Ultrasonic characterization of human cancellous bone in vitro using three different apparent backscatter parameters in the frequency range 0.6-15.0 mhz.

Ultrasonic techniques based on measurements of apparent backscatter may provide a useful means for diagnosing bone diseases such as osteoporosis. The term "apparent" means that the backscattered signals are not compensated for the frequency-dependent effects of attenuation and diffraction. We performed in vitro apparent backscatter measurements on 23 specimens of human cancellous bone prepared from the left and right femoral heads of seven donors. A mechanical scanning system was used to obtain backscattered signals from each specimen at several sites. Scans were performed using five different ultrasonic transducers with center frequencies of 1, 2.25, 5, 7.5, and 10 MHz. The -6 dB bandwidths of these transducers covered a frequency range of 0.6-15.0 MHz. The backscattered signals were analyzed to determine three ultrasonic parameters: apparent integrated backscatter (AIB), frequency slope of apparent backscatter (FSAB), and time slope of apparent backscatter (TSAB). Linear regression analysis was used to examine the correlation of these ultrasonic parameters with five measured physical characteristics of the specimens: mass density, X-ray bone mineral density, Young's modulus, yield strength, and ultimate strength. A total of 75 such correlations were examined (3 ultrasonic parameters x 5 specimen characteristics x 5 transducers). Good correlations were observed for AIB using the 5 MHz (r = 0.70 - 0.89) and 7.5 MHz (r = 0.75-0.93) transducers; for FSAB using the 2.25 MHz (r = 0.70 - 0.88), 5 MHz (r = 0.79 - 0.94), and 7.5 MHz (r = 0.80 - 0.92) transducers; and for TSAB using the 5 MHz (r = 0.68 - 0.89), 7.5 MHz (r = 0.75 - 0.89), and 10 MHz (r = 0.75 - 0.92) transducers.

Office assessment of patient outcome of pharmacologic therapy for urge incontinence.

Thirty adult women with urge urinary incontinence were included in this study. After completing the basic evaluation, including a self-administered incontinence questionnaire, patients were treated with a bladder relaxant preparation for 6-8 weeks. At follow-up the incontinence questionnaire and a global assessment of outcome scale were administered. Data were analyzed using the Mann-Whitney and Kruskal-Wallis tests, with a subsequent Tukey's test. After 6-8 weeks of therapy, 63% of patients reported that they were greatly or moderately improved, with a significant mean decrease in their total urge score of 51%. Subjects slightly improved (12%) and unimproved/worse (20%) had no significant change in their mean urge score. Most patients with great or moderate improvement continued with their initial drug treatment. Successful pharmacotherapy for urinary urge incontinence may be assessed by a simple global scale which correlated well with response to the MESA questionnaire. Moderate and greatly improved patients correlated with a 50% mean decrease in urge score and continued their initial drug therapy.

Varicocele in adolescence induces left and right testicular volume loss.

OBJECTIVE: To determine the effect of a palpable left-sided varicocele (which in adolescent patients can adversely affect left testicular volume) on right testicular volume with progressive Tanner development and increasing varicocele grade. PATIENTS AND METHODS: The right and left testicular volumes were measured with a standard orchidometer in 70 control patients (mean age 14.6 years, SD 2.2) with no palpable testicular abnormality and in 434 (mean age 14.3 years, SD 2.3) with a palpable left-sided varicocele. Patients with bilateral and right-sided varicoceles were excluded from the study. RESULTS: There was no significant difference between the left and right testicular volumes in the control patients. The testicular volumes of patients with a grade I varicocele were similar to those in control patients. Patients with a grade II varicocele had a significantly smaller left testis than the controls at Tanner stages 4 and 5 (P < or = 0.05). Patients with a grade III varicocele had a significantly smaller left testis than controls at each Tanner stage (P < or = 0.05) and significantly smaller right testis than controls at Tanner stages 4 and 5 (P < or = 0.05). CONCLUSION: The presence of a grade I varicocele in adolescence appears to have no effect on normal testicular growth. Some patients with a grade II varicocele are at risk of left testicular volume loss with time and should have their testicular volume measured annually. Patients with grade III varicocele are at risk of bilateral testicular volume loss; a careful evaluation and early surgical intervention are recommended in this group of patients.

