Infrapopliteal drug-eluting stents for chronic limb ischemia.

OBJECTIVE: We report our experience with the elective placement of below-knee, drug-eluting stents in patients with chronic limb ischemia. BACKGROUND: Infrapopliteal percutaneous transluminal angioplasty has been associated with a lower rate of procedural success and high rate of restenosis because of the small size of the tibial vessels and the prevalence of calcified and diffuse atherosclerotic disease. Prior published data reports 3-year patency rates below 25%. Bare metal stents have been reported in bailout situations. Drug-eluting stents have markedly reduced restenosis compared to bare metal stents in the coronary vasculature, but there is little data supporting the use of these devices below the knee. METHODS: Elective placement of drug-eluting stents in infrapopliteal lesions was performed on 10 patients with severe (> or =Fontaine Stage IIb) claudication (n = 1) or limb-threatening ischemia (n = 9) (rest pain, nonhealing ulcers and gangrene). RESULTS: A total of 17 drug-eluting stents were electively placed in 12 below-knee arteries in 10 patients, resulting in an average of 1.7 +/- 0.7 stents per patient. The mean lesion length was 24.8 +/- 10.9 mm, the mean total stent length was 38.3 +/- 19.1 mm, and the mean nominal stent diameter was 2.8 +/- 0.3 mm. One patient required target vessel revascularization (TVR) at 3 weeks because of stent thrombosis. TVR was 10% at 12.4 +/- 6.5 months of follow-up. Clinically driven angiography in three different patients was performed at 4, 15, and 16 months and confirmed drug-eluting stent patency in each case. CONCLUSIONS: The use of below-knee drug-eluting stents is feasible and appears to be safe in our small series of complex infrapopliteal lesions causing chronic limb ischemia. The occurrence of a single case of stent thrombosis warrants continued observation in this cohort. Prospective clinical trials will be necessary to confirm the benefits and justify the costs of this strategy for treating patients with infrapopliteal culprit lesions and chronic limb ischemia.

Membrane specific carbonic anhydrase (CAIV) expression in human tissues.

Membrane-bound carbonic anhydrase IV (CAIV) expression has been evaluated in a range of fetal and adult human tissues and in cell culture. All tissues tested showed expression of CAIV, assessed by Western blotting, with a single immunodetected band at 55 kDa. The levels varied in fetal lung and liver during development and in various zones of the fetal brain. CAIV was clearly expressed in lung, pancreatic tumour and skin cell cultures.

Epithelial markers in pancreatic carcinoma: immunoperoxidase localisation of DD9, CEA, EMA and CAM 5.2.

Paraffin wax embedded, formalin fixed sections of 22 adenocarcinomas of the exocrine pancreas were stained with four mouse monoclonal antibodies: DD9-E7, an antibody raised against a human pancreatic tumour xenograft; carcino-embryonic antigen (CEA); epithelial membrane antigen (EMA); and cytokeratin (CAM 5.2). An indirect immunoperoxidase technique without enzyme pre-digestion and an affinity-purified sheep anti-mouse peroxidase conjugate were used. All of the tumours were positive for DD9-E7, EMA, and CAM 5.2. Twenty out of 22 were focally positive for CEA and the staining was often weak. As all of these adenocarcinomas were DD9-E7 positive, absence of staining for DD9-E7 in a tumour makes the diagnosis of adenocarcinoma of the exocrine pancreas very unlikely, and this is of value in distinction from endocrine carcinomas with a marked acinar pattern. The weak CEA staining distinguished pancreatic carcinomas from colorectal tumours. Because the distribution of staining for EMA and CAM 5.2 was no different from that previously seen in adenocarcinomas from other sites, these markers are likely to be of limited value in the differential diagnosis of abdominal adenocarcinomas of uncertain origin.

Pharmacodynamics of torsemide administered as an intravenous injection and as a continuous infusion to patients with congestive heart failure.

