Antifibrinolytic agents reduce tissue plasminogen activator-mediated neuronal toxicity in vitro.

INTRODUCTION: Serine proteases and their inhibitors play an important role in physiological homeostasis including neuronal activity, hemostasis, and wound healing. Tissue plasminogen activator (tPA) is involved in normal neuronal plasticity and memory formation but can also be neurotoxic. We hypothesized that the serine protease inhibitor aprotinin confers neuronal protection by inhibiting tPA activity. METHODS: Using cultured rat dopaminergic neuroblasts (N27 line), tPA-induced cytotoxicity was quantitated by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay and flow cytometry using propidium iodide DNA staining. The anti-apoptotic effects of aprotinin and other protease inhibitors were also evaluated using these systems. RESULTS: Treatment of cultured neuroblasts with tPA (10-20 microg/ml) caused a dose-dependent decrease in cell viability (71.3+/-2.4 at 10 microg/ml down to 52.7+/-2.5% at 20 microg/m tPA, 24-h treatment), which was potentiated in the absence of serum in the culture medium (59.5+/-6.3% at 10 microg/ml down to 47.9+/-4.7% at 20 microg/ml). Aprotinin was effective in ameliorating cell death when administered 30 min before tPA exposure as shown by increased cell viability (91.8+/-0.6% at tPA at 20 microg/ml), but this protection was significantly reduced when aprotinin was administered after tPA. The efficacy of aprotinin as a neuroprotectant was equivalent or superior to other direct tPA antagonist peptides Glu-Gly-Arg-chlormethylketone (EGRck) and Phe-Pro-Arg-chlormethylketone (FPRck) in this setting. CONCLUSION: These data suggest that one of the mechanisms of neuroprotection afforded by aprotinin may be inhibition of tPA-mediated neurotoxicity.

Decreased alloreactivity to human islets secreting recombinant viral interleukin 10.

The objective of this study was to analyze allogeneic lymphocyte proliferative responses to cultured human pancreatic islets after gene transfer of viral interleukin (IL)-10 to the islets using replication-defective adenoviral vector. Human islets, either whole or dispersed into single cells, were cocultured with adenovector containing an expression cassette encoding the viral IL-10 gene under control of an SV40 promoter, this sequence replacing viral E1A and part of E1B early viral protein sequences. Subsequent production of recombinant protein by islets was determined by ELISA, and was found dependent on the multiplicity of infection (or ratio of vector to target cells). Protein was secreted by transfected islets at high levels 3-7 days after gene transfer. At high multiplicity of infection (100:1), islet viability was normal, but insulin secretion in response to glucose stimulation was blunted by 50%. Low-level recombinant viral IL-10 secretion by the islets was associated with increased allogeneic lymphocyte proliferation in mixed islet lymphocyte reactions. At protein levels in islet supernatant above 5 ng/ml, lymphocyte proliferation was significantly reduced. This pattern of viral IL-10 effect on lymphocyte proliferation correlated well with mixed lymphocyte reaction assays using purified protein. We conclude that transferred cytokine sequences are secreted by transfected islets as a function of the initial vector inoculum. The functional effect of the secreted cytokine viral IL-10 on allogeneic lymphocyte proliferation is dose dependent. Low-level recombinant protein secretion tended to augment lymphocyte proliferation, whereas high-level secretion significantly down-regulates this response.

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.

Efficient gene transfer to pancreatic islets mediated by adenoviral vectors.