Preliminary study on urinary cytokine levels in interstitial cystitis: does intravesical bacille Calmette-Guérin treat interstitial cystitis by altering the immune profile in the bladder?

OBJECTIVES: To obtain preliminary urinary cytokine data on subjects with active interstitial cystitis (IC), subjects with IC in remission after bacille Calmette-Guérin (BCG), and control (non-IC) subjects. IC is a severe, debilitating bladder disease of unknown etiology and no cure. In controlled clinical trials, intravesical BCG has been shown to be an effective and durable treatment for IC. The durability of this treatment led us to speculate on the mechanism by which intravesical BCG may treat IC. Evidence exists that IC may be mediated by an abnormal immune profile within the bladder. Intravesical BCG is known to stimulate the immune system of the bladder. METHODS: Fresh voided urine was collected from 15 subjects with active IC, 9 subjects with IC who received intravesical BCG and had been in remission for an average of 2.6 years, and 11 non-IC subjects. The urine was immediately centrifuged, aliquoted, and frozen in liquid nitrogen. At the time of urine collection, a validated IC questionnaire was completed. The enzyme-linked immunosorbent assay technique was used to determine levels of urinary cytokines interleukin (IL)-2, IL-4, IL-6, IL-8, IL-10, IL-12, tumor necrosis factor (TNF), human granulocyte-macrophage colony stimulating factor (hGM-CSF), IL-1beta, and interferon-gamma (IFN-gamma). RESULTS: Cytokines IL-4, IL-10, IL-12, TNF, hGM-CSF, IL-1beta, and IFN-gamma were not detected. Significant elevations in symptom scores and IL-2, IL-6, and IL-8 were found in the urine of subjects with active IC compared with subjects with IC in remission and control subjects. The urinary cytokine levels and symptom scores were identical in the IC group who had received BCG and the control group. CONCLUSIONS: Elevations in symptom scores and urinary cytokine levels were seen in subjects with active IC, suggesting an abnormal immune profile in this disease. Subjects with IC in remission after receiving BCG had identical cytokine levels and symptom scores as non-IC control subjects. Intravesical BCG may be effective in treating IC by correcting an aberrant immune imbalance in the bladder, leading to long-term symptomatic improvement. A prospective study is ongoing to further investigate the role of the immune system in IC.

Office based criteria for predicting type II stress incontinence without further evaluation studies.

PURPOSE: The 1996 Agency for Health Care Policy and Research Clinical Practice Guidelines for Urinary Incontinence suggested that surgery may be performed after basic evaluation without further testing in cases of uncomplicated nonrecurrent type II stress urinary incontinence. However, they failed to recommend explicit office based criteria to identify this condition without further invasive testing. We establish such criteria and test the efficacy. MATERIALS AND METHODS: Records of 101 women with urinary incontinence who underwent complete evaluation from June 1995 to September 1997 were reviewed. Basic evaluation consisted of medical history, physical/pelvic examination and the validated Medical, Epidemiologic and Social Aspects of Aging Urinary Incontinence Questionnaire, while further testing included cystourethroscopy, urodynamics and lateral stress cystogram. All cases diagnosed as pure type II stress urinary incontinence after basic and further testing were identified, and office based findings were characterized, which included medical history, questionnaire scores, vaginal speculum examination results and post-void residual urine volume. These criteria were applied in a blinded fashion to the initial group of 101 patients and then to a new group of 45 women with incontinence who recently underwent complete evaluation. RESULTS: Specificity and positive predictive value were 100% in both groups for nonsurgical cases, compared to 81 and 71% in the initial, and 75 and 60% in the new group, respectively, for surgical cases. CONCLUSIONS: Our office based criteria are 100% specific for predicting pure type II stress urinary incontinence in women with no suspension surgery and, thus, have the potential of reducing the need for further invasive testing and consequently the cost of preoperative evaluation.

The efficacy of intravesical bacillus Calmette-Guerin in the treatment of interstitial cystitis: long-term followup.

