Biomedical subjects

S E Raper

Publications and source records attributed to S E Raper.

At least 55 records · Page 3Linked to original sources

PubMed1995

Hepatocyte transplantation and gene therapy.

Trials of liver-directed gene therapy are a clinical reality. Technology is rapidly developing for the isolation of large numbers of human hepatocytes. Recombinant DNA techniques are available to engineer recombinant retroviruses which express genes of clinical relevance. Familial hypercholesterolemia serves as the clinical paradigm for liver-directed gene therapy, and the clinical protocol is underway. Early results are promising. Techniques for hepatocyte delivery, optimization of gene transfer, and safety considerations are important considerations in designing a human trial. Other diseases which are likely to be among the first treated with this new approach are ornithine transcarbamylase deficiency, hemophilia B, and Crigler-Najjar syndrome.

Amino Acid Metabolism, Inborn Errors↗

PubMed1995

Gene therapy for human liver disease.

Investigators working in the area of gene therapy believe the potential for advances in all medical disciplines is enormous. It is humbling, however to appreciate how far we need to go, as the field is truly in its infancy. Gene transfer technologies currently under evaluation in clinical trials have major limitations. Vector systems used in the clinics by the year 2000 probably have yet to be discovered. An additional lesson learned is that efforts at gene therapy are hampered by a lack of knowledge of the basic biology of the target organ and pathogenesis of the underlying disease. Successful gene therapy programs will critically evaluate the field and through fundamental research move steadily forward toward the long-term goal of truly effective therapy for a wide spectrum of disease. In the near future, two liver diseases are the most likely to be treated with gene therapy. The evaluation of patients with familial hypercholesterolemia is ongoing, and once approved, more candidates will be enrolled for therapy. Progress also has been made in creating vectors for the treatment of ornithine transcarbamylase deficiency. It remains to be seen whether adenoviruses or retroviruses will be used first in attempts to control this disease. Although the inflammatory response noted with current recombinant viruses is a formidable problem, the efficiency of gene transfer into the liver with these vectors makes continued study worthwhile.

Adenoviridae↗

PubMed1994

In vivo correction of low density lipoprotein receptor deficiency in the Watanabe heritable hyperlipidemic rabbit with recombinant adenoviruses.

A rabbit animal model of the human disease familial hypercholesterolemia (FH), which is the result of low density lipoprotein (LDL) receptor deficiency, was used to develop an in vivo approach to gene therapy based on recombinant adenoviruses. Recombinant, replication-defective adenoviruses expressing the lacZ gene under the control of different promoters were infused into the portal circulation of New Zealand White (NZW) rabbits. Expression of lacZ could be obtained in virtually all hepatocytes within 3 days post-infusion, but was undetectable by 3 weeks. This was not associated with liver pathology. An LDL receptor expressing adenovirus was constructed using the most active promoter and was infused into the portal vein of rabbits deficient in LDL receptor. Analysis of liver tissues harvested 3 days after virus infusion demonstrated human LDL receptor protein in the majority of hepatocytes that exceeded the levels found in human liver by at least 10-fold. Transgene expression was stable for 7-10 days and diminished to undetectable levels within 3 weeks. Infusion of LDL receptor expressing virus led to substantial reductions in serum cholesterol that returned to base line within 3 weeks; this acute reduction in serum cholesterol was associated with accumulations of lipid in hepatocytes. The development of neutralizing antibodies to the recombinant adenovirus markedly diminished the effectiveness of a second dose. These studies illustrate the advantages of recombinant adenoviruses for the treatment of liver metabolic diseases and define issues, such as viral genome instability and blocking immune response, that need to be overcome before the promise of this technology can be fully realized.

Adenoviruses, Human↗

PubMed1994

Plasminogen activator inhibitor-1 is an immediate early response gene in regenerating rat liver.