The natriuretic and diuretic effects of a 100-mg dose of torsemide administered as a continuous infusion of torsemide and as a single bolus were compared in a group of patients with stable mild-to-moderate congestive heart failure (CHF). Patients received in random order 100 mg of torsemide as an intravenous bolus and as a 75-mg infusion over 24 hours started simultaneously with a 25-mg loading bolus. Administration of torsemide to patients with CHF as a continuous infusion was an effective dosing regimen, resulting in 24-hour diuresis and natriuresis that was numerically but not statistically greater than that observed with bolus administration. The response with continuous infusion occurred with less torsemide in the urine, resulting in a significantly greater efficiency of torsemide with this regimen. The effectiveness of torsemide as a continuous infusion does not mean that this mode of administration should be used in all patients. The response to 100 mg of torsemide in patients with mild-to-moderate CHF is the same whether administered as an intravenous bolus, a continuous intravenous infusion, or by mouth. This is consistent with the high bioavailability demonstrated in previous studies. The mode of therapy used should be dictated by each individual patient's needs. This study shows that continuous infusion is a viable option for administration of torsemide, and dosing guidelines for use of such a strategy are presented.

The identification of a novel NCA-related pancreatic tumour-associated antigen, DD9-antigen: a comparison with the expression of other tumour antigens by the pancreatic tumour cell line GER.

Pancreatic tumour-associated monoclonal antibody DD9E7, raised against the GER pancreatic adenocarcinoma cell line, recognises a protein epitope on a novel family of membrane-bound cell surface glycoproteins (Mr 80-115,000). Western blot analysis of SDS/PAGE gels of tumour biopsies and of normal adult pancreas has shown that these glycoproteins are highly expressed in most pancreatic tumours but cannot be detected in normal adult pancreas. Using monoclonal antibodies directed against other antigens that have been associated with pancreatic adenocarcinoma (Du-Pan-2, Ca 19-9, CEA, NCA-95/55, EMA, and FAP), we have been able to show that although some of the antigens are also expressed by the GER pancreatic tumour cell line, the glycoproteins identified by monoclonal antibody DD9E7 are distinct from those other antigens in both molecular weight and antibody binding characteristics. Neuraminidase, periodic acid, and tunicamycin treatment of cultured cells has shown that monoclonal antibody DD9E7 recognises an epitope on the protein core of the antigen. This epitope is also present in NCA-1, but not in CEA, which suggest that there may be an association between DD9-antigen and other members of the NCA/CEA supergene family.

A study of pancreatic secretory and intracellular enzymes in pancreatic cancer tissue, other gastrointestinal cancers, normal pancreas and serum.

Serum and tumour tissue extracts from patients with pancreatic exocrine adenocarcinoma and a number of other gastrointestinal tumours were examined for digestive and intracellular enzymes or tumour associated variants by cellogel electrophoresis. Enzymes were identified within the gel by their specific catalytic activities. Normal serum and extracts of normal pancreas were also studied. All the pancreatic secretory enzymes were present in extracts of normal pancreas; their levels were markedly reduced in tumour extracts and no characteristic isozymes were found. Alkaline phosphatase, carbonic anhydrase and a number of carbohydrate metabolising enzymes were present in all extracts; tumour associated isozymes were not consistently identified. These findings suggest that most human pancreatic exocrine cancers do not produce pancreatic secretory enzymes or intracellular isozyme variants, and that the identification of these enzymes or their isozymes is unlikely to be of practical diagnostic value.

The isolation and characterization of a bile ductule cell population from normal and bile-duct ligated rat livers.

The separation of bile ductule cells from Kupffer and sinusoidal endothelial cells in a suspension of non-parenchymal cells has been attempted. Bile duct ligation was performed so that a four-fold increase in the total number of non-parenchymal cells isolated from rat liver was attained and the proportions of both Kupffer and bile ductule cells were elevated. Rate zonal centrifugation, through a Ficoll gradient, partially separated the cells into two populations: (1) small cells (4-6 micrometer diameter) probably originating from the sinusoidal endothelium and (2) larger bile ductule and Kupffer cells (8-12 micrometer diameter). A more successful separation was achieved by isopycnic centrifugation through a linear metrizamide gradient. Bile duct ligation (14 days) altered the distribution of cells on the gradient and the peak containing bile ductule and Kupffer cells partially subdivided into the separate cell types. Antiserum raised against the bile ductule fraction was shown to be compatible with that raised against common bile duct tissue. gamma-glutamyl transpeptidase and leucine aminopeptidase activity were preferentially located in the rate zonal fraction containing bile ductule cells. Their specific activity increased after bile duct ligation as did that of beta-glucuronidase, which was raised in all cells.

