Inhibited growth of colon cancer carcinomatosis by antibodies to vascular endothelial and epidermal growth factor receptors.

Vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) regulate colon cancer growth and metastasis. Previous studies utilizing antibodies against the VEGF receptor (DC101) or EGF receptor (C225) have demonstrated independently that these agents can inhibit tumour growth and induce apoptosis in colon cancer in in vivo and in vitro systems. We hypothesized that simultaneous blockade of the VEGF and EGF receptors would enhance the therapy of colon cancer in a mouse model of peritoneal carcinomatosis. Nude mice were given intraperitoneal injection of KM12L4 human colon cancer cells to generate peritoneal metastases. Mice were then randomized into one of four treatment groups: control, anti-VEGFR (DC101), anti-EGFR (C225), or DC101 and C225. Relative to the control group, treatment with DC101 or with DC101+C225 decreased tumour vascularity, growth, proliferation, formation of ascites and increased apoptosis of both tumour cells and endothelial cells. Although C225 therapy did not change any of the above parameters, C225 combined with DC101 led to a significant decrease in tumour vascularity and increases in tumour cell and endothelial cell apoptosis (vs the DC101 group). These findings suggest that DC101 inhibits angiogenesis, endothelial cell survival, and VEGF-mediated ascites formation in a murine model of colon cancer carcinomatosis. The addition of C225 to DC101 appears to lead to a further decrease in angiogenesis and ascites formation. Combination anti-VEGF and anti-EGFR therapy may represent a novel therapeutic strategy for the management of colon peritoneal carcinomatosis.

Tyrosine kinase inhibition of multiple angiogenic growth factor receptors improves survival in mice bearing colon cancer liver metastases by inhibition of endothelial cell survival mechanisms.

Redundant mechanisms mediate colon cancer angiogenesis. Targeting multiple angiogenic factors simultaneously may improve survival of mice with colon cancer metastases. BALB/c mice underwent splenic injection with CT-26 colon cancer cells to generate liver metastases and received administration of either vehicle alone or a tyrosine kinase inhibitor for vascular endothelial growth factor, basic fibroblast growth factor, and platelet-derived growth factor receptors (SU6668). Mice were sacrificed when they became moribund as determined by a blinded observer. In a parallel experiment, groups of mice were sacrificed at earlier time points to better define the kinetics of the effect of SU6668 on angiogenic parameters over time. SU6668 increased median survival by 58% (P < 0.001) and led to a progressive increase in tumor cell and endothelial cell apoptosis that increased over time. In addition, pericyte vessel coverage and tumor vascularity were significantly decreased in mice treated with SU6668. Based on current knowledge of endothelial cell survival, these data suggest that SU6668 may prevent tumor endothelial cell survival directly (vascular endothelial growth factor) and indirectly (pericyte coverage) by affecting endothelial cell survival mechanisms.

Effects of an antibody to vascular endothelial growth factor receptor-2 on survival, tumor vascularity, and apoptosis in a murine model of colon carcinomatosis.

Vascular endothelial growth factor (VEGF) is the predominant regulator of colon cancer angiogenesis and is associated with a poor prognosis and the development of metastases. We hypothesized that DC101, an antibody against the VEGF receptor-2 (flk-1), may be efficacious in the therapy of colon cancer peritoneal carcinomatosis in a murine model. BALB/c mice underwent intraperitoneal injection of CT-26 colon cancer cells to generate peritoneal metastases. Mice received control solvent or DC101 for up to 60 days. In parallel studies, mice were sacrificed at sequential time points to determine the effect of DC101 on tumor angiogenesis, tumor cell proliferation and apoptosis, and endothelial cell apoptosis. Mice treated with DC101 demonstrated a 30% increase in mean survival. In addition, DC101 also led to a significant decrease in tumor vascularity, growth and tumor cell proliferation. In sequential studies, anti-VEGF-R therapy led to a progressive increase in endothelial cell apoptosis followed by an increase in tumor cell apoptosis. These findings suggest that anti-flk-1 therapy may prolong survival in patients with colon cancer carcinomatosis. The temporal studies demonstrating that anti-flk-1 therapy lead to an increase in endothelial cell apoptosis that in turn lead to an increase in tumor cell apoptosis confirms the role of VEGF as an endothelial cell survival factor.

Molecular biology of hepatitis C infection.

Hepatitis C infection (HCV) is an emerging epidemic. Liver specialists are managing this disease with limited scientific information about the underlying pathogenesis and treatment. The current review offers a molecular dissection of infection, a snapshot of the HCV life cycle, and emerging strategies for antiviral therapy.

Orthotopic liver transplantation for hepatitis C: outcome, effect of immunosuppression, and causes of retransplantation during an 8-year single-center experience.

