and monkey cultured cells by positron emission tomography (PET) imaging when transduced cells are incubated with a fluoride-18 labelled penciclovir analogue. In our hands, PET images of cell cultures estimate the number of transduced cells rather than intensity of transgene expression once a threshold of TK per cell is reached. Interestingly, in vivo systemic administration of a clinical grade recombinant adenovirus expressing TK into macaques gives rise to an intense retention of the radiotracer in the liver parenchyma, providing an experimental system to visualize transgene expression that ought to be similar in human and macaques. Such imaging methodology might contribute to improve strategies based on adenoviral vectors.

LDL Cholesteryl Oleate

Cholesteryl esters (CE) are synthesized by 2 enzymes, lecithin:cholesterol acyltransferase (LCAT) and acylCoA:cholesterol acyltransferase (ACAT). LCAT functions in the plasma and forms CE in HDL by transferring polyunsaturated fatty acid from phosphatidylcholine to cholesterol. ACAT, which functions intracellularly, uses fatty acid from acylCoA and forms CE enriched in monounsaturated fatty acid. Two isoforms of ACAT are expressed.1 ACAT1 is present in many tissues and produces the CE that are stored intracellularly, whereas ACAT2 is expressed in the intestine and liver and produces the CE that are secreted in chylomicrons and VLDL.2 See page 1396 In this issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Bell et al report that ACAT2 gene deletion prevents hypercholesterolemia and atherosclerosis induced by diets containing 10% saturated or monounsaturated fat in a murine model of high LDL apoB100 only, LDL receptor-deleted (LDLr−/−) mice.3 These mice also had larger LDL particle size and hypertriglyceridemia. They attribute the protection of ACAT2 deficiency to a decrease in LDL particles enriched in CE derived from oleate (monounsaturated). When the diet contains large amounts of monounsaturated fats, ACAT2 activity appears to favor production of VLDL particles containing an excessive amount of cholesteryl oleate,4 and these monounsaturated CE remain associated with the lipoprotein particle when it undergoes conversion to LDL. These findings remind us that CE synthesized in the liver can be a source of CE for LDL, a fact that tends to be overlooked because of the current emphasis on the role of the reverse cholesterol transport pathway and cholesteryl ester transport protein (CETP) in supplying CE to VLDL and ultimately LDL. The Figure illustrates how LCAT, CETP, and ACAT2 function to supply monounsaturated and polyunsaturated CE that can accumulate in LDL. A schematic summary of the pathways that provide cholesteryl esters (CE) for incorporation …

720. Evaluating AAV-Mediated Transduction of Primate Liver as a Therapeutic Strategy for Lysosomal Storage Disorders

720. Evaluating AAV-Mediated Transduction of Primate Liver as a Therapeutic Strategy for Lysosomal Storage Disorders

964. Balloon Occlusion Catheter-Based Delivery of HDAd into the Nonhuman Primate Liver Results in Stable, High Level Transgene Expression with Minimal Toxicity

964. Balloon Occlusion Catheter-Based Delivery of HDAd into the Nonhuman Primate Liver Results in Stable, High Level Transgene Expression with Minimal Toxicity

1086. Transgene Immunity Dictates the Stability of Efficient EGFP Gene Transfer by Conventional (ss) and Self Complementary (sc) AAV7 Vectors in Nonhuman Primate Liver

1086. Transgene Immunity Dictates the Stability of Efficient EGFP Gene Transfer by Conventional (ss) and Self Complementary (sc) AAV7 Vectors in Nonhuman Primate Liver

Pseudo-hydrodynamic Delivery of Helper-dependent Adenoviral Vectors into Non-human Primates for Liver-directed Gene Therapy

Helper-dependent adenoviral vectors (HDAds) are attractive for liver-directed gene therapy because they can mediate long-term, high-level transgene expression without chronic toxicity. However, systemic delivery requires high vector doses for efficient hepatic transduction, resulting in dose-dependent acute toxicity. Clearly, strategies to improve hepatic transduction with low vector doses are needed. In this regard, we have previously shown that hydrodynamic injection of helper-dependent adenoviral vectors into mice results in increased hepatic transduction, reduced systemic vector dissemination, and reduced pro-inflammatory cytokines compared with conventional injection and thus has the potential to improve dramatically the therapeutic index of helper-dependent adenoviral vectors. Unfortunately, the rapid, large-volume injection used in this method cannot be applied to larger animals. Therefore, we have developed a novel balloon occlusion catheter-based method to mimic hydrodynamic injection of helper-dependent adenoviral vectors into non-human primates that does not require rapid, large-volume injection. Using a low, clinically relevant vector dose, this minimally invasive method results in high-efficiency hepatic transduction with minimal toxicity and stable long-term transgene expression for at least 413 days.