The effect of dietary protein deprivation on protein synthesis in the isolated liver parenchymal cell.

1. Parenchymal cells were isolated from the liver of rats that had been deprived of dietary protein for 3 weeks. 2. The cells were two-thirds the diameter of those derived from livers of normal animals and consumed oxygen at a rate of 16-2 +/- 2-7 mu1/h per 10(6) viable cells, half the normal value. 3. Albumin and transferrin were synthesized at rates of 0-94 +/- 0-12 and 0-60 +/- 0-07 microgram/h per 10(6) viable cells respectively and urea at a rate of 0-77 +/- 0-12 microgram/h per 10(6) cells. This represents a 25-50% decrease in the rates of synthesis measured in cells isolated from normal livers. 4. The results are discussed in relation to the long-term effects of malnutrition on liver cell function.

Production of antibodies against antigens released from human pancreatic tumour xenografts.

Antibodies directed against the antigen released from viable tumour cells during growth have been raised by cross-immunization of immunocompetent hairy litter-mates with serum from nude mice bearing pancreatic tumour xenografts. Antisera raised against the components released from a primary pancreatic tumour xenograft (WB) or from a tumour cell-line xenograft (GER) showed a titre greater than 1:625 against cultured pancreatic tumour cells by an I125-binding assay. Five out of 14 of the hairy litter-mates immunized with serum from the same tumour (GER) produced antisera that bound more strongly to pancreatic cancer cells than to 13 other non-pancreatic cell lines (binding ratio greater than 2). Absorption of the antisera with pure CEA reduced the level of binding by 11-25% without affecting the specificity for pancreatic tumour cells. The antibody response could be completely removed by absorption with pancreatic tumour cells, whereas 50% and 18% of the activity remained after absorption with normal pancreas homogenate and a mixed non-pancreatic tumour-cell pool, respectively. Immunofluorescent staining of pancreatic tumour sections indicated that the antibody was localized on the membrane of ductular epithelial cells. Challenge of immunocompetent mice using this procedure has produced a polyspecific antiserum with signs of selectivity for the circulating antigens released from pancreatic cancer cells, and may provide a route to the production of antibody against specific tumour components.

An investigation of cellular components released from human renal cancer and foetal kidney xenografts in nude mice (nu/nu) by cross-immunization of hairy littermate relatives.

The release of components from human kidney tumour xenografts (GYL) and human foetal kidney explants maintained in nude mice has been studied. The GYL tumour released antigens into the serum which could be detected by the generation of antibodies following cross-immunisation of closely related hairy litter mate (HLM) mice. The production of anti-GYL antibody was monitored by an I125 binding assay using viable GYL tumour cells. In 2/16 hairy litter mate mice, cell surface antibody binding by GYL cells was twice that found with 8 other human tumour cell lines (including 2 other kidney cancer cell lines). Absorption of these antisera with 10(7) GYL tumour cells completely abolished this response, where 50%, 38% and 25% of activity remained following absorption with; a normal kidney cell line, a homogenate of normal kidney and a mixed pool of human tumour cells. Six out of 8 GYL tumour bearing nude mice tested had elevated plasma levels of HCG. Absorption of the HLM antisera with an excess of commercial HCG abrogated I125 binding by only 15%, suggesting that antibody production was not directed primarily against ectopic HCG.

Establishment and characterization of primary human pancreatic carcinoma in continuous cell culture and in nude mice.

Primary human panceratic exocrine adenocarcinoma has been established in tissue culture and as xenografts in immune-deficient nu/nu mice. The cell line has a doubling time of 36 h and grows as a confluent monolayer together with a constant population of free-floating cells. Evidence of tumourigenicity was provided by growth on an early diploid fibroblast monolayer and in soft agar, and as solid tumours in immune-deficient nu/mu mice. Chromosome analysis of the cultured cells confirmed their tumour origin. Xenografts established from the cell line or directly from primary tumour tissue have retained a similar histology to the original tumour on serial transplantation. An electrophoretic study of exportable pancreatic digestive enzymes and a number of intracellular enzymes has shown that the cell line and xenografts maintain a human intracellular enzyme profile, but do not produce pancreatic digestive enzymes.