OBJECTIVE: To determine the outcome of orthotopic liver transplantation (OLT) for end-stage liver disease caused by hepatitis C virus (HCV). SUMMARY BACKGROUND DATA: HCV has become the leading cause of cirrhosis and hepatic failure leading to OLT. Recurrent HCV after OLT is associated with significant complications and may lead to graft loss that requires retransplantation (re-OLT). The authors studied the outcome of transplantation for HCV, the effect of primary immunotherapy, and causes of retransplantation. METHODS: The authors conducted a retrospective review of their experience during an 8-year period (1990-1997), during which 374 patients underwent transplants for HCV (298 [79.6%] received one OLT; 76 [20.4%] required re-OLT). Median follow-up was 2 years (range 0 to 8.3). Immunosuppression was based on cyclosporine in 190 patients and tacrolimus in 132 patients. In a third group of patients, therapy was switched from cyclosporine to tacrolimus or from tacrolimus to cyclosporine (cyclosporine/tacrolimus group). RESULTS: Overall, 1-, 2-, and 5-year actuarial patient survival rates were 86%, 82%, and 76%, respectively. The 2-year patient survival rate was 81 % in the cyclosporine group, 85% in the tacrolimus group, and 82% in the cyclosporine/tacrolimus group. In patients receiving one OLT, overall 1-, 2-, and 5-year patient survival rates were 85%, 81%, and 75%, respectively. The 2-year patient survival rate was 79% in the cyclosporine group, 84% in the tacrolimus group, and 80% in the cyclosporine/tacrolimus group. The overall graft survival rates were 70%, 65%, and 60% at 1, 2, and 5 years, respectively. The graft survival rate at 2 years was similar under cyclosporine (68.5%), tacrolimus (64%), or cyclosporine/tacrolimus (60%) therapy. Re-OLT was required in 42 (11.2%) patients for graft dysfunction in the initial 30 days after OLT. Other causes for re-OLT included hepatic artery thrombosis in 10 (2.6%), chronic rejection in 8 (2.1%), and recurrent HCV in 13 (3.4%) patients. The overall survival rates after re-OLT were 63% and 58% at 1 and 2 years. The 1-year survival rate after re-OLT was 61 % for graft dysfunction, 50% for chronic rejection, 60% for hepatic artery thrombosis, and 60% for recurrent HCV. At re-OLT, 85.3% of the patients were critically ill (United Network for Organ Sharing [UNOS] status 1); only 14.7% of the patients were UNOS status 2 and 3. In re-OLT for chronic rejection and recurrent HCV, the 1-year survival rate of UNOS 1 patients was 38.4%, compared with 87.5% for UNOS 2 and 3 patients. In patients requiring re-OLT, there was no difference in the 1-year patient survival rate after re-OLT when cyclosporine (60%), tacrolimus (63%), or cyclosporine/tacrolimus (56%) was used for primary therapy. With cyclosporine, three patients (1.5%) required re-OLT for chronic rejection versus one patient (0.7%) with tacrolimus. Re-OLT for recurrent HCV was required in four (3%) and seven (3.6%) patients with tacrolimus and cyclosporine therapy, respectively. CONCLUSIONS: Orthotopic liver transplantation for HCV is performed with excellent results. There are no distinct advantages to the use of cyclosporine versus tacrolimus immunosuppression when patient and graft survival are considered. Re-OLT is an important option in the treatment of recurrent HCV and should be performed early in the course of recurrent disease. Survival after re-OLT is not distinctively affected by cyclosporine or tacrolimus primary immunotherapy. The incidence of re-OLT for recurrent HCV or chronic rejection is low after either tacrolimus or cyclosporine therapy.

A simple model to estimate survival after retransplantation of the liver.

To formulate a model predicting survival after liver retransplantation, we analyzed in detail the last 150 cases of hepatic retransplantation at UCLA. Cox proportional hazards regression analysis identified five variables that demonstrated independent simultaneous prognostic value in estimating patient survival after retransplantation: (1) age group (pediatric or adult), (2) recipient requiring preoperative mechanical ventilation, (3) donor organ cold ischemia > or =12 hr, (4) preoperative serum creatinine, and (5) preoperative serum total bilirubin. The Cox regression equation that predicts survival based on these covariates was simplified by assigning individual patients a risk classification based on a 5-point scoring system. We demonstrate that this system can be employed to identify a subgroup of patients in which the expected outcome is too poor to justify retransplantation. These findings may assist in the rational selection of patients suitable for retransplantation.

Gene transfer into nerve and muscle by isolated limb perfusion or during replantation.

