Binding of adenovirus species C hexon to prothrombin and the influence of hexon on vector properties in vitro and in vivo.

The majority of adenovirus (Ad) vectors are based on human Ad type 5, which is a member of Ad species C. Species C also includes the closely-related types 1, 2, 6, 57 and 89. It is known that coagulation factors bind to Ad5 hexon and play a key role in the liver tropism of Ad5 vectors, but it is unclear how coagulation factors affect vectors derived from other species C Ads. We evaluated species C Ad vectors both in vitro and following intravenous injection in mice. To assess the impact of hexon differences, we constructed chimeric Ad5 vectors that contain the hexon hypervariable regions from other species C types, including vectors with hexon mutations that decreased coagulation factor binding. After intravenous injection into mice, vectors with Ad5 or Ad6 hexon had strong liver tropism, while vectors with chimeric hexon from other Ad types had weaker liver tropism due to inhibition by natural antibodies and complement. In addition, we discovered a novel ability of hexon to bind prothrombin, which is the most abundant coagulation factor in blood, and we found striking differences in the affinity of Ads for human, mouse and bovine coagulation factors. When compared to Ad5, vectors with non-Ad5 species C hexons had considerably higher affinity for both human and mouse prothrombin. Most of the vectors tested were strongly dependent on coagulation factors for liver transduction, but vectors with chimeric Ad6 hexon showed much less dependence on coagulation factors than other vectors. We found that in vitro neutralization experiments with mouse serum predicted in vivo behavior of Ad5 vectors, but in vitro experiments did not predict the in vivo behavior of vectors based on other Ad types. In sum, hexons from different human Ad species C viruses confer diverse properties on vectors, including differing abilities to target the liver.

Hexons from adenovirus serotypes 5 and 48 differentially protect adenovirus vectors from neutralization by mouse and human serum.

Adenovirus vectors are widely used in gene therapy clinical trials, and preclinical studies with these vectors are often conducted in mice. It is therefore critical to understand whether mouse studies adequately predict the behavior of adenovirus vectors in humans. The most commonly-used adenovirus vectors are derived from adenovirus serotype 5 (Ad5). The Ad5 hexon protein can bind coagulation factor X (FX), and binding of FX has a major impact on vector interactions with other blood proteins. In mouse serum, FX protects Ad5 vectors from neutralization by natural antibodies and complement. In the current study, we similarly find that human FX inhibits neutralization of Ad5 vectors by human serum, and this finding is consistent among individual human sera. We show that human IgM and human IgG can each induce complement-mediated neutralization when Ad5 vectors are not protected by FX. Although mouse and human serum had similar effects on Ad5 vectors, we found that this was not true for a chimeric Ad5 vector that incorporated hexon regions from adenovirus serotype 48. Interestingly, this hexon-chimeric vector was neutralized by human serum, but not by mouse serum. These findings indicate that studies in mouse serum accurately predict the behavior of Ad5 vectors in human serum, but mouse serum is not an accurate model system for all adenovirus vectors.

Antagonism of the Neurokinin-1 Receptor Improves Survival in a Mouse Model of Sepsis by Decreasing Inflammation and Increasing Early Cardiovascular Function.

OBJECTIVES: Sepsis remains a serious clinical problem despite intensive research efforts and numerous attempts to improve outcome by modifying the inflammatory response. Substance P, the principal ligand for the neurokinin-1 receptor, is a potent proinflammatory mediator that exacerbates inflammatory responses and cardiovascular variables in sepsis. DESIGN: The current study examined whether inhibition of the neurokinin-1 receptor with a specific antagonist (CJ-12,255) would improve survival in the cecal ligation and puncture model of sepsis in adult female outbred mice. SETTING: University basic science research laboratory. MEASUREMENTS AND MAIN RESULTS: Neurokinin-1 receptor treatment at the initiation of sepsis improved survival in cecal ligation and puncture sepsis (neurokinin-1 receptor antagonist survival = 79% vs vehicle = 54%). Delaying therapy for as little as 8 hours postcecal ligation and puncture failed to provide a survival benefit. Neurokinin-1 receptor antagonist treatment did not prevent the sepsis-induced decrease in circulating WBCs, augment the early (6 hr postcecal ligation and puncture) recruitment of inflammatory cells to the peritoneum, or improve phagocytic cell killing of pathogens. However, the neurokinin-1 receptor antagonist significantly reduced both circulating and peritoneal cytokine concentrations. In addition, the cardiovascular variable, pulse distension (a surrogate for stroke volume) was improved in the neurokinin-1 receptor antagonist group during the first 6 hours of sepsis, and there was a significant reduction in loss of fluid into the intestine. CONCLUSION: These data show that early activation of the neurokinin-1 receptor by substance P decreases sepsis survival through multiple mechanisms including depressing stroke volume, increasing fluid loss into the intestine, and increasing inflammatory cytokine production.