The growth of human bladder and kidney cancers as xenografts in nude mice and rats.

Primary human kidney and bladder tumours were implanted into both nude rats (rnu/rnu) and nude mice (nu/nu). 40% of bladder tumours and 27% of kidney tumours were established as xenografts. Histological sections of primary tumour and xenografts confirmed their similarities. Two patients with renal tissues were hypercalcaemic prior to nephrectomy, and animals in which these tumours grew as xenografts also became hypercalcaemic. Cell lines were established in tissue culture from two of the kidney tumour xenografts, and chromosome studies have confirmed their human karyotype.

Rapid determination of 5-fluorocytosine levels in blood.

Levels of 5-fluorocytosine in blood were rapidly calculated by determining the amount of competition in the creatinine iminohydrolase (creatinine deiminase; EC 3.5.4.21) assay for creatinine with the Kodak Ektachem system (Eastman Kodak Co., Rochester, N.Y.). The correlation with bioassay values was extremely high (r = 0.982). Standards and samples were highly stable over time.

Antibody response of nude (RNU/RNU) and hairy (RNU/+) rats to circulating cell surface components from human pancreatic cancer xenografts.

Homozygous nude rats (rnu/rnu) injected s.c. with 3 X 10(7) human pancreatic cancer cells from the GER cell line developed circulating antibody to GER cell surface, detected in a 125I binding assay against viable GER cells in vitro. Antibody titre rose with progressive xenograft growth. These antibodies showed no selectivity for GER cells when compared with a panel of other human cell lines. Heterozygous nude rats (rnu/+) immunised with serum from their GER xenograft-bearing nude relatives (rnu/rnu) also developed anti-GER cell surface antibodies. These antibodies showed some selectivity for GER and WAD (a second human pancreatic cancer cell line) when compared with other human cancer cells and lymphocytes. These findings show that some human pancreatic cancer cell surface components may persist independently in the circulation of xenograft bearing rnu/rnu rats despite the presence of antibody excess to other surface determinants from the same cells. It is suggested that differences in the relative immune competence of rnu/rnu and rnu/+ rats may offer a biological opportunity for enhancing the recognition of weak antigenic determinants which may have some useful selectivity for different types of human tumour cells.

Tumor cachexia and hypercalcemia in nude rats and mice bearing human renal carcinomas.

Human hypercalcemia-inducing renal tumors have been established as xenografts in nude mice and nude rats. Animals bearing these tumors became cachexic and hypercalcemic and these changes could be reversed by excision of xenograft tumors. Metabolic studies carried out in nude rats suggested that the hypercalcemia-inducing factor was effecting renal excretion of calcium but was probably not parathyroid hormone. Although the hypercalcemia-inducing property of the tumor was stable throughout serial transplantation of tumor from animal to animal, it was lost after passage through cells in culture.

Differential screening of a human pancreatic adenocarcinoma lambda gt11 expression library has identified increased transcription of elongation factor EF-1 alpha in tumour cells.

A human pancreatic adenocarcinoma lambda gt11 expression library was differentially screened with mRNA derived from normal and cancerous pancreatic tissues. Five clones preferentially hybridized with pancreatic adenocarcinoma mRNA. cDNA inserts from 4 of these clones were amplified by PCR, labelled with alpha 32P and used in Northern blot analysis against mRNA prepared from a variety of tumour and normal tissues. lambda GER-4 identified a pancreas-associated mRNA (greater than 10 kb) with no homology with known sequences at either the nucleic or amino-acid level. lambda GER-2 identified a 1.7-kb mRNA transcript that was over-expressed in mRNA prepared from pancreas, colon, breast, lung and gastric tumours relative to normal tissues. Sequence analysis and restriction-enzyme mapping showed that this clone was completely homologous with the active form of human elongation factor EF-1 alpha. This high level of EF-1 alpha-mRNA expression in tumour tissues lends support to the increasing evidence that EF-1 alpha is an important regulator of the cell cycle.

Growth of human digestive-tumour xenografts in athymic nude rats.