Genetic manipulation of pancreatic islets before transplantation has the potential to alter cellular immunity as well as islet function. The purpose of this study was to examine the feasibility of gene transfer to islets, using replication-defective adenoviral vectors. Newborn mouse islets were infected with AdHCMVsp1LacZ vector encoding Escherichia coli beta-galactosidase (beta-gal). Islets were cocultured with vector, at virus-to-target cell ratios of 10:1, for 1 hr. Gene transfer was assessed by specific histochemical stain for beta-gal (X-gal). Islet DNA and RNA were analyzed by Southern and PCR for beta-gal and adeno sequences, and recombinant protein production by western and ONPG assays. Islet integrity after gene transfer was assessed by static incubations and transplantation to nondiabetic and to diabetic mice. Southern analysis and PCR confirmed the presence of E coli beta-galactosidase and the E4 adeno DNA in infected islets, but not in controls. Reverse-transcription PCR and western analysis demonstrated expression and protein production of inserted E coli beta-galactosidase, but not E4 message. Insulin release in response to static incubations was unimpaired in infected islets. Syngeneic islet grafts stained positively for insulin for up to 7 days. Transplanted, genetically manipulated islets functioned similarly to control islets in reversing murine drug-induced diabetes. Thus, gene transfer into islets can be accomplished using adenovirus-based vectors. The capacity of this virus to infect non-dividing cells allows insertion of cDNA into pancreatic islets, with potential application to the transplant setting.

In vivo adenoviral-mediated human p53 tumor suppressor gene transfer and expression in rat liver after resection.

BACKGROUND: The aim of this study was to establish a clinically relevant model for gene transfer to liver with an adenoviral vector encoding wild-type p53 as a first step toward use of this class of gene products in the treatment of primary and metastatic liver tumors. METHODS: Full-size or 50% hepatectomized rat livers were subjected to asanguineous portal perfusion with a replication-defective adenoviral vector encoding wild-type p53 (Ad5p53), whereas control animals received adenoviral vector encoding Escherichia coli beta-galactosidase (beta-gal) (Ad5LacZ) or Ringer's lactate only. Liver biopsy specimens, blood samples, and liver weight were serially obtained. Gene transfer and expression were confirmed by X-Gal staining for gamma-gal, DNA/RNA polymerase chain reaction, (PCR) and Western blots for p53 and beta-gal. Liver integrity was assessed by histologic findings, serum transaminase levels, and synthetic function. RESULTS: The gene transfer rate in whole liver and after hepatectomy ranged from 20% to 40%. DNA PCR showed Ad sequences in livers transduced with Ad5p53 and Ad5LacZ. RNA PCR and Western blot confirmed expression and production of recombinant wild-type p53. Liver regeneration was not affected by p53 gene transduction. Liver histologic findings and synthetic function were not different between transduced and control groups. CONCLUSIONS: Ad5p53 gene transfer to full-size or hepatectomized livers is efficient. Liver regeneration and hepatocyte function are unaffected by overexpression of p53. Adenovirus-mediated tumor-suppressor transduction of the liver is a safe and promising adjuvant in cancer gene therapy.

Adenoviral-mediated gene transfer to bladder in vivo.

This study was designed to examine the potential for gene therapy in bladder in vivo using adenoviral vectors. Gene transfer to rat bladders was accomplished via direct intravesical instillation using a replication-defective adenoviral vector containing a marker gene encoding for Escherichia coli beta-galactosidase (beta-gal). Successful gene transfer was confirmed by analyzing bladder samples for DNA and RNA using polymerase chain reaction (PCR) with primers specific for beta-gal and adeno sequences, detecting beta-gal in full-thickness bladder wall using specific histochemical staining (X-gal) and documenting recombinant protein production. Bladder architecture was preserved, without evidence of distant spread of virus as assessed by PCR. Gene expression was evident for at least 7 days. In summary, bladder cells can be genetically altered using replication-deficient adenoviral vectors via simple intravesical instillation of vector. Introduction of exogenous genetic material is a potentially powerful therapeutic modality for immunomodulation of bladder neoplasms.

Adenovirus-mediated gene transfer in the transplant setting. I. Conditions for expression of transferred genes in cold-preserved hepatocytes.

