Vladimir Subbotin

Hypothesis: naked plasmid DNA is taken up by cells in vivo by a receptor-mediated process.

Following the initial demonstration that intramuscularly‐injected naked plasmid DNA (pDNA) is expressed in myofibers, it was shown that pDNA can be used for vaccination purposes. More recent studies have indicated that naked pDNA can also achieve high levels of transgene expression in vivo. This efficiency of naked pDNA expression, especially via intravascular route, is truly astounding. In this prospective review, we examine the possible mechanisms of naked pDNA uptake. The possible mechanisms; (a) large membrane disruption, (b) small membrane pores, and (c) receptor‐mediated endocytosis, are considered in turn. Some recent original laboratory data relevant to these hypotheses are also presented. Copyright

Efficient Expression of Naked DNA Delivered Intraarterially to Limb Muscles of Nonhuman Primates

We have previously shown that the intraarterial delivery of naked plasmid DNA (pDNA) into the femoral artery of rats leads to high levels of foreign gene expression throughout the muscles of the hindlimb. The present study shows that the procedure can also enable high levels of foreign gene expression throughout the limb skeletal muscles in rhesus monkeys. The average luciferase expression in the target muscle was 991.5 +/- 187 ng/g for the arm and 1692 +/- 768 ng/g for the leg; compared with 780 ng/g in rat hindlimb. Large numbers of beta-galactosidase-positive myofibers were found in both leg and arm muscles, ranging from less than 1% to more than 30% in various muscles, with an average of 6.9%. The nonhuman primates tolerated the procedure without significant adverse effects in skeletal muscles, arteries, or other organs. Other studies in immunosuppressed rats indicated that stable expression is possible. These results suggest that the procedure is likely to enable efficient and stable gene expression in human muscle without substantial toxicity.

Time course of gene expression after plasmid DNA gene transfer to the liver

High levels of expression in hepatocytes can be achieved after intraportal delivery of plasmid DNA vectors with up to 10% of all liver cells transfected. CMV promoter‐driven expression is very high on Day 1 after injection, but is diminished strongly by Day 2. Expression slowly declines after 1 week. We describe experiments aimed at elucidating the reasons for this rapid decline in transgene expression.

Interleukin-17 antagonism inhibits acute but not chronic vascular rejection.

BACKGROUND Blocking the action of interleukin (IL) 17 with an IL-17 receptor (R):Fc fusion protein inhibits T-cell proliferative responses to alloantigens and prolongs vascularized heart graft survival. In this study, we examined whether IL-17 antagonism could suppress the development of chronic rejection. METHODS A 0.6-cm section of C57BL10 (H2b) thoracic aorta was transplanted to recipient C3H (H2k) abdominal aorta. IL-17R:Fc or control human immunoglobulin G was administered i.p. (500 microg/day) from days 0 to 6 or from days 0 to 29. Mice were killed on days 7 or 30. Grafts were examined histologically and stained for alpha-smooth muscle actin (alpha-smA). Antidonor mixed leukocyte reaction, cytotoxic T cell, and alloantibody responses were quantified. RESULTS On day 7, control grafts showed mononuclear cell (MNC) infiltration, pronounced endothelial damage, and apoptosis of intimal and medial cell compartments. By day 30, there was concentric intimal thickening, accumulation of alpha-smA+ cells, and collagen deposition. Patchy destruction of the elastic membranes and loss of alpha-smA expression in media were evident. IL-17R:Fc for 6 days decreased MNC infiltration in the intimal and medial compartments at day 7. The endothelium was preserved (completely or partially) in all grafts. The medial compartment showed normal alpha-smA expression. Irrespective of IL-17R:Fc treatment for either 6 days or continuously, allografts harvested at day 30 showed circumferential intimal thickening, with accumulation of alpha-smA+ cells and collagen deposition. There was no effect on circulating alloantibody levels. CONCLUSIONS These findings support a role for IL-17 in the immunopathogenesis of acute vascular rejection and demonstrate the potential of IL-17 antagonism for therapy. By contrast, IL-17 antagonism does not appear to prevent ensuing chronic graft vascular disease, in particular neointimal formation.