PURPOSE: Interstitial cystitis is a severe debilitating bladder disease characterized by unrelenting pelvic pain and urinary frequency. A prospective, double-blind, placebo controlled study of the use of intravesical bacillus Calmette-Guerin (BCG) in the treatment of interstitial cystitis was recently completed with a mean followup of 8 months. Results demonstrated a 60% BCG response rate, compared to a 27% placebo response rate. We now report the long-term followup results of those patients who received intravesical BCG. MATERIALS AND METHODS: Subjects randomized to receive BCG were followed at routine intervals with questionnaires and voiding diaries identical to those in the blinded study. Adverse events were closely monitored in the treatment and followup phases of the study. Subject baseline values were compared to followup data. RESULTS: Of the BCG responders mean followup was 27 months (range 24 to 33), and 8 of 9 (89%) continue to have an excellent response in all parameters measured. The global interstitial cystitis survey improved 70%, daily voids decreased 31%, nocturia improved 54%, mean voided volume increased 61%, pelvic pain decreased 81%, vaginal pain decreased 71%, urgency decreased 71% and dysuria decreased 82%. Overall well-being improved 54% and the Rand-36 quality of life survey overall improved 64%. In 86% of the patients (6 of 7) dyspareunia resolved. Of the initial BCG nonresponders there was no significant difference in interstitial cystitis symptomatology from baseline to last followup, suggesting that BCG does not worsen interstitial cystitis symptoms. No long-term adverse events from BCG were noted. CONCLUSIONS: Intravesical Tice BCG is safe, effective and durable in the treatment of interstitial cystitis. Of those patients who received only 6 weekly treatments and responded favorably 89% continue to have an excellent response with followup ranging from 24 to 33 months.

Low-molecular-weight inhibitor of in vitro fibroblast colony formation from human urine.

The role of urinary toxins in interstitial cystitis (IC) has been suggested. This report describes the partial purification of a substance from human urine that inhibited in vitro colony formation by mouse fibroblasts. Urine samples from 15 women with IC and 17 healthy women serving as volunteers were fractioned by ultrafiltration and chromatography methods and tested by the inhibition of Swiss 3T3 fibroblast colony formation. The fibroblasts were cultured at low density with varying concentrations of whole or fractioned urine. Colonies were counted at 10 days. Colony formation was reduced by incubation with whole urine, ultrafiltrate, and nonadsorbed C18 fractions. Inhibition of colony formation by urine from healthy volunteers or women with IC was not significantly different. In vitro colony formation by Swiss 3T3 cells was inhibited by a component of human urine. The toxicity of urine from IC patients was not different from that of urine from healthy controls.

Semiautomated quantitation of urinary protein and creatinine.

The application of personal computers to routine tasks such as the recording and manipulation of data can greatly increase the speed and accuracy of the analyses. This report describes the use of a Macintosh computer and a BioTek microplate reader for the analysis of urinary protein and creatinine. Previously published assay methods for protein and creatinine were adapted for analysis using 96-well microplates. The adapted assays had excellent linearity (r2 = 0.99), with reduced variability compared to the previous methods. This resulted in increased sensitivity as demonstrated by 95% confidence limits. In addition, reductions of both assay materials and sample sizes were realized. The direct connection of the computer and microplate reader reduced the time needed to record absorbance measurements and perform analyses when compared to the more common protocols and methods of analysis. The assay protocols, equipment and interface, data file management, and methods of analysis are described.

Are pelvic computed tomography, bone scan and pelvic lymphadenectomy necessary in the staging of prostatic cancer?

OBJECTIVE: To investigate the effectiveness and economy of pelvic computed tomography (CT), bone scan and pelvic lymphadenectomy as staging modalities in patients undergoing radical prostatectomy. The use of prostate specific antigen (PSA) and Gleason's score as adjuncts to predict extracapsular disease were also evaluated and their economic implications examined. PATIENTS AND METHODS: Between January 1990 and June 1993, 861 men were newly diagnosed with prostate cancer, of whom 409 underwent surgery. All patients underwent pelvic CT scans and PSA analysis. Patients undergoing surgery had pre-operative bone scans and Gleason's scoring of their pathological tissue. RESULTS: Only 13 (1.5%) of 861 men had positive pelvic CT scans. Of the 409 patients who underwent surgery, all had negative pelvic CT and bone scans, and all underwent a modified pelvic lymphadenectomy; 192 (47%) had extracapsular disease. Only 15 (3.7%) patients who underwent surgery were found to have positive nodes. CONCLUSIONS: The use of pelvic CT and bone scans for clinical staging in patients with a PSA level of < or = 20 ng/mL should not be advocated because they have a very low yield and are not cost effective. We question the role of a modified pelvic lymphadenectomy for staging purposes, either by an open or laparoscopic procedure, because the yield of positive diagnoses is very low.