Plasminogen activator inhibitor-1 (PAI-1), a M(r) 50,000 serine protease inhibitor, is the major physiological inhibitor of plasminogen activation. Quiescent rat hepatocytes do not express the PAI-1 gene in vivo; however, PAI-1 is synthesized both by primary cultures of rat hepatocytes and by hepatoma cells in vitro. Furthermore, PAI-1 is expressed by fibroblastic cells in vitro, in response to mitogen stimulation, suggesting a possible connection between hepatocyte PAI-1 expression and cell proliferation. To determine whether PAI-1 is an early growth response gene in hepatocytes in vivo, we analyzed its expression in regenerating rat liver. Male rats underwent partial (70%) hepatectomy (PH) or sham operation (SO), and liver samples were analyzed by Northern blot analysis and in situ hybridization. PAI-1 mRNA was not present at time 0 h, nor at any other time in SO rats but was induced rapidly in regenerating livers, peaking at 2 h and declining to negligible levels by 8 h posthepatectomy. This induction was not inhibited by cycloheximide. In situ hybridization analysis localized PAI-1 transcripts to hepatocytes. Immunohistochemical analysis demonstrated PAI-1-specific staining in hepatocytes in the livers of both PH and SO rats, but the temporal and spatial distribution profiles differed between PH and SO rats. Our studies demonstrate that PAI-1 is an immediate early response gene, transiently expressed in regenerating liver, expression of which may be important in hepatocyte growth and proliferation in vivo.

Animals↗

PubMed1994

Vasopressin stimulates DNA synthesis in cultured rat hepatocytes.

Liver regeneration following partial hepatectomy is significantly impaired in rats with hereditary vasopressin (AVP) deficiency. This suggested that AVP might have a direct effect on cultured rat hepatocytes. Hepatocytes from male Sprague-Dawley rats were isolated using a two-step collagenase perfusion technique and plated at a density of 10(5)/16-mm Primaria plate. After a suitable attachment period, hepatocytes were incubated with minimal essential media, AVP, AVP plus a specific AVP antagonist, or oxytocin. Hepatocyte proliferation was measured by [3H]thymidine incorporation ([3H]Thy) into hepatocyte DNA. AVP (10 nM) increased [3H]Thy significantly (and this effect was blocked by an AVP-specific antagonist (50 nM). Oxytocin had no effect on hepatocyte DNA synthesis. To further investigate the influence of AVP on hepatocyte proliferation, the effect of AVP on transforming growth factor-alpha (TGF-alpha)-stimulated hepatocyte proliferation was also studied. This combination was chosen based on the ability of AVP to inhibit the biologic effects of EGF (a TGF-alpha analog). There was significant attenuation of TGF-alpha (50 nM)-stimulated [3H]Thy in the presence of AVP (10 nM). In summary: (1) AVP stimulates proliferation of cultured rat hepatocytes. (2) The effect of AVP can be significantly abolished by a specific AVP antagonist. (3) The proliferative response of AVP is specific. (4) AVP significantly attenuates TGF-alpha-stimulated hepatocyte hepatic DNA synthesis. Further studies should elucidate the mechanisms for the effects of AVP on hepatic proliferation alone or in combination with other factors.

Animals↗

PubMed1994

Successful ex vivo gene therapy directed to liver in a patient with familial hypercholesterolaemia.

An ex vivo approach to gene therapy for familial hypercholesterolaemia (FH) has been developed in which the recipient is transplanted with autologous hepatocytes that are genetically corrected with recombinant retroviruses carrying the LDL receptor. We describe the treatment of a 29 year old woman with homozygous FH by ex vivo gene therapy directed to liver. She tolerated the procedures well and in situ hybridization of liver tissue four months after therapy revealed evidence for engraftment of transgene expressing cells. The patient's LDL/HDL ratio declined from 10-13 before gene therapy to 5-8 following gene therapy, improvements which have remained stable for the duration of the treatment (18 months). This represents the first report of human gene therapy in which stable correction of a therapeutic endpoint has been achieved.

Adult↗

PubMed1994

Enucleation combined with hepatic vascular exclusion is a safe and effective alternative to hepatic resection for liver cell adenoma.