Transplantation of genetically modified hepatocytes has been suggested as a therapeutic modality for impaired hepatocellular function. This study examined adenoviral-mediated gene transfer to isolated hepatocytes, under conditions mimicking clinical transplant preservation. Isolated rat hepatocytes were infected using replication-defective adenoviral vectors with an expression cassette containing the beta-galactosidase gene driven by a CMV promoter. Hepatocytes were infected in suspension immediately after isolation, then either cultured or transplanted immediately into a syngeneic host. Gene transfer efficiency was assessed by histochemical staining and FACS analysis for the gene product. The presence of viral DNA and mRNA, as well as viral-derived protein production, were assayed. Efficiency of gene transfer was examined as a function of several preservation conditions. Infection efficiency was best in cells preserved in UW solution, correlated directly with virus:hepatocyte ratio and with length of exposure to virus. Successful infection resulted in significant viral-derived protein production, persisting for at least two weeks in culture. These results demonstrate the versatility of adenoviral vectors in accomplishing rapid and efficient gene transfer into nondividing hepatocytes during cold preservation. Such genetically modified hepatocytes have potential use for immediate transplantation, without the need for further manipulation.

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.

Regeneration-induced accelerated rejection in reduced-size liver grafts.

Liver regenerative processes are associated with enhanced expression of alloantigens. Accordingly, we tested the hypothesis that such enhanced surface expression of alloantigens during regeneration of reduced-size liver grafts is associated with accelerated rejection. Our OLT model was LEW (RT1) to BN (RT1n), with donor liver resected by 50%. The study group consisted of reduced-size allografts. Control groups were syngeneic reduced-size isografts and full-size allografts. Reduced-size isograft recipients survived indefinitely. Both isografts and allografts regenerated to their prereduction size within 12 days. Recipients of reduced-size allografts died of accelerated rejection within 12.2 +/- 0.8 days, significantly earlier than recipients receiving full-size allografts (36.2 +/- 4.1 days, P < 0.01). The accelerated rejection in the regenerating allografts was mediated both by cellular and humoral mechanisms, evidenced by earlier lymphocytic invasion of the graft, enhanced donor MHC class II expression, and the emergence of IgM antibodies, directed specifically against donor endothelial antigens. These data suggest that regeneration of reduced-size allografts is accompanied by accelerated cellularly and humorally mediated alloreactivity. Recipients of reduced-size allografts may, therefore, benefit from more potent immunosuppression during the period of active liver regeneration.

Role of 99mTc-labeled DISIDA scan in the assessment of marginal liver grafts after orthotopic transplantation.

Accurate prognostic indicators are lacking for livers with early marginal graft function, making the decision to re-transplant a difficult one. Therefore, we studied 99mTc-labeled DISIDA scanning as a predictor of recovery of marginal grafts. Records of 28 liver transplant recipients with prolonged periods of marginal graft function after liver transplantation were analyzed. Twelve of 28 (Group I) had delayed PNF and were re-transplanted within 3-8 days (mean 5.3) of surgery. The remaining 16 (Group II) recovered slowly, with normal graft function at 1 month. All patients received DISIDA scans 2 to 5 d after surgery. Clearance of tracer from the blood pool was slower in Group I patients (77S +/- 241 sec) than in Group II (260 +/- 38 sec; p < 0.01). Qualitative differences in the pattern of parenchymal uptake were also noted. Homogenous uptake, consistent with cholestasis, was seen in 15/16 (94%) Group II patients, with improved uptake after 7-35 d. In contrast, 11/12 Group I patients had non-homogenous uptake, consistent with multiple liver infarctions. This pattern correlated with higher peak SGOT in Group I (4358 +/- 658 U/dl vs 1636 +/- 127 U/dl p < 0.01), and PT (20 +/- 0.7 sec vs. 16.5 +/- 0.36 sec; p < 0.01). In summary, delays in DISIDA tracer clearance from blood, and non-homogenous hepatic uptake correlate with elevated liver function tests and with delayed PNF. Homogenous uptake correlates with graft recovery. DISIDA scans may, therefore, be useful in predicting recovery of marginal grafted livers.