Impact of natural IgM concentration on gene therapy with adenovirus type 5 vectors.

Natural IgM inhibits gene transfer by adenovirus type 5 (Ad5) vectors. We show that polyreactive natural IgM antibodies bind to Ad5 and that inhibition of liver transduction by IgM depends on Kupffer cells. By manipulating IgM concentration in vivo, we demonstrate that IgM inhibits liver transduction in a concentration-dependent manner. We further show that differences in natural IgM between BALB/c and C57BL/6 mice contribute to lower efficiency of Ad5 gene transfer in BALB/c mice.

Adenosine receptor antagonists effect on plasma-enhanced killing.

Previous studies demonstrated that naive plasma has inherent capabilities to enhance bacterial opsonization and phagocyte killing, but not all plasma is equally effective. This raised the question of whether plasma constituents other than opsonins may play a role. Adenosine receptor antagonists have been shown to modulate cytokine response and survival in mice after a bacterial challenge. We investigated whether selective adenosine receptor blockade would influence the ability of naive plasma to effectively control bacterial growth. Colonic bacteria- and thioglycollate-elicited peritoneal macrophages and neutrophils were obtained from naive mice. Stock murine plasma from naive was purchased and categorized as having high plasma-enhanced bacterial killing capacity using our previously described methods. Bacteria and plasma were incubated to allow for opsonization and then added to macrophages previously exposed to selected adenosine receptor antagonists: ZM 241385: A2A, MRS1754: A2B, DPCPX: A1, and MRS1220: A3. The final mixture was plated on blood agar plates in aerobic and anaerobic conditions and bacterial colony-forming units quantified after 24 h. This study demonstrated that exogenous adenosine was able to significantly decrease phagocyte killing of cecal bacteria. Blocking adenosine receptors with selective antagonists altered the bacterial killing capacity of plasma. Selectively blocking the A1, A2A, or A2B receptors proved most beneficial at reversing the effect of adenosine. Consistent with previous work, only macrophage killing of bacteria could be modulated by adenosine receptor blockade because neutrophils were unaffected. These data demonstrate that adenosine decreases macrophage killing of enteric bacteria and that this effect is mediated through the adenosine receptors.

Cecal ligation and puncture-induced murine sepsis does not cause lung injury.

OBJECTIVE: The cause of death in murine models of sepsis remains unclear. The primary purpose of this study was to determine if significant lung injury develops in mice predicted to die after cecal ligation and puncture-induced sepsis compared with those predicted to live. DESIGN: Prospective, laboratory controlled experiments. SETTING: University research laboratory. SUBJECTS: Adult, female, outbred Institute of Cancer Research mice. INTERVENTIONS: Mice underwent cecal ligation and puncture to induce sepsis. Two groups of mice were euthanized at 24 and 48 hrs postcecal ligation and puncture and samples were collected. These mice were further stratified into groups predicted to die (Die-P) and predicted to live (Live-P) based on plasma interleukin-6 levels obtained 24 hrs postcecal ligation and puncture. Multiple measures of lung inflammation and lung injury were quantified in these two groups. Results from a group of mice receiving intratracheal normal saline without surgical intervention were also included as a negative control. As a positive control, bacterial pneumonia was induced with Pseudomonas aeruginosa to cause definitive lung injury. Separate mice were followed for survival until Day 28 postcecal ligation and puncture. These mice were used to verify the interleukin-6 cutoffs for survival prediction. MEASUREMENTS AND MAIN RESULTS: After sepsis, both the Die-P and Live-P mice had significantly suppressed measures of respiratory physiology but maintained normal levels of arterial oxygen saturation. Bronchoalveolar lavage levels of pro- and anti-inflammatory cytokines were not elevated in the Die-P mice compared with the Live-P. In addition, there was no increase in the recruitment of neutrophils to the lung, pulmonary vascular permeability, or histological evidence of damage. In contrast, all of these pulmonary injury and inflammatory parameters were increased in mice with Pseudomonas pneumonia. CONCLUSIONS: These data demonstrate that mice predicted to die during sepsis have no significant lung injury. In murine intra-abdominal sepsis, pulmonary injury cannot be considered the etiology of death in the acute phase.

Presence of preexisting antibodies mediates survival in sepsis.