Liver cell adenomas are benign, rare tumors that occur primarily in women with a history of oral contraceptive use. Surgical treatment is recommended to reduce the risk of sudden, unpredictable hemorrhage or malignant transformation, but mortality rates of 5 to 8 per cent are reported for major hepatic resections. This report summarizes an experience of eight patients with liver cell adenoma treated by enucleation or resection, alone or combined with hepatic vascular exclusion (HVE). The latter technique, originally described in the mid-1960s, markedly reduces operative blood loss associated with hepatic resection. All eight patients were women of child-bearing age, and seven reported previous oral contraceptive use. The majority of patients were asymptomatic at the time of presentation. Six patients (75%) had a single adenoma and the remaining two had multiple lesions. HVE was used successfully in four patients with no perioperative mortality and reduced by nearly 50 per cent the intraoperative blood loss compared to conventional hepatic resection (1635 mL versus 3875 mL, respectively). Six evaluable patients were followed for an average of 65 months, with no evidence of liver dysfunction or adenoma recurrence. Enucleation of liver cell adenoma appears to be a safe alternative to formal hepatic resection and provides excellent long-term results. HVE markedly reduces operative blood loss, has no adverse effect on metabolic function, decreases the potential health risk associated with transfusion, and should be used whenever possible.

Adenoma, Liver Cell↗

PubMed1993

An approach for treating the hepatobiliary disease of cystic fibrosis by somatic gene transfer.

Cystic fibrosis (CF) is an inherited disease of epithelial cell ion transport that is associated with pathology in multiple organ systems, including lung, pancreas, and liver. As treatment of the pulmonary manifestations of CF has improved, management of CF liver disease has become increasingly important in adult patients. This report describes an approach for treating CF liver disease by somatic gene transfer. In situ hybridization and immunocytochemistry analysis of rat liver sections indicated that the endogenous CFTR (cystic fibrosis transmembrane conductance regulator) gene is primarily expressed in the intrahepatic biliary epithelial cells. To specifically target recombinant genes to the biliary epithelium in vivo, recombinant adenoviruses expressing lacZ or human CFTR were infused retrograde into the biliary tract through the common bile duct. Conditions were established for achieving recombinant gene expression in virtually all cells of the intrahepatic bile ducts in vivo. Expression persisted in the smaller bile ducts for the duration of the experiment, which was 21 days. These studies suggest that it may be feasible to prevent CF liver disease by genetically reconstituting CFTR expression in the biliary tract, using an approach that is clinically feasible.

Adenoviridae↗

PubMed1993

The effects of transforming growth factor alpha and somatostatin on regenerating hepatocytes in the rat.

Transforming growth factor alpha (TGF alpha) stimulates DNA synthesis in adult rat hepatocytes, and plays a physiological role after partial hepatectomy by an autocrine mechanism. Somatostatin (SS-14) is a potent inhibitor of gastrointestinal function and inhibits proliferation in various cell types. We examined the proliferative effect of TGF alpha and the inhibitory effect of SS-14 on hepatocytes isolated at various times after partial hepatectomy. To study the mechanism of SS-14 further, we treated rats with the long acting SS-14 analog, octreotide, before or after 70% hepatectomy to determine whether or not a differential effect could be seen. We confirmed the proliferative effects of TGF alpha, and the inhibitory action of SS-14 in the early phase of liver regeneration in vitro. Regenerating hepatocytes isolated from hepatectomized livers respond to TGF alpha only at early time points (2 h) but do not respond to SS-14. In addition, the long acting SS-14 analog, octreotide, inhibited hepatic regeneration only when administered prior to hepatectomy. We conclude that exogenous peptide stimulation is effective only in the early phase of the hepatic proliferative response. After the initial changes brought about by hepatectomy, subsequent steps of the regenerative process appear refractory to external stimuli.

Animals↗

PubMed1993

Hepatectomy impairs hepatic processing of somatostatin-14.

Somatostatin-14 (SS-14) inhibits the hepatotrophic effect of a variety of growth factors in cultured hepatocytes. We hypothesized that hepatic somatostatin processing might be altered during regeneration. Male Sprague-Dawley rats underwent the intraportal injection of radiolabeled SS-14 after sham or 70% hepatectomy. To study the mechanisms of hepatic SS-14 transport in the rat, the lysosomal enzyme inhibitors, chloroquine and leupeptin, and the microtubule inhibitors, vinblastine and colchicine, were administered 1 to 2 hours prior to the intraportal injection of SS-14. Bile was collected, organs were weighed, and radioactivity was quantitated. The analysis of serial timed collections of bile revealed that, for saline, chloroquine, and leupeptin, peak biliary radioactivity appeared at 20 minutes. Pretreatment with vinblastine and colchicine abolished the 20-minute peak of radioactivity. The appearance of biliary and hepatic iodine 125-SS-14 (125I-[tyr11]-SS-14) at various times after 70% hepatectomy showed a significant decrease starting at 2 hours, which persisted for up to 24 hours. In regenerating liver, both vinblastine and chloroquine decreased 125I-[tyr11]-SS-14 in bile and the liver. In summary, after sham or 70% hepatectomy, vinblastine and colchicine inhibit biliary and increase hepatic 125I-[tyr11]-SS-14 accumulation. After 70% hepatectomy was performed, chloroquine also inhibited 125I-[tyr11]-SS-14 accumulation. We concluded that an important mechanism for hepatic regeneration is decreased responsiveness to SS-14, by decreased SS-14 uptake and increased SS-14 degradation.

