Novel apparatus to measure hyperthermal heavy ion damage to DNA: strand breaks, base loss, and fragmentation.

We have developed a novel apparatus that allows us to irradiate nonvolatile organic films of high mass (1-100 microg range) spread out over a large surface area (42 cm(2)) with low energy (kT-100 eV) heavy ions and to quantitatively analyze the film substance via standard biochemical techniques afterwards. Here we discuss the details of the apparatus and method and show that it allows us to measure substantial damage to double stranded DNA molecules (plasmids) and its fundamental subunits induced by heavy ions with unprecedented low energies, i.e., 2.5 eV/amu; these energies correspond to track end energies of stopping ions or secondary ions created along primary ion tracks. We find that hyperthermal Ar(+) ions interacting with plasmid DNA will lead to the formation of single and double strand breaks, as well as fragmentation of nucleosides, which also involve chemical modifications and site specific rupture along the N1-C1 glycosidic bond, resulting in base release. In cells, such localized clustered damage will enhance the severity of DNA strand lesions, thus making them harder to repair.

Transjugular intrahepatic portosystemic shunt with an autologous vein-covered stent: results in a swine model.

PURPOSE: To investigate the feasibility, safety, and efficacy of an autologous vein-covered stent (AVCS) to prevent shunt stenosis in a porcine transjugular intrahepatic portosystemic shunt (TIPS) model. MATERIALS AND METHODS: TIPS were created with an AVCS in 12 healthy domestic swine and with a bare stent in 10 additional swine. Tissue response was compared with use of venography, histology, and computerized morphometry analysis 2 weeks after implantation. Differences between AVCS and noncovered stents (established by a t-test), as well as regional differences within a single stent (established by an f test), were considered significant at P <.05. RESULTS: Twenty of 22 TIPS procedures were technically successful. Ten of 12 shunts with an AVCS (83%) and two of 10 with bare stents (20%) remained patent (<50% diameter narrowing) at euthanasia 2 weeks later (P <.01). Histologic evaluation of harvested bare stents showed marked intimal hyperplasia (IH), composed of smooth muscle cells, myofibroblasts, and fibroblasts. In contrast, the AVCS were remarkably free of IH and thromboses. In patent TIPS in both groups, endothelial coverage of the luminal surface was present histologically. IH accounted for 57% (26.27/45.79) of total stent cross-sectional lumen area in the control group and 21% (8.34/39.54) in the AVCS group (P <.01), with no intrashunt differences (P >.05). CONCLUSION: Based on short-term follow-up, AVCS significantly improved TIPS patency by prevention of both IH and in-stent thrombosis. TIPS created with an AVCS was feasible and safe in our porcine model.

Radiation sensitivity and apoptosis in human lymphoma cells.

PURPOSE: The impact ofapoptosis on radiation-induced eradication of clonogenic tumour cells is uncertain. The aim was to analyse the relationship of different functional stages during the apoptotic process to cell death and clonogenic cell eradication. MATERIALS AND METHODS: Apoptosis in Jurkat T-cells was studied by morphology, light scatter and caspase activation. Mitochondrial integrity was determined by the mitochondrial membrane potential (delta(phi)m). Cell death was quantified using propidium iodide exclusion. Clonogenic cell death was determined using a dilution survival assay. The influence of Bcl-2 was tested using a Bcl-2 transfected Jurkat clone. RESULTS: Irradiation induced profound apoptosis within 48 h associated with caspase activation and breakdown of delta(phi)m. Inhibition of caspases abrogated the apoptotic morphology with no influence on breakdown of delta(phi)m and survival. Over-expression of Bcl-2 abrogated all hallmarks of apoptosis; delayed cell death, however, had no influence on clonogenic survival after irradiation. CONCLUSION: Based on Bcl-2 as a positional marker, radiation-induced apoptosis can be divided into two stages: the initiation/decision phase, characterized by a breakdown of the mitochondrial membrane potential, and the execution phase, characterized by caspase activation. The execution phase had no influence on survival, whereas the initiation/decision phase controls immediate survival. However, abrogation of both phases did not influence radiation sensitivity.

A specific inhibitor of apoptosis decreases tissue injury after intestinal ischemia-reperfusion in mice.

