Viral IL-10 gene therapy inhibits TNF-alpha and IL-1 beta, not IL-6, in the newborn endotoxemic mouse.

INTRODUCTION: Modulation of the inflammatory cascade within the liver of critically ill infants may improve the chance of survival. Using gene therapy, the authors hypothesized that augmented local production of the counter-regulatory cytokine viral interleukin-10 (IL-10) in vivo will modulate the critical cytokines in the inflammatory response. The purpose of the present study was to determine whether replication-defective adenovirus-mediated viral IL-10 (vIL-10) gene transfer and expression within the liver can achieve this goal in newborn mice. MATERIALS AND METHODS: Four-week-old Balb/c mice were administered (intraperitoneally) 1 x 10(9) plaque-forming units (pfu) per milliliter of an adenovirus vector (E1a/b-deleted) than encodes the sv40 promoter and the BCRF1 cDNA, or of control vector dl434 that expresses no foreign gene. Forty-eight hours later the mice were challenged with 50 micrograms/kg of lipopolysaccharide (LPS) they were killed 1, 2, 6, or 24 hours later (six at each time point). Southern blot analysis was performed on genomic DNA isolated from the liver, lung, and kidney to assess gene transfer of BCRF1. Homogenized liver protein was analyzed for tumor necrosis factor-alpha (TNF-alpha), IL-1 beta, IL-6, and recombinant vIL-10. RESULTS: Southern blot analysis confirmed successful gene transfer to the liver but not to the lung, kidney, or dl434-transduced liver in mice that received adenovectors. Viral IL-10 levels within the liver ranged from 14 to 18 ng/mL. In controls, TNF-alpha production was elevated at early time points, to 18,000 pg/mL, but decreased rapidly by 24 hours after LPS challenge. The TNF-alpha levels of animals treated with Ad5svBCRF1 were significantly lower than those of controls throughout the course of study (P < .0001). After the LPS challenge, hepatic IL-1 beta decreased, from a maximum of 800 pg/mL (2 hours) to 411 pg/mL (24 hours). Inhibition of IL-1 beta by vIL-10 occurred at 1 hour (P > .016) and 2 hours (P < .001) only. Hepatic production of IL-6 after LPS challenge ranged from 7 to 8,000 pg/mL in all groups and was not altered by vIL-10 gene therapy. CONCLUSION: In vivo administration of adenovectors encoding BCRF1 to newborn mice results in efficient hepatic transduction and expression of recombinant vIL-10. The Kupffer cell response to LPS is suppressed with respect to TNF-alpha and IL-1 beta, but not IL-6. In vivo modulation of hepatic cytokine responses is achievable using gene products that mimic cellular cytokines. This is an effective model for the selective evaluation of therapeutic gene products for gene therapy of sepsis.

Hepatic function is preserved following liver-directed, adenovirus-mediated gene transfer.

Inherited and acquired disorders of the liver are attractive targets for gene therapy. Hepatic cells are susceptible targets for shuttle vectors because of a diversity of protein and viral receptors and accessibility of a selective afferent blood supply. Preservation of existing hepatic cell integrity and metabolic function is of paramount importance for successful whole animal gene therapy trials. In this report, we examine hepatic cell function and integrity following adenovirus-mediated reporter gene transfer to the liver in vivo. E1-deleted, replication-defective adenovectors encoding the LacZ gene driven by the human CMV promoter were delivered to the liver by isolated portal perfusion. The gene transfer rate, as determined by specific histochemical staining, approached 30% with recombinant protein detectable by Western blot throughout the course of study. Hepatic cell integrity as assessed by histology and hepatic enzyme profile (serum aspartate aminotransferase, gamma-glutamyl transpeptidase) demonstrated normal cellular architecture and no significant difference between transfected liver and controls. Hepatic synthetic and metabolic function, as determined by albumin levels, prothrombin time, and bilirubin, were similar between the two study groups. This study demonstrates that efficient adenovirus-mediated gene transfer and expression in the rat liver do not compromise hepatic cell metabolism and integrity.

Adenovirus-mediated gene transfer in the transplant setting. Part III. Variables affecting gene transfer in liver grafts.

We have established a system of efficient gene transfer to liver grafts using adenovirus vectors. The purpose of this study was to examine variables affecting gene transfer to rat liver grafts during cold preservation. Our results demonstrate that gene transfer efficiency was directly correlated with the ratio of vector to hepatic cells (multiplicity of infection [MOI] and the length of exposure to the vector. At MOIs of 10:1 and 50:1, the hepatic cell transduction rate was 25-30% and 100%, respectively. However, higher MOI was associated with significant mortality. Prolonging the cold preservation/exposure time resulted in an increased transduction rate (50% at MOI of 10:1). Similar gene transfer efficiencies were observed when the vector was diluted in lactated Ringer's or University of Wisconsin solution. Recombinant protein production was evident within 12 hr after reperfusion, and increased to a peak level within 48 hr. These results suggest a predictable pattern of gene transfer and expression after ex vivo transduction of liver grafts with adenovirus vectors. These data are essential in directing desirable levels of recombinant protein within the transplanted organ.

Adenovirus-mediated gene transfer in the transplant setting. II. Successful expression of transferred cDNA in syngeneic liver grafts.

These experiments establish a model for gene transfer to transplanted liver grafts ex vivo using adenoviral vectors. Rat liver grafts (n = 8) were harvested, and preserved in UW or lactated Ringer's. The grafts were infected ex-vivo via portal vein perfusion with replication-defective Ad vectors encoding the beta-galactosidase (beta-gal) gene diluted in UW solution. The infected grafts were stored at 4 degrees C for 1 hr, then transplanted into syngeneic hosts. Liver biopsies were taken at 1, 7, and 15 days after transplantation. Infection rate was assessed by histochemical staining for beta-gal. Liver DNA and RNA were assayed for the beta-gal sequences, and recombinant protein production measured at 24 hr and 7 days after transplantation. Under conditions mimicking liver graft cold storage, efficient gene transfer was achieved with an infection rate of 10-15%, as assessed by X-gal staining. Viral DNA and RNA presence in the graft was confirmed; gene expression with protein production were verified by western blots and a functional protein assay. All studies were negative in control livers. Gene expression persisted for at least 2 weeks after transplantation. We conclude that efficient adenovirus-mediated gene insertion and expression of gene products can be accomplished in whole-liver grafts under hypothermic preservation conditions currently used in clinical transplantation.

Limb leads of the electrocardiogram: sequencing revisited.

The six limb leads are normally presented in a format the logic of which is traditional rather than anatomical and does not allow visual interpolation such as is customary with the six chest leads. The sequence: a VL, I, -aVR, II, aVF, III was suggested years ago, and is used in some European countries, particularly Sweden. It provides a better impression of the extent of the changes of inferior infarction and makes the rather neglected lead aVR much more useful, though reversed in polarity. It also provides a more direct indication of the electrical axis, and simplifies comparisons with the frontal plane vectorcardiogram. Because modern digital electrocardiographs can provide the sequenced format, this seems a good time to review the advantages of adopting it.