Animals↗

PubMed1993

A novel approach for introducing hepatocytes into the portal circulation.

Many attempts have been made to treat metabolic diseases of the liver. One approach, called ex vivo gene therapy, is based on correcting the genetic defect in the patient's hepatocytes. In this model, the liver is resected and hepatocytes are isolated, plated in culture, and genetically corrected with recombinant retroviruses. The cells are then harvested and infused into the portal circulation, where they seed in the sinusoids of the liver. In this study, a canine model was used to develop an approach to ex vivo gene therapy that requires only one surgical procedure. The basic concept is to place a catheter in the mesenteric circulation at the time of liver resection that can be used subsequently to deliver hepatocytes into the liver. With four 18 to 28 kg mongrel dogs, a laparotomy was performed, the left lateral segment of the liver was resected, and a 9.5 Fr Hickman catheter was introduced into the inferior mesenteric vein. At 10 days, the catheter was studied angiographically and removed. The catheter remained patent and was easily removed without untoward effects. A subsequent necropsy was performed to evaluate the consequences of this procedure. There was no hematoma at the site of the catheter placement or evidence for intraportal clot. This approach has been used in subsequent studies to treat a patient with familial hypercholesterolemia.

Animals↗

PubMed1992

Identification and partial characterization of a somatostatin-14 binding protein on rat liver plasma membranes.

Binding of somatostatin-14 to rat liver plasma membranes was characterized with 125-labeled[tyr11] somatostatin-14. Binding at 24 degrees C reached a plateau at 50 min and was reversible by synthetic somatostatin-14. Scatchard analysis revealed a single class of binding sites (affinity constant = 2.4 +/- 0.2 nmol/L, binding capacity = 148 +/- 0.02 fmol/mg protein). Specificity for somatostatin-14 was demonstrated by the inhibition of 125I-[tyr11]somatostatin-14 binding by biologically active somatostatin analogs but not by a biologically inactive somatostatin analog or unrelated peptides. The radioiodinated binding site complex could be cross-linked with disuccinimidyl suberate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel autoradiography revealed a 70,000-Da band. Dithiothreitol, a reducing reagent, did not alter the mobility of the band, and the band could be abolished in the presence of 10 mumol/L synthetic somatostatin-14. Covalently cross-linked, iodinated binding protein complexes could be solubilized by the nonreducing detergents Zwittergent 3-12 and 3-([3-cholamidopropyl] diethylammonio)-1-propanesulfonic acid (CHAPS). Solubilized complex bound to wheat-germ agglutinin-agarose columns and was eluted by N,N',N"-triacetylchitotriose. Binding to wheat-germ agglutinin agarose columns was lost after pretreatment with endo-beta-N-acetylglucosaminidase F. Binding studies with liver plasma membranes, 125I-labeled[tyrosine11]somatostatin-14 and guanine nucleotides showed inhibition of binding in the presence of guanine nucleotides. These results indicate that the purified rat liver plasma membranes contain a specific binding protein for somatostatin-14, the binding protein appears to be glycosylated and somatostatin-14 binding to rat liver plasma membranes may be regulated by G proteins.

Adenosine Diphosphate↗

PubMed1992

Inhibition of DNA synthesis by somatostatin in rat hepatocytes stimulated by hepatocyte growth factor or epidermal growth factor.