PURPOSE: Apoptosis is a stereotypical pathway of cell death that is orchestrated by a family of cysteine endoproteases called caspases. This study examined the effect of apoptosis inhibition with a specific caspase inhibitor on murine intestinal viability after ischemia-reperfusion (IR). METHODS: C57Bl6 X SV129 mice underwent segmental small bowel ischemia by vascular isolation of 10 cm of terminal ileum. In separate experiments, the ischemic time was varied from 30 to 130 minutes with a reperfusion interval of 6 hours. The degree of small bowel injury was quantified from 1 to 5 (increasing severity) by standardized, blinded histologic grading. The degree of apoptosis was assessed with a specific assay (terminal deoxyamcleotydil transferase-mediated deoxyuridine triphosphate nick end labeling [TUNEL]) and quantified by calculating the apoptotic index (apoptotic cells/10 high-power fields). To evaluate for activation of interleukin-1beta converting enzyme we measured tissue mature interleukin-1beta levels using a specific enzyme-linked immunosorbent assay. To evaluate the effect of apoptosis inhibition on intestinal viability after IR, mice received 3.0 mg of the caspase inhibitor ZVAD (N-benzyloxycarbonyl Val-Ala-Asp-Ome-fluoromethylketone) subcutaneously before and after IR in five divided doses (n = 11), the same dose of ZFA (N-benzyloxycarbonyl Phe-Ala fluoromethylketone), a structurally similar molecule with no anticaspase activity (n = 9), or sham operation (n = 6). RESULTS: A linear relationship existed between ischemic interval and histologic grade (r = 0.69, P <.006). Increasing the ischemic interval from 0 to 50 minutes was associated with a fivefold increase in apoptotic index (P =.05). Ischemic bowel was measured to have an average of 57.3 +/- 7.8 pg/mL whereas normal bowel had an average of 1.8 +/- 0.5 pg/mL of mature interleukin-1beta present. Mice tolerated multiple injections of ZVAD and ZFA without signs of toxicity. Animals treated with ZVAD (apoptosis inhibitor) had little injury after 50 minutes of ischemia and 6 hours of reperfusion (injury grade 1.8) compared with sham controls (injury grade 1.2, P =.7) and had significantly less injury than mice treated with ZFA (placebo) (injury grade 3.0, P <.006). CONCLUSIONS: Increasing ischemic interval in a segmental small bowel murine IR model is associated with increased histologic injury and augmented apoptosis as evidenced by increased TUNEL staining and interleukin-1beta converting enzyme activation. Inhibition of apoptosis with a specific caspase inhibitor significantly diminishes the degree of small bowel injury.

A caspase inhibitor decreases oxidized low-density lipoprotein-induced apoptosis in bovine endothelial cells.

BACKGROUND: Apoptosis is a pathway of cell death orchestrated by a family of proteases called caspases. Oxidized low density lipoprotein (oxLDL) is a putative cause of atherogenesis. We examined the effect of oxLDL on endothelial cell (EC) apoptosis and the ability of a caspase antagonist to inhibit oxLDL-induced EC injury. METHODS: Bovine ECs were plated at a concentration of 5.0 x 10(5) cells/ml and exposed to LDL oxidized by ultraviolet radiation at a concentration of 100 microgram oxLDL/ml for 20 h. Some ECs were pretreated with an irreversible caspase inhibitor (ZVAD). Samples were analyzed histologically. Apoptosis was measured using the Annexin V assay (flow cytometry) which detects phosphatidylserine on plasma membranes and confirmed by TUNEL assay (flow cytometry). Statistical assessments were performed using ANOVA. RESULTS: ECs treated with LDL were morphologically similar to untreated cells. Cells treated with oxLDL demonstrated cytoplasmic shrinkage, plasma membrane blebbing, chromatin condensation, and loss of adhesion. These effects were diminished after pretreatment with the caspase inhibitor ZVAD. The Annexin V assay showed: (a) cells exposed to LDL had a 12 +/- 1% apoptosis rate, (b) exposure to oxLDL induced apoptosis in 30 +/- 0.3% of the cells, and (c) pretreatment with the caspase inhibitor ZVAD decreased the oxLDL-induced apoptosis to 16 +/- 1% (P < 0.05). This decrease in apoptosis was also reflected by an increase in the percentage of alive cells from 34 +/- 7% after oxLDL exposure to 55 +/- 6% after apoptosis inhibition with ZVAD. TUNEL assay demonstrated a 2.5-fold reduction in mean fluorescence intensity between cells treated with oxLDL alone and those treated with ZVAD, suggesting a significant decrease in apoptosis in the latter group. CONCLUSIONS: We conclude that treatment of bovine ECs with oxLDL induces apoptosis which can be significantly reduced by a specific caspase inhibitor.