In this study, the authors tested the feasibility of adenovirus vectors transferring functional genetic material into relevent soft-tissue structures during replantation of mouse hindlimbs. An adenovirus vector was constructed encoding the marker gene LacZ and CMV promoter and titered by plaque forming assay to 5 x 10(9) particles/ml. C3H mouse hindlimbs were divided into three groups. In Group 1 (n = 9), the femoral neurovascular bundle was divided and re-anastomosed . Group 2 (n = 9) hindlimbs were transected at mid-femur, perfused with adenovirus, and replanted. Group 3 limbs (n = 4) were perfused with saline only, followed by replantation. After 48 hr, morbidity and mortality were assessed, and the replanted limbs were assayed for gene transfer by histochemistry and polymerase chain reaction. 12/18 limbs were viable after 48 hr. Histochemical staining for adenovirus-mediated LacZ expression was positive within skeletal muscle, femoral nerve, and capillaries adjacent to the anastomoses. Distal muscle was also gene transfer positive. PCR analysis confirmed adenovirus-mediated gene transfer within the femoral nerve and skeletal muscle. This study confirms that viral-mediated gene transfer can be accomplished into the soft tissues of a replanted extremity.

Adenovirus-based transfer of wild-type p53 gene increases ovarian tumor radiosensitivity.

The p53 tumor suppressor gene product is known to be active in mediating radiation-induced G1-S cell cycle arrest and apoptosis in a number of normal cell lines. These functions are compromised by inactivation of p53, which promotes tumor progression. Because the p53 gene appears to play an important role in the cellular response to radiation, wild-type p53 gene replacement might be expected to increase the sensitivity of malignant cells with mutant p53 to the cytotoxic effects of ionizing radiation. This study demonstrates that adenovirus (AdV)-mediated transfer and expression of the wild-type p53 in malignant cells lacking the p53 gene results in an increase in cellular radiosensitivity in vitro and tumor radioresponsiveness in vivo. Cultures of the p53 double deletion mutant ovarian cell line SK-OV-3 were infected with nonreplicative adenoviral vectors containing either the wild-type p53 gene (AdVp53) or the luciferase gene (AdVluc). Cultures infected with AdVp53 efficiently expressed wild-type p53 protein and were more sensitive to radiation than uninfected cultures or cultures infected with AdVluc. The ability of AdVp53 to radiosensitize tumors in vivo was tested using SK-OV-3 tumors growing in the flanks of severe combined immune-deficient mice. Intratumoral injection with AdVp53, but not AdVluc, led to enhanced radioresponsiveness and 45% long-term tumor control. These studies demonstrate the ability of AdVp53 to effectively transfer and express p53 protein in established tumors with a resultant increase in radiation responsiveness.

Adenovirus-mediated gene transfer in the transplant setting: early events after orthotopic transplantation of liver allografts expressing TGF-beta1.

We hypothesized that adenovirus mediated gene transfer of TGF-beta1 into liver grafts would enhanced local expression of this recombinant protein and down-regulate inflammatory and alloreactive immune response. A full length DNA encoding the murine TGF-beta1 was used to replaced the E1 region of adenovirus type 5 (AdmTGF-beta1). Expression and protein production of biologically active murine TGF-beta1 was tested in AdmTGF-beta1-transduced Hep G2 cells and TGF-beta-sensitive MV1 cells. In the transplant setting, the replication-defective vector was used to perfused cold preserved ACI liver allograft prior to transplantation into Lewis recipients. Control livers were similarly perfused with cold lactated Ringer's solution and were followed without immunosuppression. Animals were sacrificed at 1, 3, and 5 days after transplantation. Intragraft cytokine levels of TNFalpha, and IFNgamma were determined using ELISA and quantitative PCR. TGF-beta1 ELISA of culture supernatants from AdmTGF-beta1 transduced hepatocyte cell line Hep G2 excreted TGF-beta1 in quantities directly correlated with multiplicity of infection (MOI, vector:hepatic cell ratio). The biological activity of the excreted recombinant protein was confirmed by growth inhibition of MV1 TGF-beta-sensitive cells. Enhanced production of TGF-beta1 in transduced allografts was associated with decreased levels of TNFalpha and IFNgamma when compared with nonimmunosuppressed controls. Adenovirus-mediated gene transfer of murine TGF-beta1 into hepatic cells results in the expression of biologically active protein. Transduction of allografts with TGF-beta1 down-regulates TNFalpha and IFNgamma production early after orthotopic transplantation. Graft transduction with TGF-beta1 offers a novel approach to study the effects of single immune modulator on alloreactive immune response, T cell function, and cytokine cascade.

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.

Etiology and management of symptomatic adult hepatic artery thrombosis after orthotopic liver transplantation (OLT).