Retroviral-mediated gene transfer into rat experimental liver transplant.

Replication-defective retroviral vectors were used for ex vivo gene transfer into rat liver grafts under conditions mimicking clinical liver transplantation. Supernatant containing single- and double-gene vectors encoding for either the human IL-7 and/or neomycin phosphotransferase genes were used to perfuse the liver grafts during cold ischemia before transplantation. Whole liver grafts were perfused with vector supernatant or medium only. Reduced-size liver grafts (50% hepatectomy) were similarly perfused either immediately after reduction or 24 hr later after induction of active hepatocyte division. After transplantation of these grafts in orthotopic position, the liver tissue was removed at specified intervals, and genomic DNA and mRNA were examined for proviral sequences and expression. Stable integration of the proviral sequences was detected only in reduced-size grafts transplanted 24 hr after hepatectomy. Proviral message of both neomycin phosphotransferase and human IL-7 were present up to 21 days after transduction. This study demonstrates efficient ex vivo gene transfer to donor liver grafts. Gene transfer to livers before transplantation carries the potential to modulate immunogenicity and alter the antigraft immune response.

Diagnosis and treatment of bowel perforation following pediatric orthotopic liver transplantation.

OBJECTIVE: Bowel perforation is a frequent cause of mortality after pediatric orthotopic liver transplantation. The aims of this study were to identify the cause of this phenomenon and to examine current methods of treatment. DESIGN: This is a retrospective analysis of 246 pediatric patients who underwent orthotopic liver transplantation at a large, urban, tertiary care medical center between 1984 and 1992. We examined the frequency of bowel perforations after transplantation and identified predisposing factors and signs. In this series, bowel perforations occurred in 24 of 246 recipients and were common in those who had previous liver-related surgery (22 patients). Clinical signs included fever (13 patients), leukocytosis (14 patients), and free air on abdominal roentgenograms (11 patients). RESULTS: Perforation occurred at the Roux-en-Y limb in 15 of 24 recipients as well as in the right transverse colon (five patients), terminal ileum (three patients), and duodenum (one patient). The repair was resection and/or primary closure (18 patients), or diversion (six patients). Recurrent perforations (nine patients) could not be attributed to the method of the repair. Perforation-related sepsis was the primary cause of death in 12 patients (50%) and was more common among patients who developed recurrent perforation (seven [78%] of nine patients). CONCLUSIONS: The occurrence and location of bowel perforation after pediatric orthotopic liver transplantation suggests that the cause is related to bowel injury during difficult hepatectomy. Mortality may be reduced by early second-look operations in high-risk patients.

Airway blood flow modifies allergic airway smooth muscle contraction.

We tested the hypothesis that airway perfusion modifies the contractile response of airway smooth muscle to allergen challenge by influencing the clearance of locally released spasmogens. In six intact, lightly sedated, sheep allergic to Ascaris suum, we measured tracheal mucosal blood flow (Qtr) with a soluble gas uptake method and tracheal dead space (Vtr), an index of airway smooth muscle tone, by helium dilution before and serially after local aerosol challenge with A. suum extract or ragweed extract (control). The former challenge was repeated during continuous intravenous infusion of either vasopressin or nitroglycerin, which by themselves had no effect on Vtr and decreased and increased Qtr, respectively. Ragweed had no effect on Qtr and Vtr, whereas A. suum increased mean (+/- SE) Qtr by 111 +/- 31% (p less than 0.05) and decreased mean Vtr by 15 +/- 2% (p less than 0.05) immediately after challenge, with Qtr returning to baseline by 40 min and Vtr by 80 min. Vasopressin infusion prevented the A. suum-induced increase in Qtr and prolonged the decrease in mean Vtr (p less than 0.05). During nitroglycerin infusion, A. suum failed to alter Qtr or Vtr. Vasopressin and nitroglycerin had no effect on the contractile responses of tracheal smooth muscle to A. suum in vitro. These results indicate that the effects of vasopressin and nitroglycerin on antigen-induced airway smooth muscle contraction in vivo were due to alterations in airway blood flow rather than to alterations in the release of or airway smooth muscle responsiveness to chemical mediators.