The antiproliferative effects of somatostatin on hepatocytes stimulated by hepatocyte growth factor (HGF) or epidermal growth factor (EGF) were investigated using primary cultures of adult rat hepatocytes. Somatostatin inhibits HGF-induced (at a dose of 10 ng/mL) or EGF-induced (at a dose of 100 ng/mL) 3H-thymidine incorporation into hepatocytes in a dose-dependent manner (10(-10) to 10(-8) M). This inhibition was confirmed by autoradiography. The effect of somatostatin was nontoxic as judged by preserved albumin synthesis, a marker for differentiated hepatocyte function. In the presence or absence of somatostatin, neither HGF nor EGF significantly altered intracellular cyclic adenosine monophosphate (cAMP). We conclude that somatostatin is a potent inhibitor of HGF- or EGF-induced deoxyribonucleic acid synthesis in adult rat hepatocytes. The mechanism of this inhibition appears to be independent of cAMP. The significance of somatostatin in liver regeneration has yet to be assessed.

Animals↗

PubMed1992

Transforming growth factor-alpha (TGF-alpha) improves hepatic DNA synthesis after hepatectomy in cirrhotic rats.

Impaired liver regeneration in cirrhosis complicates the surgical treatment of liver tumors which arise in this setting. We developed a rat model to investigate the regenerative response of cirrhotic liver after hepatectomy and studied the effect of exogenous transforming growth factor-alpha (TGF-alpha), a potent liver mitogen. Micronodular cirrhosis was established by the simultaneous administration of CCl4 and phenobarbital. Hepatic DNA synthesis ([3H]thymidine incorporation into DNA) 24 hr after partial hepatectomy in cirrhotic rats was 15.6 +/- 3.4 cpm/micrograms DNA (means +/- SEM), which was significantly lower than in normal rats (37.3 +/- 3.4 cpm/micrograms DNA, P less than 0.05). Exogenous TGF-alpha (30 nmol/kg, sc every 12 hr) significantly improved [3H]thymidine incorporation (35.6 +/- 8.2 cpm/micrograms DNA, P less than 0.05). An autoradiographic nuclear labeling index also confirmed increased DNA synthesis (6.7% vs 13.4%). TGF-alpha had no effect on normal regenerating liver (42.5 +/- 8.8 cpm/micrograms DNA, NS). Although the significance of TGF-alpha-enhanced liver regeneration in cirrhosis has yet to be assessed, this model may be useful for the study of mechanisms which control hepatic proliferation.

Animals↗

PubMed1992

Ex vivo gene therapy of familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is an autosomal dominant disorder caused by a deficiency in the receptor that clears low density lipoprotein (LDL) from the serum (reviewed in Ref. 1 and 2). Patients with one abnormal LDL receptor allele have moderate elevations in plasma LDL and suffer premature coronary artery disease (CAD). Approximately 5% of all patients under 45 who have had a myocardial infarction carry this trait. Patients with two abnormal LDL receptor genes (homozygous deficient patients) have severe hypercholesterolemia and life-threatening coronary artery disease in childhood. Strategies for treating patients with FH are directed at lowering the plasma level of LDL. In heterozygotes, this is accomplished through the administration of drugs that stimulate the expression of LDL receptor from the normal allele (2). This therapeutic approach is not effective in the treatment of homozygous deficient patients, especially those that retain less than 2% of residual LDL receptor activity. Partial amelioration of hyperlipidemia has been achieved in some homozygous deficient patients by diverting the portal circulation through a portacaval anastomosis (3) and by chronic plasmapheresis therapy (4). A more direct approach has been to correct the deficiency of hepatic LDL receptor by transplanting a liver that expresses normal levels of LDL receptor. Three patients that survived this procedure normalized their serum LDL-cholesterol (5-9). We have used an authentic animal model for FH, the Watanabe Heritable Hyperlipidemic rabbit (WHHL), to develop gene therapies for the homozygous form of FH (10-13). The WHHL rabbit has a mutation in its LDL receptor gene which renders the receptor completely dysfunctional (12) leading to severe hypercholesterolemia, diffuse atherosclerosis, and premature death. The potential efficacy of gene therapy for FH is supported by a series of studies we have performed in the WHHL rabbit in which we have achieved metabolic improvement (14-18). Liver tissue was removed from WHHL rabbits and used to isolate hepatocytes and establish primary cultures. A functional rabbit LDL receptor gene was transduced into a high proportion of hepatocytes using recombinant retroviruses, and the genetically corrected cells were transplanted into the animal from which they were derived. Transplantation of the genetically corrected, autologous hepatocytes was associated with a 30-40% decrease in serum cholesterol that persisted for the duration of the experiment (4 months, Ref. 18). Recombinant derived LDL receptor RNA was detected in liver for at least 6 months. There was no apparent immunological response to the recombinant derived LDL receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗

PubMed1992

Transplantation of genetically modified autologous hepatocytes into nonhuman primates: feasibility and short-term toxicity.