Transjugular intrahepatic portosystemic shunt: effect of bile leak on smooth muscle cell proliferation.

PURPOSE: To evaluate the effect of bile on smooth muscle cell (SMC) proliferation in vitro and in vivo in a porcine transjugular intrahepatic portosystemic shunt (TIPS) model. MATERIALS AND METHODS: In vitro, SMCs explanted from porcine thoracic aorta were cultured with standard techniques. After initial pilot studies, they were subcultured in one of three groups: 1% porcine serum plus 1% bile, 10% porcine serum plus 1% bile, and 10% porcine serum. Cells were harvested at 3, 10, or 14 days, and DNA, protein, and disintegrations per minute (an indicator of proliferation) were measured. In vivo, TIPS creation was successful in 45 swine. All pigs were euthanized at 10-16 days. The proliferative response within the stent was histologically quantified and correlated for evidence of bile leak. RESULTS: In pilot studies, 2.5%-10.0% bile solutions caused 100% SMC mortality by 3 days. In the presence of 1% bile (with or without porcine serum), both DNA and protein production decreased significantly compared with that in porcine serum alone (P < .05). In vivo, 13 of 45 specimens (29%) showed bile leak at gross or microscopic examination. SMC proliferation was less overall in animals with versus those without bile leak (difference not significant). CONCLUSION: These data suggest that the proliferative response in a TIPS is not primarily due to bile leak. Bile leak may promote thrombosis, but it appears to inhibit myointimal proliferation.

Transjugular intrahepatic portosystemic shunt in a porcine model: histologic characteristics at the early stage.

RATIONALE AND OBJECTIVES: The authors attempted to determine the histologic processes that take place during development of stenosis after transjugular intrahepatic portosystemic shunt (TIPS) creation. MATERIALS AND METHODS: TIPS were created with metallic stents in 20 healthy domestic pigs (tantalum stents in 10, stainless steel stents in 10). The animals were sacrificed 2-16 days later. All the shunts were examined by means of venography both immediately after placement of the stents and before sacrifice. All histologic sections were assessed with modified Giemsa and basic fuchsin stains. Anti-smooth-muscle-cell alpha-actin stain was used in three specimens. The stenotic reaction was quantified by using standard planimetry techniques and a computerized image-analysis system. RESULTS: Within 16 days after TIPS placement, 15 (75%) of the 20 shunts were completely occluded, four (20%) of 20 shunts were partially occluded, and one (5%) of 20 shunts remained widely patent (animal died of unknown cause 2 days after the TIPS procedure). Stent occlusion was caused primarily by pseudointimal hyperplasia, which was similar morphologically in the portal, middle, and hepatic portions of the stent. Myofibroblastic proliferation was the most striking feature of the pseudointimal hyperplasia. The average thickness of the proliferation was 2.14 mm, which was 67% of the total diameter of the stent. A mild fibrous or lymphocytic reaction occurred around the stent wires and between the pseudointimal hyperplasia and the liver parenchyma. CONCLUSION: The histologic features of pseudointimal formation in this swine TIPS model closely resemble those in humans. This model may prove useful for evaluating stents and other devices and improving the understanding of restenosis after vascular interventions.

Dehydroepiandrosterone protects low density lipoproteins against peroxidation by free radicals produced by gamma-radiolysis of ethanol-water mixtures.

Oxidized low density lipoproteins (LDL) are believed to play a central role in the events that initiate atherosclerosis. Antioxidants have been shown to decrease the oxidation of LDL, leading to the diminution of atherosclerosis. Since it is well-known that decreased levels of dehydroepiandrosterone (DHEA) are linked to the development of atherosclerosis, we studied the modulation of the oxidation of LDL by DHEA. LDL were obtained from 10 healthy subjects and oxidized by free radicals produced by gamma-radiolysis of ethanol-water mixtures. The formation of conjugated dienes and thiobarbituric acid-reactive substances (TBARS), the vitamin E content, as well as the incorporation of 4-[14C]DHEA in LDL and the chemotactic effect of oxidized LDL in the presence of DHEA towards monocytes, were investigated. It was found that DHEA was able to inhibit the oxidation of LDL by reducing over 90% of the conjugated dienes and TBARS formation, as well as by reducing the vitamin E disappearance and significantly decreasing the chemotactic activity towards monocytes. Our results suggest that DHEA exerts its antioxidative effect by protecting the endogenous vitamin E of LDL.

Coculture of endothelial cells and smooth muscle cells in bilayer and conditioned media models.