Sepsis is one of the leading causes of death in hospitals worldwide. Even with optimal therapy, severe sepsis results in 50% mortality, indicating variability in the response of individuals towards treatment. We hypothesize that the presence of preexisting antibodies present in the blood before the onset of sepsis induced by cecal ligation and puncture (CLP) in mice accounts for the differences in their survival. A plasma-enhanced killing (PEK) assay was performed to calculate the PEK capacity of plasma, that is, the ability of plasma to augment polymorphonuclear neutrophil killing of bacteria. Plasma-enhanced killing was calculated as PEK = [1 / log (N)] × 100, where N = number of surviving bacteria; a higher PEK indicated better bacterial killing. A range of PEK in plasma collected from mice before CLP was observed, documenting individual differences in bacterial killing capacity. Mortality was predicted based on plasma IL-6 levels at 24 h after CLP. Mice predicted to die (Die-P) had a lower PEK (<14) and higher peritoneal bacterial counts at 24 h after sepsis compared with those predicted to live (Live-P) with a PEK of greater than 16. Mice with PEK of less than 14 were 3.1 times more likely to die compared with the group with PEK of greater than 16. To understand the mechanism of defense conferred by the preexisting antibodies, binding of IgM or IgG to enteric bacteria was documented by flow cytometry. To determine the relative contribution of IgM or IgG, the immunoglobulins were specifically immunodepleted from the naive plasma samples and the PEK of the depleted plasma measured. Compared with naive plasma, depletion of IgM had no effect on the PEK. However, depletion of IgG increased PEK, suggesting that an inhibitory IgG binds to antigenic sites on bacteria preventing optimal opsonization of the bacteria. These data demonstrate that, before CLP, circulating inhibitory IgG antibodies exist that prevent bacterial killing by polymorphonuclear neutrophils in a CLP model of sepsis.

Correction of hyperoxaluria by liver repopulation with hepatocytes in a mouse model of primary hyperoxaluria type-1.

BACKGROUND: Primary hyperoxaluria type-1 (PH1) is an autosomal recessive disease characterized by excessive oxalate production by hepatocytes caused by the deficiency of peroxisomal alanine-glyoxylate aminotransferase (AGT) activity. Persistent hyperoxaluria causes nephrocalcinosis and urolithiasis, leading to renal failure, followed by tissue oxalosis with life-threatening complications. Combined liver-kidney transplantation is the only definitive treatment of PH1. Hepatocyte transplantation, which is much less invasive, could have offered an attractive alternative. However, because the AGT-deficient hepatocytes overproduce oxalate, a large fraction of the mutant host hepatocytes must be replaced by AGT-competent cells, which is beyond the capacity of current hepatocyte transplantation procedures. Here, we have evaluated a preparative irradiation-based method of liver repopulation in an Agxt-deleted mouse model of PH1 (Agxt-/-). MATERIALS AND METHODS: Hepatocytes (10(6) viable cells) isolated from congeneic mice ([ROSA]26 C57BL/6J) expressing Escherichia coli beta-galactosidase were transplanted into Agxt-/- mice by intrasplenic injection. The preparative regimen consisted of X-irradiation of the host liver and mitotic stimulation of the hepatocytes by adenovector-based expression of hepatocyte growth factor. RESULTS: The procedure resulted in progressive replacement of the mutant host hepatocytes with the AGT-competent hepatocytes, leading to correction of urinary oxalate excretion. Oral ethylene glycol challenge (0.7% for 1 week) resulted in nephrocalcinosis and microlithiasis in untreated Agxt-/- mice, but not in the mice after hepatic repopulation. CONCLUSION: The results indicate that hepatocyte transplantation after appropriate preparative regimens may permit sufficient repopulation of the liver to ameliorate hyperoxaluria, and therefore should be evaluated further as a potential treatment of PH1.

Hepatic repopulation with stably transduced conditionally immortalized hepatocytes in the Gunn rat.

BACKGROUND/AIMS: Conditionally immortalized hepatocytes offer a renewable source of hepatocytes, but although preparative maneuvers have been developed for hepatic repopulation with primary hepatocytes, extensive proliferation of transplanted immortalized hepatocytes has not been accomplished heretofore. Our aim was to achieve ex vivo gene therapy of uridinediphosphoglucuronate glucuronosyltransferase-1A1 (UGT1A1)-deficient jaundiced Gunn rats (model of Crigler-Najjar syndrome type-1) by hepatic repopulation with genetically modified and conditionally immortalized hepatocytes. METHODS: Gunn rat hepatocytes were conditionally immortalized by stable transduction with a thermolabile mutant simian virus 40 T-antigen ((ts)Tag(A58)) and further transduced with UGT1A1. These hepatocytes proliferate at 33 degrees C, but at 37 degrees C the (ts)Tag(A58) is degraded and the cells become quiescent. The cells were transplanted into Gunn rat livers after preparative hepatic irradiation (50 Gy) and 66% hepatectomy. RESULTS: The engrafted UGT1A1-positive immortalized hepatocytes replaced approximately 80% of the host hepatocytes in 20 weeks, leading to normalization of hyperbilirubinemia. Liver histology, and serum albumin and alanine aminotransferase levels remained normal. CONCLUSIONS: We achieved complete cure of hyperbilirubinemia in Gunn rats by ex vivo gene therapy via genetically modified and conditionally immortalized hepatocytes.