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Biomedical subjects

R W Busuttil

Publications and source records attributed to R W Busuttil.

At least 163 records · Page 9Linked to original sources
PubMed1994

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.

Adenoviridae↗
PubMed1994

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.

Adenoviridae↗
PubMed1994

Intragraft expression of messenger RNA for interleukin-6 and tumor necrosis factor-alpha is a predictor of rat small intestine transplant rejection.

Detection of rejection after small intestine transplantation (SIT) is difficult, relying largely on histopathology. The purpose of this study was to determine if the intragraft expression of messenger RNA (mRNA) for interleukin-2 receptor (IL-2R), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF) correlated with rejection in a unidirectional, heterotopic rat SIT model. Graft samples were obtained on postoperative day (POD) 3, 5, 7, 8, 9, 10, 12, and 14. After staining, formalin-fixed samples were blindly evaluated for rejection. Reverse transcriptase polymerase chain reaction (rtPCR) using primers specific for beta-actin, IL-2R, IL-6, and TNF was performed on liquid nitrogen-frozen samples. Semiquantitation was accomplished using radionuclide incorporation and beta-scintillation counting. Intestinal histopathology in all isografts (ISO) and POD 3 allografts (ALLO) was normal. Rejection progressed in ALLO from mild on POD 5 to severe by POD 8. rtPCR analysis revealed constitutive expression of IL-2R mRNA in both ISO and ALLO. TNF and IL-6 demonstrated significant increases in mRNA expression in ALLO compared to ISO beginning on POD 5. In summary, intragraft expression of IL-2R mRNA demonstrated late up-regulation in ALLO which did not correlate with rejection. TNF and IL-6 mRNA expression predicted rat SIT rejection. rtPCR analysis of TNF and IL-6 may serve as a useful diagnostic adjunct for rat SIT rejection.

Animals↗
PubMed1994

Current treatment modalities for hepatocellular carcinoma.

OBJECTIVE: This study evaluated the currently available treatment modalities for hepatocellular carcinoma (HCC). SUMMARY BACKGROUND DATA: One of the most common tumors worldwide, HCC has several known risk factors. Untreated HCC typically has a dismal prognosis. Early detection remains the key to successful treatment of this malignancy. Surgical resection has been the mainstay of treatment for HCC, but newer modalities have been recently introduced. METHODS: The authors evaluated the treatment modalities for HCC. RESULTS: Surgical resection affords 5-year survival rates as high as 45% with more favorable subgroups having 1) small tumors, 2) well-differentiated tumors, 3) unifocal tumors, 4) lack of vascular invasion, 5) absence of cirrhosis, and 6) the fibrolamellar variant (FL-HCC). Resection has been limited primarily by low resectability rates and recurrent disease. Newer therapeutic modalities that appear the most promising are transarterial chemoembolization and percutaneous ethanol injection. Neither therapy has been evaluated in a prospective randomized manner. Combination chemotherapy and surgical intervention may provide the best results, but randomized controlled trials with long-term follow-up are needed. As single-treatment modalities, radiation therapy, intravenous chemotherapy, intra-arterial chemotherapy, and immunotherapy play limited palliative roles. CONCLUSIONS: Surgical resection in the form of partial or total hepatectomy is the preferred treatment for HCC. The early detection of tumors by screening high-risk populations is crucial. Randomized trials of combinations of chemotherapy and surgical resection are needed to demonstrate their potential utility for treatment.

Carcinoma, Hepatocellular↗
PubMed1994

One thousand liver transplants. The lessons learned.

OBJECTIVE: To evaluate the first 1000 liver transplants performed at UCLA Medical Center to determine factors responsible for improved results. SUMMARY BACKGROUND DATA: Liver transplant has evolved impressively since the first case was performed in 1963. The 1980s have highlighted this progress with the development of better organ preservation, standardization of operative procedure, improved immunosuppressive agents, and better understanding of patient selection. METHODS: The first 100 consecutive liver transplants (group 1) performed from February 1984 through October 1986 were compared with the last 200 (group 2) performed between September 1991 and June 1992. An analysis was made of donor use; changes in patient candidacy; patient care variables; morbidity and mortality; survival data; and hospital resource use. RESULTS: In group 1, 31% of donors were refused because of medical unsuitability compared with 4% in group 2 (p < 0.0001). In group 1, alcoholic patients comprised 1% of liver transplant candidates compared with 20% group 2 (p < 0.0001). High-risk patients (United Network for Organ Sharing criteria 4) only comprised 11% of patients in group 1 compared with 37% in group 2 (p < 0.0001). Operative time (7.6 hours compared with 5.4 hours), packed cell replacement (17 units compared with 9.5 units), intensive care unit stay (10 days compared with 5 days), and hospital stay (50 days compared with 31 days) were all significantly improved (p < 0.05). In group 1, the 1-year survival rate was 73% and improved to 88% in group 2 (p < 0.0001). CONCLUSIONS: Despite unfavorable donor characteristics (obesity, cause of death, age, hypotension), most organs function well and should not be refused based on history alone. The older and high-risk patient (renal failure, ventilator dependence, portal vein pathology, and so on) is routinely transplanted with good success. Despite liberalization of both donor and recipient criteria, patient survival after liver transplant is improved, use of hospital resources is maximized, and cost reduction is achieved.