Hepatic artery thrombosis (HAT) after adult orthotopic liver transplantation (OLT) is associated with fulminant sepsis and irretrievable loss of the graft. The purpose of this study was 1) to identify recipients at risk for the development of HAT; 2) to define early signs and methods for diagnosis; 3) to determine surgical treatment strategies following diagnosis of HAT. The charts of 680 adults who underwent primary OLT were reviewed. Eleven patients were symptomatic from HAT. Operative data revealed problematic arterial reconstruction in 9/11, and were related to inadequate recipient inflow, necessitating an interposition allogeneic iliac graft in seven patients, or anastomosis to aberrant right hepatic artery in two recipients. Early HAT in 4/11 occurred within 4 weeks after transplantation, whereas late thrombosis in 7/11 was identified 30 days to 1 year after OLT. The postthrombosis course was manifested by elevated liver transaminases (7/11), sepsis and recurrent cholangitis (9/11), or gas gangrene of the liver (4/11). The treatment modalities included thrombectomy and revision of the arterial anastomosis (1/11), emergency hepatectomy with temporary portocaval shunt (2/11), and urgent retransplantation (5/11). Antibiotic therapy and elective retransplantation was the treatment in 4/11. Overall 1-year patient survival and satisfactory graft function was 45 percent.

Adenoviral-p53 gene transfer to orthotopic and peritoneal murine bladder cancer.

PURPOSE: This study was designed to examine the potential for adenoviral-mediated gene therapy in primary and metastatic bladder cancer. MATERIALS AND METHODS: Orthotopic and intraperitoneal bladder tumors were established after delivery of 1 x 10(6) MBT-2 cells into syngeneic mice. Gene transfer was accomplished via intravesical or intraperitoneal instillation by using an E-1 deleted adenovirus encoding LacZ or human p53. Successful tumor transduction was confirmed in tumor DNA and mRNA by polymerase chain reaction. Detection of recombinant gene product was detected by histochemical staining (X-gal) and Western blot. RESULTS: Palpable tumors developed 18 days following implantation. LacZ and p53 mRNA were present in tumor and adjacent normal tissue after bladder and intraperitoneal vector administration. Recombinant gene products were identified by histochemistry and Western blot. CONCLUSION: Bladder tumor-directed gene transfer using adenoviral vectors is an efficient and powerful tool for evaluating the adjuvant role of therapeutic gene products.

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.

Transduction of hepatic allografts achieves local levels of viral IL-10 which suppress alloreactivity in vitro.

The application of gene therapy in transplantation might be targeted at immunoregulation within the donor graft. Viral IL-10 (vIL-10) down-regulates antigen presenting cells (APC) and effector functions in in vitro and in vivo models of alloreactivity. In the current study, we have constructed a replication-defective adenovirus bearing the cDNA encoding viral IL-10 and examined the level and chronicity of its expression in rat liver allografts up to 7 days after orthotopic transplantation. The results demonstrate that liver allografts may be efficiently transfected with adenovirus expressing viral IL-10. Detection of the recombinant viral cytokine was limited to the allograft without measurable levels in peripheral blood. In parallel, the effect of vIL-10 on mixed leukocyte reaction is also assessed using peripheral blood lymphocytes obtained from naive donor and recipient animals. Equivalent levels of viral IL-10 (5-10 ng/ml) achieved after adenovirus-mediated gene transfer suppress the in vitro alloreactivity of peripheral blood lymphocytes up to 70%. Adenovirus-mediated gene transfer of viral IL-10 offers the promise of effectively and favorably altering the alloreactive immune response.

Adenovirus-mediated p53 gene delivery inhibits 9L glioma growth in rats.

Adenoviral vectors have recently been shown to effectively deliver genes into a variety of tissues. Since these vectors have some advantages over the more extensively investigated retroviruses, we studied the effect of two replication-defective adenovectors bearing human wild type tumor suppressor gene p53 (Adp53) and Escherichia coli beta-galactosidase gene (AdLacZ) on 9L glioma cells. Successful in vitro gene transfer was shown by DNA polymerase chain reaction (PCR), and expression was confirmed by reverse transcriptase RNA PCR and Western blot analyses. Transduction of 9L cells with the Adp53 inhibited cell growth and induced phenotypic changes consistent with cell death at low titers, while AdLacZ caused cytopathic changes only at high titers. Stereotactic injection of AdLacZ (10(7) plaque forming units) into tumor bed stained 25 to 30% of tumor cells at the site of vector delivery. Injection of Adp53 (10(7) plaque forming units), but not AdLacZ (controls), into established 4-day old 9L glioma brain tumors decreased tumor volume by 40% after 14 days. As a step toward gene therapy of brain tumors using replication-defective adenoviruses, these data support the use of tumor suppressor gene transfer for in vivo treatment of whole animal brain tumor models.

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.