Prevention of chronic rejection in mouse aortic allografts by combined treatment with CTLA4-Ig and anti-CD40 ligand monoclonal antibody

BACKGROUND In this study, using a murine model of aortic allotransplantation, the role of blockade of signaling through CD28/B7 and CD40/CD40 ligand costimulatory pathways in the evolvement of posttransplant vasculopathy was examined. METHODS Aortic allografts were transplanted across C57BL/1OJ (H2b)-->C3H (H2k) strain combinations. Transient or more stable blockade of second signaling was achieved by either a single injection or multiple injections of CTLA4-Ig fusion protein (200 microg/dose i.p.) and/or anti-CD40 ligand (CD40L) monoclonal antibody (250 microg i.m.). At day 30 after transplantation, the grafts were harvested for histopathological and immunohistochemical examination. RESULTS Similar to allografts of untreated animals, aortic allografts obtained from recipients treated with either CTLA4-Ig or anti-CD40L monoclonal antibody alone exhibited marked narrowing of the lumen primarily due to concentric intimal thickening caused by proliferation of alpha-smooth muscle actin-positive cells. Contemporaneous treatment, however, with either a single injection or multiple injections of CTLA4-Ig and anti-CD40L monoclonal antibody resulted in marked diminution of intimal thickening. Interestingly, concurrent prolonged inhibition of CD28/B7 and CD40/CD40L pathways resulted in complete abrogation of the development of posttransplant arteriopathy. CONCLUSION These data suggest that a more stable disruption of signaling through costimulatory pathways may be required to obviate the development of posttransplant vasculopathy.

Recovery of the Endogenous β Cell Function in the NOD Model of Autoimmune Diabetes

In light of accumulating evidence that the endocrine pancreas has regenerative properties and that hematopoietic chimerism can abrogate destruction of β cells in autoimmune diabetes, we addressed the question of whether recovery of physiologically adequate endogenous insulin regulation could be achieved in the nonobese diabetic (NOD) mice rendered allogeneic chimerae. Allogeneic bone marrow (BM) was transplanted into NOD mice at the preclinical and overtly clinical stages of the disease using lethal and nonlethal doses of radiation for recipient conditioning. Islets of Langerhans, syngeneic to the BM donors, were transplanted under kidney capsules of the overtly diabetic animals to sustain euglycemia for the time span required for recovery of the endogenous pancreas. Nephrectomies of the graft‐bearing organs were performed 14 weeks later to confirm the restoration of endogenous insulin regulation. Reparative processes in the pancreata were assessed histologically and immunohistochemically. The level of chimerism in NOD recipients was evaluated by flow cytometric analysis. We have shown that as low as 1% of initial allogeneic chimerism can reverse the diabetogenic processes in islets of Langerhans in prediabetic NOD mice, and that restoration of endogenous β cell function to physiologically sufficient levels is achievable even if the allogeneic BM transplantation is performed after the clinical onset of diabetes. If the same pattern of islet regeneration were shown in humans, induction of an autoimmunity‐free status by establishment of a low level of chimerism, or other alternative means, might become a new therapy for type 1 diabetes.

ISOLATION, PHENOTYPE, AND ALLOSTIMULATORY ACTIVITY OF MOUSE LIVER DENDRITIC CELLS

Donor liver-derived dendritic cells (DC) have recently been identified within various lymphoid and nonlymphoid tissues of organ allograft recipients, including nonimmunosuppressed mice transplanted with and permanently accepting major histocompatibility complex (MHC)-disparate hepatic allografts. These findings have raised questions about the basis of the tolerogenicity of the liver--and, in particular, about the properties of liver-derived DC. To study further the structure, immunophenotype and allostimulatory activity of leukocytes resident in normal mouse (B10.BR;H-2k, I-Ek) liver, a procedure was developed to maximize the yield of viable, nonparenchymal cells (NPC) obtained following collagenase digestion of perfused liver fragments and density centrifugation (Percoll). These cells comprised populations expressing lymphoid and myeloid cell surface antigens. As compared with spleen cells, they proved good allostimulators of naive (B10; H-2b, I-E-) splenic T cells when tested in primary mixed leukocyte reactions (MLR). After overnight (18-hr) incubation of the NPC, enrichment for transiently adherent, low-density (LD) cells on metrizamide gradients permitted the recovery of low numbers of cells (approx. 2-5 x 10(5) per liver), many of which displayed distinct DC morphology. Flow cytometric analysis revealed that these cells were CD3-, CD4-, CD8-, and B220-, but strongly expressed CD45 (leukocyte-common antigen), and mild-to-moderate levels of CD11b, heat-stable antigen, and CD44. The cells also expressed moderate intensity of NLDC 145 but not 33D1, DC restricted markers which have been shown to be differentially expressed on mouse DC isolated from various organs. This DC-enriched population was more strongly MHC class II(I-Ek)+ than NPC, as determined by immunocytochemistry and flow cytometry and exhibited much more potent allostimulatory activity for naive T cells. These findings demonstrate that freshly isolated murine liver NPC, and perhaps their counterparts in situ, exhibit allostimulatory activity that is enhanced in the non-adherent, low-density (DC-enriched) fraction after overnight culture. They further suggest that the maturation of liver DC may play a key role in determining the immunogenicity and or tolerogenicity of hepatic allografts.