Smooth muscle cell (SMC) growth characteristics are affected by endothelial cells (ECs) in vivo and in vitro. In this study, we compare a bilayer EC/SMC coculture model that allows cell contact with a model of SMCs growing in media continuously conditioned by ECs, but without physical contact. Bovine aortic SMCs were plated on one side of a 13-microns-thick, semipermeable membrane. Three models were compared: (1) SMCs cultured alone (with no cells on the opposite side of the membrane, O/SMC); (2) SMCs cultured with ECs on the opposite side of the membrane in a bilayer coculture system that allows physical contact between ECs and SMCs (EC/SMC); and (3) SMCs cultured in media continuously conditioned by adjacent ECs, without contact (conditioned media, CM). After cultures were established, SMCs were harvested at 7 and 14 days after plating (n = 5 cultures/day/group). SMC DNA and protein content and [3H]thymidine incorporation were measured in each group. On Days 7 and 14 after plating, ECs in both the EC/SMC and CM models stimulated SMC proliferation > 50% compared to O/SMC controls (P < 0.05). SMC density was similar for the EC/ SMC and CM models at Day 7, but SMC density was higher in the EC/SMC group at Day 14 in culture (P < 0.05). At Day 7, protein synthesis was similar in the three groups, but by Day 14, SMCs in the EC/SMC group had produced significantly less cellular protein/ DNA than SMCs in the CM group (P < 0.05), which in turn had less protein/DNA than the control (O/SMC) group (P < 0.05). SMCs in the EC/SMC and CM groups retained a thin, spindle shape with filamentous projections, compared to the hypertrophic appearance of SMCs in the absence of ECs. Electron microscopy revealed projections from SMCs which traversed the pores in the coculture membrane and made intimate contact with ECs. The degree of EC/SMC contact increased from 7 to 14 days (P < 0.05). Compared to SMCs alone, ECs in bilayer coculture or conditioned media altered SMCs growth characteristics similarly after 7 days in culture. By 14 days, however, the bilayer coculture had a significantly greater effect on SMC density and protein synthesis. The bilayer model is unique in terms of luminal/abluminal orientation of the cells, the proximity of the cell layers, and the presence of physical cell contact. Since the bilayer model amplifies the effect of ECs on SMCs, it may be more useful than conditioned media to study EC-SMC interactions.

In vitro and in vivo secretion of human ISG15, an IFN-induced immunomodulatory cytokine.

ISG15, a 15-kDa protein of unique primary amino acid sequence, functions intracellularly as a ubiquitin homologue and a cytokine that induces production of IFN-gamma and augments NK/lymphokine-activated killer cell proliferation and function. ISG15 is secreted from monocytes and lymphocytes, and in this study we have characterized in vitro and in vivo production of ISG15 in response to IFN-alphabeta. Low levels of ISG15 were present constitutively in PBMCs; dose-dependent ISG15 synthesis was observed in response to IFN-alpha or IFN-beta, but not IFN-gamma. High m.w. conjugates, present in PBMC extracts constitutively, were enhanced after IFN-alpha or IFN-beta treatment. Metabolic labeling experiments demonstrated that IFN-beta-induced ISG15 was released from primary cultures of peripheral blood CD3+ (including both CD4+ and CD8+ subpopulations). Furthermore, ISG15 was released from viable cell lines of monocyte, T lymphocyte, B lymphocyte, and epithelial origins. Since ISG15 was secreted in response to IFN treatment in vitro, its levels in the serum of healthy human volunteers treated with IFN-beta(ser) were quantitated by asymmetric sandwich ELISA. Both single and multiple doses of IFN-beta(ser) increased serum ISG15 levels significantly (p < 0.01) over baseline. A maximum 7.3-fold enhancement of serum ISG15 was obtained after multiple injections of 8 million units of IFN-beta(ser). Significant change was observed at 24 and 48 h of multiple 0.02-million-unit injections, yielding 1.2- and 1.7-fold increases over basal levels, respectively. These studies suggest that ISG15 is a novel member of the cytokine cascade that is synthesized and released in response to IFN-beta both in vitro and in vivo.

Endothelial cells modulate smooth muscle cell morphology by inhibition of transforming growth factor-beta 1 activation.