Ex vivo gene therapy directed to the liver is being developed for the treatment of inherited metabolic diseases. Transplantation of hepatocytes that have been transduced with a low-density lipoprotein (LDL) receptor gene is a potential form of therapy for familial hypercholesterolemia (FH). We have demonstrated efficacy of ex vivo gene therapy for familial hypercholesterolemia in a rabbit animal model of this disease. In preparation for human trials, we describe in this report experiments in baboons for documentation of the feasibility and safety of autologous hepatocyte transplantation. Three baboons underwent a partial hepatectomy and their hepatocytes were isolated, cultured, and transduced with a retrovirus containing the human LDL receptor gene. The hepatocytes were harvested and infused into an indwelling catheter that had been placed into the inferior mesenteric vein at the time of liver resection. The baboons tolerated the procedures well and are being maintained and clinically evaluated for an indefinite time period. Follow-up evaluations have ranged from 3 to 8 months. Clinical evaluations have been unremarkable and blood chemistry and hematology determinations have stayed within normal limits.

Animals↗

PubMed1992

Retroviral-mediated gene transfer in human hepatocytes.

BACKGROUND: The ability to modify human hepatocytes genetically is an essential first step in the development of liver-directed ex vivo gene therapy for inherited metabolic disease. The purpose of these studies was to prove that the genome of human hepatocytes can be altered successfully to express foreign genetic material. METHODS: Human hepatocytes were plated at 2 or 4 x 10(6) cells/10 cm Primaria (Falcon, Oxnard, Calif.) plates. Fresh virus from the amphotropic viral producer cell line BAG, containing the Escherichia coli beta-galactosidase gene lacZ, was placed directly onto hepatocyte cultures and quantitative analysis of cells staining positive for the lacZ gene was undertaken. In a different human liver, a variety of viruses from producer cell lines containing clones of the human low-density lipoprotein (LDL) receptor were plated directly on cultures of human hepatocytes, and gene transfer was demonstrated by increased uptake of fluorescent-labeled LDL. RESULTS: Beta-galactosidase production in hepatocytes was assayed histochemically with the chromogenic substrate X-gal. The highest percentage of cells staining positive for expression of enzyme was seen at 4 x 10(6) cells/plate (43.66% +/- 1.02% vs 27.99% +/- 2.31%). Gene transfer was also documented by the uptake of fluorescent-labeled LDL with a variety of different vectors containing the human LDL receptor. CONCLUSIONS: (1) Human hepatocytes can be cultured in vitro and are susceptible to retroviral infection, (2) functional gene transfer is demonstrated by intracellular function of foreign genes, and (3) the level of expression appears dependent on plating density. We conclude that human hepatocytes are suitable targets for genetic manipulation and may play an important role in human gene therapy trials.

Carbocyanines↗

PubMed1991

Extracorporeal lithotripsy. An important adjunct in the nonoperative management of retained or recurrent bile duct stones.

Retained or recurrent bile duct stones can be successfully removed in up to 80% to 85% of patients with the use of percutaneous or endoscopic techniques. However, problems related to difficult biliary access, large stones, and biliary strictures may decrease the success rate of this approach. We evaluated the safety and efficacy of extracorporeal shock-wave lithotripsy (ESWL) in 16 patients with complicated biliary stones treated prospectively over a 24-month period. Successful stone fragmentation was achieved in 15 patients (94%) using a Dornier HM3 lithotripter (average of 2290 shocks at 22 kV). Three patients (19%) required a second ESWL treatment. Biliary clearance of stone fragments was spontaneous in seven (43%) of the patients and required additional treatment in eight (57%) of the patients. Complications from ESWL were minor and included transient hematuria and ecchymoses at the skin entry site. Extracorporeal shock-wave lithotripsy failed in one patient (6%) with a biliary stricture and surgery was required. At hospital discharge, all patients were asymptomatic and stone free. Treatment with ESWL appears to be a safe and effective adjunct for selected patients with complex biliary stone disease.

Adult↗