Increased hepatic endothelin-1 levels and endothelin receptor density in cirrhotic rats

Endothelin-1 (ET-1), the most powerful agent to cause constriction of the hepatic vasculature, is synthesized in the liver by sinusoidal endothelial cells. Circulating ET-1 levels have been shown to increase in liver cirrhosis. As liver could be a major source of increased plasma ET-1 as well as a target for its pathologic actions, this study was designed to determine hepatic ET-1 and ET receptor(s) in experimental liver cirrhosis. Cirrhosis was induced in rats by intraperitoneal administration of carbon tetrachloride for 8 weeks. Hepatic ET-1 was measured by radioimmunoassay and ET receptors were determined by radioligand competition binding procedure. A four fold increase in ET-1 concentration accompanied by a 65% increase in ET-receptor density was observed in the cirrhotic liver. There was no change in the ET receptor affinity. The capacity of the liver to metabolize ET-1 was reduced significantly in cirrhosis. Interestingly, transforming growth factor-beta, hepatic levels of which increase in cirrhosis, stimulated ET-1 synthesis in cultured Ito cells. It has been shown that ET-1 is a potent constrictor of Ito cells that proliferate and transform into highly contractile myofibroblasts in liver cirrhosis. Thus, interactions between ET-1 and Ito cells may have significant implications in the pathogenesis and complications of liver cirrhosis.

Acute pancreatitis and bacterial translocation.

Infectious complications are the most frequent and severe complications of acute narcotizing pancreatitis (AP) with a mortality rate up to 80%. Although experimental and clinical studies suggest that the microbiologic source of pancreatic infection could be enteric, information in this regard is scant. This study evaluated bacterial translocation (BT) using mild and severe models of AP. Mild AP was induced by 6-hr continuous intravenous infusion of cerulein, while severe AP was induced by additional infusion of glycodeoxycholic acid into the biliopancreatic duct. BT was evaluated with organ cultures performed when animals were killed (24 hr). To confirm the gastrointestinal origin of the translocating microorganisms, fluorescent microspheres were also given to the animals in drinking water 24 hr before induction of AP. At the time of death beads were counted with a (fluorescence-activated cell sorter) (FACS) in peritoneal lavages and with fluorescent microscopy in frozen sections of the pancreata. Morphology of the distal small bowel showed significant changes in the animals with AP compared to controls, such as reduction of villus high and altered microvasculature. Mild AP induced BT to the pancreas in 100% of the animals, compared to pancreata from control groups. Severe AP induced increased BT to the pancreas. BT to liver and spleen was also significantly increased with AP. The presence of fluorescent microspheres confirmed their enteric derivation. This study provides evidence for the enteric origin of microorganisms responsible for pancreatic infectious complications during AP. The evidence of BT after laparotomy suggests an increased risk of infections with the association of these conditions. This could provide an explanation for the high mortality associated with laparotomy in course of AP.

Mechanism of plasmid delivery by hydrodynamic tail vein injection. I. Hepatocyte uptake of various molecules

The hydrodynamic tail vein (HTV) injection of naked plasmid DNA is a simple yet effective in vivo gene delivery method into hepatocytes. It is increasingly being used as a research tool to elucidate mechanisms of gene expression and the role of genes and their cognate proteins in the pathogenesis of disease in animal models. A greater understanding of its mechanism will aid these efforts and has relevance to macromolecular and nucleic acid delivery in general.