BACKGROUND: We have previously demonstrated in a coculture model that endothelial cells (ECs) exert regulatory control over smooth muscle cell (SMC) morphology. This study was performed to test the hypothesis that ECs inhibit transforming growth factor-beta 2 (TGF-beta 1) activation through the release of plasminogen activator inhibitor (PAI-1). METHODS: Bovine SMCs were cultured on a thin, semipermeable membrane, either alone or opposite ECs in coculture (SMC/EC). Conditioned media and cell lysates at 1, 5, and 21 days were assayed for TGF-beta 1 and PAI-1 by enzyme-linked immunoabsorbent assay. Cell proliferation rates, protein, and DNA content were measured and compared with SMC morphology. RESULTS: Activation of TGF-beta 1 was significantly decreased (1.2% versus 18.9% active TGF-beta 1 p < 0.05) and PAI-1 was increased (659 pg/ml versus 343 pg/ml p < 0.05) in SMC/EC medium on day 1, compared with the medium of SMC alone. Significantly higher levels of PAI-1 were measured in cell lysates of cocultured ECs (128 pg/micrograms DNA) than in cocultured SMCs (5.8 pg/micrograms DNA, p < 0.05). SMC/EC coculture prevented the SMC hill-and-valley growth morphology seen in SMCs cultured alone. CONCLUSIONS: In a model designed to study SMC/EC interactions, it was seen that ECs can alter growth characteristics of SMCs by producing PAI-1, which interferes with the plasminogen pathway of TGF-beta 1 activation. This suggests that reduced EC PAI-1 production could play a role in alteration of SMC phenotype in vivo.

Acetylator genotype (NAT2)-dependent formation of aberrant crypts in congenic Syrian hamsters administered 3,2'-dimethyl-4-aminobiphenyl.

Some but not all human epidemiological studies suggest a higher incidence of colon cancer in rapid acetylator individuals. Aberrant crypts, the earliest morphologically evident preneoplastic lesions in chemical colon carcinogenesis, were measured in rapid and slow acetylator congenic Syrian hamsters administered 3,2' -dimethyl-4-aminobiphenyl, an aromatic amine colon carcinogen, to investigate the specific role of the acetylator genotype (NAT2) in colon carcinogenesis. Age-matched rapid (Bio. 82.73/H-Patr) and slow (Bio. 82.73/ H-Pat(s) acetylator female Syrian hamsters congenic at the NAT2 locus received a s.c. injection of 3,2' -dimethyl-4-aminobiphenyl (100 mg/kg) at the start of weeks 1 and 2. After 10 and 14 weeks, the hamsters were sacrificed, and each whole cecum, colon, and rectum was stained with 0.2% methylene blue, fixed in 4% paraformaldehyde, and examined under a dissecting microscope for the presence of aberrant crypts. Aberrant crypts were identified in the cecums and colons of both rapid and slow acetylator congenic hamsters treated with 3,2' -dimethyl-4-aminobiphenyl but not in vehicle controls. The size of the aberrant crypt foci was larger in the colon than in the cecum, and the highest frequency of aberrant crypt foci was observed in the cecum. No aberrant crypts were detected in the rectum. The frequency of aberrant crypt foci was significantly higher (2-3-fold) in rapid versus slow acetylator congenic hamsters in both cecum (P = 0.0352) and colon (P = 0.0006). These results support human epidemiological studies that suggest the rapid acetylator genotype is associated with higher risk of colon cancer induced by aromatic amines.

Endothelial cell modulation of smooth muscle cell morphology and organizational growth pattern.

Intimal hyperplasia is characterized by smooth muscle cell (SMC) dedifferentiation from a contractile to a synthetic phenotype prior to migration and proliferation. Regulatory mechanisms controlling SMC phenotype are not well known. This study examined the effect of endothelial cells (ECs) on SMC morphology in coculture. Subcultured bovine ECs and SMCs were plated on opposite sides of a 13 microns thick, semipermeable membrane (0.45 micron pores, Cyclopore) to allow potential humoral and cellular cross-membrane communication. SMCs were studied (5 wells/group) in coculture opposite confluent ECs (EC/SMC) and alone (SMC controls). After 4 days of culture in Dulbecco's modified Eagle medium/2.5% calf serum, SMCs were harvested. The ratio of protein/DNA was measured as an index of SMC hypertrophy (synthetic SMC phenotype). SMCs were examined with light and scanning electron microscopy to evaluate cell surface area, cellular morphology, and macroscopic growth characteristics. Flow cytometry was used to determine the cellular RNA/DNA ratio. SMC control cultures had a significantly greater protein-to-DNA content than SMCs cocultured with ECs (175 +/- 9 vs. 115 +/- 7 micrograms protein/micrograms DNA; p < 0.001). SMC control cultures also had 6.5 times greater cell surface area (5.8 +/- 0.3 x 10(3) microns2) than cocultured SMCs (0.9 +/- 0.1; p < 0.001). In SMC control cultures, SMC hypertrophy and rapid "hill and valley" formation were observed. In contrast, SMCs from the EC/SMC group exhibited a more spindle-shaped, contractile-appearing phenotype with more uniform, evenly distributed cells and no hill and valley formation. SMC control cultures also had a higher RNA/DNA ratio. Thus the presence of confluent ECs substantially altered the morphology and growth characteristic normally observed for SMCs in vitro. This coculture system provides a model to further study EC-SMC interaction, which could have important in vivo consequences.