The athymic nude rat rnu/rnu has been established as an in vivo model for the acceptance of human digestive-tumour xenografts. We report the successful xenografting of 7/12 (58%) primary explants from patients with digestive cancer. Successful xenografting also occurred in 21/25 (84%) pancreatic tumours derived from a pancreatic exocrine adenocarcinoma (GER) maintained in cell culture; 2 of those have been successfully passaged in nude rats. The simultaneous implantation of these tumours into nude mice led to an almost identical take rate. Passage of one colonic and one pancreatic xenograft from nude rats into nude mice, and transplantation back into nude rats, increased the take rates. The critical period for the establishment of primary tumour growth was usually 28-42 days. The xenografts maintained histological and cytological characteristics of the primary explants or of the original tumour from which the cell line derived. The karyotype of the cell line was also maintained in the solid tumour. Three murine tumours were successfully grown as xenografts. Despite their immunoincompetence, the rats in this study showed no increased morbidity or mortality when kept in conventional conditions, compared with animals housed in isolators. The athymic nude rat will become a valuable complementary tool to the nude mouse for the establishment and maintenance of human digestive tumours and for surgical and serial serological studies.

The generation of monoclonal antibodies against human pancreatic exocrine cancer: a study of six different immunisation regimes.

Six different immunisation regimes have been used to generate spleen cells with reactivity against human pancreatic exocrine cancer. Immunised spleen cells were fused with an NSO/1 myeloma line and supernatants from these hybridomas selectively screened for monoclonal antibodies which bound predominantly to a pancreatic cancer cell line (GER). The spleen cells from hairy litter mates immunised with pancreatic cancer xenograft homogenates and viable GER cells generated 13% of hybridoma supernatants which showed some selectivity for GER pancreatic cancer cells in a fixed cell ELISA assay. The other methods produced only 4% of hybrids with selectivity for GER cells. The antigen distribution on gluteraldehyde fixed cells was similar to that found for viable cell monolayers but many antigens were unstable on formalin fixation. Immunohistochemical staining of GER cells grown on glass slides showed a heterogeneity of antigen distribution with up to 70% of the cells exhibiting a vesicular pattern of staining. Fifty percent of the antibodies which bound to GER cells were also reactive against antigens in formalin-fixed paraffin-embedded tissue sections of the original GER tumour. Monoclonal antibody DD9E7 identified an antigen expressed on 12/14 pancreatic adenocarcinomas. The antibody showed strong staining of malignant luminal membranes and cytoplasm. The antigen was also present in normal salivary and sweat glands, and colon and breast carcinomas, but its tissue distribution was unlike that of CEA or EMA. The expression of this antigen in 12/14 of pancreatic carcinomas suggests that DD9E7 may be a useful reagent for pancreatic tumour detection.

Vitamin D receptors and anti-proliferative effects of vitamin D derivatives in human pancreatic carcinoma cells in vivo and in vitro.

The GER human pancreatic carcinoma cell line possesses receptors for 1,25-dihydroxyvitamin D3. We report that the vitamin D analogue EB 1089 inhibits the growth of these cells in vitro and when grown as tumour xenografts in immunodeficient mice. Tumour-bearing mice were given EB 1089 at a dose of 5 microg kg(-1) body weight i.p. thrice weekly for 4-6 weeks. Tumour growth was significantly inhibited in treated animals compared with controls in the absence of hypercalcaemia. These findings may have therapeutic implications in pancreatic cancer.

The immunoperoxidase localization of tumour markers in ovarian cancer: the value of CEA, EMA, cytokeratin and DD9.

Primary tumours from 40 patients with epithelial ovarian cancer, treated at St Thomas's Hospital over a 10-year period, were studied for the immunocytochemical expression of the following tumour markers in formalin-fixed paraffin embedded material: carcinoembryonic antigen (CEA), epithelial membrane antigen (EMA), cytokeratin (CAM 5.2), and DD9. An indirect immunoperoxidase staining technique was used. All of the tumours were positive for EMA and CAM 5.2, and 30% of them were positive for both CEA and DD9. The absence of CEA and DD9 may be of value in differentiating between metastatic abdominal adenocarcinomas of ovarian origin and those of gastrointestinal origin, but no indication of prognosis was obtained using these epithelial markers. The strong and widespread staining of all the tumours for EMA suggests that this may be a useful marker for detecting metastatic or recurrent disease by immunoscintigraphy.