Adult↗
PubMed1994

Surgical anatomy of the hepatic arteries in 1000 cases.

OBJECTIVE: Anatomic variations in the hepatic arteries were studied in donor livers that were used for orthotopic transplantation. SUMMARY BACKGROUND DATA: Variations have occurred in 25% to 75% of cases. Donor livers represent an appropriate model for study because extrahepatic arterial anatomy must be defined precisely to ensure complete arterialization of the graft at time of transplantation. METHODS: Records of 1000 patients who underwent liver harvesting for orthotopic transplantation between 1984 and 1993 were reviewed. RESULTS: Arterial patterns in order of frequency included the normal Type 1 anatomy (n = 757), with the common hepatic artery arising from the celiac axis to form the gastroduodenal and proper hepatic arteries and the proper hepatic dividing distally into right and left branches; Type 3 (n = 106), with a replaced or accessory right hepatic artery originating from the superior mesenteric artery; Type 2 (n = 97), with a replaced or accessory left hepatic artery arising from the left gastric artery; Type 4 (n = 23), with both right and left hepatic arteries arising from the superior mesenteric and left gastric arteries, respectively; Type 5 (n = 15), with the entire common hepatic artery arising as a branch of the superior mesenteric; and Type 6 (n = 2), with the common hepatic artery originating directly from the aorta. CONCLUSIONS: These data are useful for the planning and conduct of surgical and radiological procedures of the upper abdomen, including laparoscopic operations of the biliary tract.

Hepatic Artery↗
PubMed1994

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.

Animals↗
PubMed1994

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.

Animals↗
PubMed1994

High one-month liver graft failure rates in flow cytometry crossmatch-positive recipients.

In a retrospective study of 84 primary cadaver donor orthotopic liver transplants (OLTs), we investigated the damaging effect of preformed donor-specific antibodies by a standard T-cell warm crossmatch test (TWXM) and a more sensitive flow cytometry crossmatch test (FCXM). The incidence of a positive crossmatch was 12% (10/84) by TWXM and 24% (20/84) by FCXM, respectively. The incidence of 1-month graft failure was 30% (3/10) for patients with a positive TWXM, compared with 23% (17/74) for a negative TWXM. The deleterious effect was more pronounced when the patients were examined by FCXM. Nine of 20 (45%) FCXM-positive recipients lost their grafts within 1 month (p = 0.01) and 8 of those 9 failures occurred within 2 weeks posttransplant. A comparison of graft survival rates at 6 months demonstrated that FCXM-positive patients had a 25% lower survival rate (p = 0.01) than those with a negative FCXM. The positive rate of FCXM was 4 times higher in sensitized patients (PRA 11-100). However, 9 patients in the non-sensitized group (PRA 0-10) had a positive FCXM, and 5 of 9 patients had graft failure within 1 month, suggesting that even a low level of antibodies or non-complement fixing antibodies may have a very damaging effect. This study shows that preformed donor-specific antibodies do have a deleterious effect on allografts. Although the effect is not necessarily expressed as hyperacute rejection, the antibody response is capable of damaging the allografted organs within 1 month. Consideration should be given to crossmatching OLT patients in order to avoid transplantation over a positive crossmatch.

Adult↗
PubMed1994

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.

Adenoviridae↗
PubMed1994

Metastatic hepatocellular carcinoma to bone in a liver transplant patient. A case report.

Although previously unreported in the orthopaedic literature, hepatocellular carcinoma metastasizes to bone in 2 to 20% of cases. The poor prognosis associated with this tumor often results in patient demise before the need for orthopaedic intervention. A liver transplant recipient with no known history of hepatocellular carcinoma was diagnosed with this tumor when open reduction and internal fixation of an impending pathologic femoral fracture were performed. Metastatic hepatocellular carcinoma should be considered in the differential diagnosis of a lytic osseous metastasis in appropriate patient populations.

Adult↗
PubMed1993

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.

Child↗
PubMed1993

FK506 conversion for intractable rejection of the liver allograft.

Twenty-seven liver transplant recipients with intractable, biopsy-proven, acute or chronic rejection (defined as vanishing bile duct syndrome) were converted from cyclosporin to FK506. Successful conversion was achieved in 9 of 15 patients with acute rejection and in 6 of 12 patients with vanishing bile duct syndrome. A normal bilirubin was achieved more quickly in those with acute rejection (within 1 month) than in those with chronic rejection (within 3 months). A preconversion total bilirubin of less than 12 mg/dl was considered significant with regard to a successful outcome (P = 0.002). Graft survival was 66.7% and patient survival 73% in the case of acute rejection, and 50% and 66.7%, respectively, in the case of chronic rejection. Nephrotoxicity, neurotoxicity, and gastrointestinal side effects were the most serious complications of FK506 conversion. Six of ten patients had a drop in GFR that was 50% or greater after a minimum of 1 month of FK506 exposure. The mean maintenance dose of FK506 to maintain FK506 serum levels of 0.5-1.5 ng/ml was 0.07 mg/kg per 12 h for adults (half the recommended dose), compared to 0.15 mg/kg per 12 h for pediatric patients. This study demonstrates that FK506 can be used successfully to convert patients with intractable acute and chronic rejection. Careful adjustments of FK506 dosages and levels are required to minimize side effects.

Acute Disease↗