Effect of intravenous glucagon on intestinal viability after segmental mesenteric ischemia.

PURPOSE: We have previously shown that intravenous glucagon significantly improves rat survival if given after release of superior mesenteric artery occlusion. The purpose of this study was to isolate the effects of glucagon on the intestine by creating a rat model of segmental mesenteric ischemia that avoids the systemic shock associated with total superior mesenteric artery occlusion and reperfusion. METHODS: In 18 anesthetized Sprague-Dawley rats, an 8 cm segment of midileum was made totally ischemic for 110 minutes by occluding its vascular arcades and the bowel ends with microvascular clamps. Control animals (n = 8) received normal saline solution (10 ml/kg/hr intravenously) during ischemia and for 2 hours after declamping. Ten animals also received glucagon (1 microgram/kg/min intravenously) during 2 hours after declamping. After a 24-hour recovery, the ischemic bowel segment, plus nonischemic proximal ileum, was examined histologically by computerized planimetry to measure wall thickness (muscularis, mucosa, and transmural), percent epithelial coverage, and villar surface ratio (villar surface length/bowel circumference). All observations were blinded. Comparisons were made by Student's t test. RESULTS: Compared with nonischemic ileum, the ischemic segment in control animals showed severe mucosal injury with a reduction in mucosal thickness to 25%, epithelial coverage to 23%, and villar surface ratio to 33% of that seen in nonischemic ileum (p < 0.01). Substantial preservation of ileal mucosal viability was seen in glucagon-treated animals. Mucosal thickness and epithelial coverage were twice as well preserved in glucagon-treated rats compared with control rats (p < 0.05), and villar surface ratio was also increased significantly (p < 0.05). The muscularis was not significantly injured in this model. CONCLUSIONS: By use of quantitative histologic measurements, we found that intravenous glucagon significantly improved ileal mucosal viability when given early during reperfusion after segmental ischemia. The mechanism of this effect and its potential clinical application merit further study.

Concurrent radiation therapy and chemotherapy in the treatment of primary squamous cell carcinoma of the vulva.

BACKGROUND: Interest in combined modality treatment and in quality of life issues may affect the choice of radical vulvectomy as the treatment of choice in many vulvar carcinomas. To evaluate the potential role of combined radiation and chemotherapy with or without local excision as primary treatment for squamous cell carcinoma of the vulva, the outccomes of 19 patients with this disease treated with combination therapy were reviewed. METHODS: Nineteen patients were treated between September 1987 and October 1992. Fifteen patients had American Joint Committee on Cancer Stage III disease; 4 had Stage II. All had clinically negative inguinal lymph nodes with the exception of two patients who had positive ipsilateral inguinal nodes that were removed before treatment. The patients received 45-50 Gy to the pelvis and inguinal nodes with concurrent chemotherapy that consisted of 5-fluorouracil given as a 96-hour continuous infusion (1000 mg/m2/d) during weeks 1 and 5 of radiation. A single dose of mitomycin-C (10 mg/m2) during the first day of chemotherapy has been used since November 1991. Ten patients were boosted with implants or electrons and 6 others underwent local excision. RESULTS: The median follow-up was 34 months. Responses were determined clinically 1 month after completion of the radiation and chemotherapy. Clinically, complete responses were obtained in 10 patients (53%), partial responses in 7 (37%), and no response in 1; 1 patient progressed during treatment. The combined modality therapy (radiation/chemotherapy/with or without wide local excision) resulted in a local control rate of 74% (14/19). All five treatment failures occurred within 6 months of treatment. Four of these patients were rendered disease free by radical vulvectomy and/or exenteration, for an overall local control rate of 95% (18/19). CONCLUSION: Concurrent radiation therapy and chemotherapy with local excision performed as needed, appears to be a reasonable alternative to radical vulvectomy in patients with primary squamous cell carcinoma of the vulva. Radical surgery remains a viable option for patients in whom primary therapy has failed.