Effect of histamine on tracheal mucosal perfusion, water content and airway smooth muscle in sheep.

We examined the response of tracheal mucosal blood flow normalized for systemic arterial pressure (Qtrn), water content (VH20) and luminal dead space (Vtr) to nebulized histamine in intact, lightly anesthetized sheep. Nebulized histamine produced rapid increases in mean Qtrn (+84%) and VH2O (+85%), and a decrease in mean Vtr (-17%) (P less than 0.05) within 5 min post completion of challenge. Mean Vtr rapidly returned to baseline, while mean Qtrn and VH2O remained elevated for 60 and 90 min after challenge, respectively. Pretreatment with chlorpheniramine (H1-antagonist) blocked the changes in Vtr and VH2O, and attenuated the increase in Qtrn. Metiamide (H2-antagonist) pretreatment abolished the increase in Qtrn and blunted the increase in VH2O, but had no effect on the decrease in VTR. 2-methylhistamine (H1-agonist) decreased mean Qtrn and Vtr (P less than 0.05) and dimaprit (H2-agonist) increased mean Qtrn (P less than 0.05) without changing Vtr. Neither 2-methylhistamine nor dimaprit significantly altered VH2O. Atropine blocked histamine induced decreases in Vtr and slightly attenuated the increases in Qtrn and VH2O. Thus, histamine increased airway smooth muscle tone and mucosal water content principally via H1 receptors, and mucosal perfusion via H2 receptors. The airway smooth muscle contraction involved muscarinic pathways.

Airway mucosal blood flow: response to autonomic and inflammatory stimuli.

A major portion of airway blood flow is distributed to the subepithelial tissue space and is strategically located to influence both epithelial and airway smooth muscle functions. To assess the magnitude and responsiveness of blood flow through the subepithelial microvasculature, we measured tracheal mucosal blood flow with a soluble gas method in intact sheep. We found responses of tracheal mucosal blood flow to pharmacological stimuli alpha- and beta- (adrenoceptor agonists) and inflammatory stimuli (antigen and histamine), and demonstrated that alterations in mucosal blood flow influence the magnitude and duration of allergic airway smooth muscle contraction in the trachea. Mucosal blood flow, which under certain circumstances is regulated independently of total airway blood flow, could play a critical role in the manifestations of and recovery from airway disease.

Vasomotion influences airflow in peripheral airways.

The purpose of this study was to test the hypothesis that blood flow, by its effect on blood volume, influences airflow resistance in peripheral airways. In conscious ewes, forced sinusoidal flow oscillations (5 Hz) were applied through a balloon-tipped, dual-channel fiberoptic bronchoscope placed in a segmental bronchus, and peripheral airflow resistance (Rp) was determined from flow and bronchial pressure. Drugs with predominant vascular or airway smooth muscle effects were administered locally through the bronchoscope. Nitroglycerin (NTG) produced a dose-dependent increase in mean Rp (+288% at 1,000 micrograms), which was blocked by methylene blue (p less than 0.05) and not reversed by atropine. Carbachol (CARB) also increased mean Rp in a dose-dependent manner (+605% at 400 micrograms); this effect was not blocked by methylene blue, but it was reversed by atropine (p less than 0.05). The increase in mean Rp after a single dose of NTG (250 micrograms) was sustained for at least 20 min and transiently reversed by vasopressin (0.2 units, p less than 0.05) but not by isoproterenol (100 micrograms). Conversely, the sustained increase in Rp after a single dose of CARB (50 micrograms) was transiently reversed by isoproterenol (p less than 0.05) but not by vasopressin. We conclude that NTG increased Rp by vasodilation and CARB by bronchoconstriction. This supports the hypothesis that vasodilation limits airflow in the lung periphery, presumably because of vascular congestion.