Quantitating intestinal ischemia with nitroblue tetrazolium salts.

We evaluated a spectrophotometric nitroblue tetrazolium (NBT) reduction assay as a technique to quantitate experimental intestinal ischemic injury. NBT is a tetrazolium salt that is reduced by mitochondrial coenzymes to form a blue formazan dye which can be measured on a spectrophotometer. We used a rat model of progressive intestinal ischemia to compare NBT reduction with standard histologic grading by light microscopy. Isolated segments of small intestine were made ischemic for periods of 15, 30, 60, 90, and 120 min in each of five rats (no reperfusion). A portion of each segment was prepared for both NBT reduction assay and blinded histologic grading. The reproducibility of these results was then tested in a second identical study of four rats (Part 2). When compared to nonischemic segments of intestine, NBT reduction was significantly decreased after 30 min of ischemia (P < 0.05, ANOVA) and continued to decrease as ischemic time increased. These findings were reproduced in the second experimental group. Overall, NBT reduction correlated closely with duration of ischemia (r = 0.81, P < 0.001) and histologic grade (r = 0.77, P < 0.001). Based on criteria developed in Part 1, NBT reduction had a sensitivity of 100% and a specificity of 94% for detecting ischemia > or = 30 min in Part 2. We conclude that the spectrophotometric NBT assay in an accurate technique for quantitating small intestinal ischemic injury which also gives useful information about the functional status of mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo priming effects of interferon-beta ser on NK activity of peripheral blood mononuclear cells in cancer patients.

Natural killer (NK) activity was assayed in fresh peripheral blood mononuclear cells (PBMs) from cancer patients receiving interferon (IFN)-beta ser. Patients received a single intravenous injection of IFN-beta ser (90 x 10(6) IU m-2) on alternate days for 2 weeks, followed by a higher dose (180 x 10(6) IU m-2) on the same schedule. PBM NK lysis of K562 target cells was significantly increased in PBMs sampled 24 h after the initial injection (P < 0.05). At the end of the first 2 weeks of the protocol, NK cytotoxic activity of PBMs had fallen below the original baseline levels; the higher IFN dose subsequently given was without effect. However, significant increases in the proportion of CD16+ cells were seen following each injection. A positive correlation was also seen between the increased lytic activity of CD16+ NK cells and the proportion of CD38+ NK cells, but not the proportion of CD56+ NK cells. In vitro IFN-treatment of these in vivo-treated PBMs resulted in a further increase in NK activity. Pre-exposure in vivo to IFN-beta ser seems to prime the PBMs to respond to in vitro stimulation by IFN-gamma, which otherwise had no effect. Phenotypic analysis of PBMs after in vitro exposure to IFN-beta ser showed that the levels of CD16+, CD38+ and CD56+ cells did not change. All the NK activity responding to IFN-beta ser was found in the CD16+ enriched population of PBM, suggesting that it is unlikely that in vivo redistribution of CD16+ subsets representative of NK cells has occurred in the peripheral blood.

Effect of endothelial cells and transforming growth factor-beta 1 on cultured vascular smooth muscle cell growth patterns.

Smooth muscle cells (SMCs) cultured alone exhibit characteristic "hill and valley" macroscopic growth features. We studied smooth muscle cells cocultured with endothelial cells and the effect of transforming growth factor beta 1 on smooth muscle cells: Bovine smooth muscle cells were plated on 13 microns-thick semipermeable membranes. Smooth muscle cells were cultured either alone (in Dulbecco's Modified Eagles Media/2.5% calf serum, four wells/group); with neutralizing anti-transforming growth factor-beta 1 antibody (10 micrograms/ml); with the protease inhibitor aprotinin (prevents plasmin-mediated activation of transforming growth factor-beta 1, 200 mg/ml); or in the presence of confluent bovine endothelial cells cocultured on the opposite side of the membrane before plating smooth muscle cells. After 72 hours in culture smooth muscle cell organizational growth characteristics were examined by light microscopy. Hill and valley formation by smooth muscle cells resulted in areas of the membrane becoming devoid of smooth muscle cells, whereas other areas developed multilayered densely populated smooth muscle cells. Computed planimetry was used to measure this bare surface area to quantitate the extent of hill and valley growth, which was compared between groups by analysis of variance. Smooth muscle cells cultured alone demonstrated prominent hill and valley formation with a bare surface area of 2.64 +/- 0.51 mm2. Smooth muscle cells exposed to transforming growth factor-beta 1 antibody had much less hill and valley formation (bare surface area 0.92 +/- 0.29, p < 0.01), whereas aprotinin virtually prevented hill and valley formation (bare surface area 0.0, p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of endothelial cell coculture on smooth muscle cell proliferation.

PURPOSE: Smooth muscle cell (SMC) growth kinetics are often studied in culture without consideration of endothelial cell (EC) influences that occur in vivo. This study examined the time-dependent effect of EC on SMC in a new type of coculture system. METHODS: Bovine aortic EC and SMC were harvested from fresh specimens, grown to four passages from primary cultures, and plated on either side of a porous 13 microns thick polyethylene terephthalate membrane. SMC were studied in coculture opposite from confluent EC (EC/SMC). Controls included SMC cultured opposite SMC (SMC/SMC) or SMC alone (with no cells on the opposite side of the membrane, phi/SMC). After cocultures were established, SMC were harvested from 1 to 4 days after release from growth arrest (n = 5 cultures/day/group). SMC DNA and protein content and 3H-thymidine incorporation were measured in each group. SMC proliferation was indexed by 3H-thymidine incorporation per cellular DNA content. RESULTS: EC stimulated SMC proliferation 56% more than SMC/SMC cultures and 244% more than SMC alone on day 1 after growth arrest (p < 0.05). This effect decreased with time so that by day 4, EC seemed to inhibit SMC proliferation (49% less proliferation than SMC/SMC and 76% less than SMC alone, p < 0.05). SMC opposite EC had significantly less protein/DNA than control SMC, and they retained a thin, spindle shape compared with the hypertrophic appearance of SMC in the absence of EC. Electron microscopy revealed EC gap junctions and cytoplasmic projections from SMC of sufficient length to transverse the pores in the coculture membrane. CONCLUSIONS: This coculture method has several useful features, including an appropriate luminal/abluminal EC/SMC orientation, a short distance between the cell layers, the potential for cell-to-cell contact, and the ability to separate the cell types for assays. It is clear that EC markedly affect SMC proliferation, protein/DNA ratio, and structure in coculture with dynamic interactions occurring for at least 4 days. These effects must be considered when attempting to model in vivo phenomena in tissue culture.

Differential response of arteries and vein grafts to blood flow reduction.

PURPOSE: This study was undertaken to evaluate the effect of blood flow on the dimensions and cellular composition of normal arteries and freshly placed vein grafts (VG). METHODS: Bilateral jugular vein interposition grafts were placed in the common carotid arteries of 12 New Zealand white rabbits, and blood flow was reduced on one side by external carotid artery ligation. Shear stress, tangential stress, vessel dimensions, and smooth muscle cell (SMC) proliferation of reduced-flow arteries and VG were compared with these measures in contralateral controls (CON). RESULTS: A sustained reduction in blood flow was documented at 4 weeks (13 +/- 4 ml/min reduced-flow vs 21 +/- 4 ml/min CON; p < 0.05). Reduced-flow carotid arteries had a smaller lumen radius and greater medial thickness compared with normal-flow arteries, but there was no difference in medial cross-sectional area or medial SMC volume and no differences in any intimal measurements. These changes resulted in normalization of shear stress (15.2 +/- 4.6 dynes/cm2 reduced-flow vs 13.6 +/- 2.5 dynes/cm2 CON; p = NS). All VG underwent a marked postimplantation hyperplasia in intima and media, but the major effect of flow reduction on VG dimensions occurred in the intima. Intimal thickness in reduced-flow VG was 60% greater than that in control VG (57 +/- 12 microns vs 35 +/- 5 microns; p = 0.05), and intimal area was 70% greater than that in controls (0.83 +/- 0.24 microns 2 vs 0.48 +/- 0.08 microns 2; p > 0.05). Smaller differences were found in medial thickness (74 +/- 4 microns reduced-flow vs 63 +/- 4 microns CON; p = 0.02) and medial area (1.03 +/- 0.36 microns 2 reduced-flow vs 0.84 +/- 0.22 microns 2 CON; p = 0.05). Intimal SMC volume in reduced-flow VG was 37% greater than that in control VG (p = 0.07). Tangential stress in VG equaled that in ipsilateral arteries, whereas shear stress in VG remained much lower than that in arteries. CONCLUSIONS: In this model, arteries and VG responded to flow reduction by wall thickening, but the mechanism differed. Arteries underwent medial remodeling, lumen caliber reduction, and shear stress normalization, whereas VG responded by an upward modulation of the proliferative response that follows graft placement. These data support a primary role for tangential stress and a secondary role for shear stress in determination of VG dimensions.