Testicular growth after successful varicocele correction in adolescents: comparison of artery sparing techniques with the Palomo procedure.

Testicular growth after varicocele surgery was evaluated in 116 boys 9 to 20 years old. A total of 88 boys was available for followup testicular examination 3 to 60 months after successful varicocele repair (mean 25). Left testicular volume loss of 2 cc or greater was present preoperatively in 72 of the 88 patients. The Palomo procedure was performed in 36 cases and repair using artery sparing techniques was done in 36. Mean relative left testicular volume increased 18% in the artery sparing group and 21% in the Palomo group. The increase in relative testicular volume compared to preoperative volumes was statistically significant in both groups (p < 0.05). There was no significant difference in testicular growth between the groups and no postoperative testicular atrophy was observed. A comparison group of 8 boys with uncorrected varicoceles demonstrated a mean relative volume increase of 3% (mean followup 22 months). The increase in testis volume in successfully corrected cases was statistically different (p < 0.05) from that of uncorrected cases. We conclude that reversal of varix induced testicular growth failure occurs only after successful surgical correction. The Palomo procedure resulted in equivalent testicular growth compared to the artery sparing techniques with fewer complications and no testicular atrophy despite intentional ligation of the testicular artery. Based on our data, we believe that the Palomo procedure should be the procedure of choice for adolescent varicocele correction.

Complement C3, eosinophil cationic protein and symptom evaluation in interstitial cystitis.

Interstitial cystitis is a painful, irritative voiding dysfunction of unknown etiology. In this study 19 women undergoing treatment for interstitial cystitis and 6 healthy women of similar age provided 2 urine and serum specimens with at least a 3-month interval between collections. Complement C3 and eosinophil cationic protein were determined by immunoassay methods, and symptom severity was quantitated with a visual analog scale questionnaire. Concentrations of complement C3 and eosinophil cationic protein from either serum or urine were not significantly different between interstitial cystitis patients and controls at either determination, although substantial differences were noted even between individual initial and followup determinations. Normalization of urine osmolality did not alter these results. Symptom severity scores were significantly greater in interstitial cystitis patients compared to controls but failed to correlate with the concentrations of complement C3 or eosinophil cationic protein. Therefore, the hypothesis that complement C3 and eosinophil cationic protein may be etiological factors of interstitial cystitis is not supported.

Pituitary gonadal dysfunction in adolescents with varicocele.

Gonadotropin-releasing hormone stimulation testing was performed on 104 adolescent males with a unilateral left varicocele to determine the frequency of testicular dysfunction in this group of patients. An abnormal test result was noted in approximately 30 percent of varicocele patients. We believe that an abnormal test result indicates a higher risk for future fertility problems, and early treatment may reduce this risk.

Phenotypic properties of cultured tumor cells: integrin alpha IIb beta 3 expression, tumor-cell-induced platelet aggregation, and tumor-cell adhesion to endothelium as important parameters of experimental metastasis.

The present study was undertaken to investigate the factors involved in determining the metastatic potential of cultured cells derived from solid tumors. We first investigated the effects of cell source and culture conditions on lung colony formation by i.v. injected B16a (B16 amelanotic melanoma) cells and inhibition of tumor colony formation by the thromboxane A2 synthase inhibitor, CGS14854. Prolonged culture resulted in a 10-fold decrease in the incidence of B16a lung colonies, whereas passage in vivo for 150 days did not affect lung colony formation by tumor cells isolated from enzymatic dispersates by centrifugal elutriation. Cultured B16a cells maintained at low density (LD) and harvested at low passage (LP) formed significantly more lung colonies than B16a cells harvested at high densities (HD) or high passage (HP). Over-confluent tumor cells produced even lower number of lung colonies. Lung colony formation by elutriated B16a cells (i.e., cells freshly isolated from tumor tissue) was consistently inhibited by CGS14854, whereas inhibition of lung colony formation by cultured B16a cells was dependent upon culture conditions. CGS14854 was ineffective or less effective against HD/HP B16a cells. The differences in lung colony formation between LD, HD and elutriated B16a cells were not due to differential cell-cycle distribution. Mechanistic studies indicated that LD/LP tumor cells induced aggregation of homologous platelets, whereas HD/HP B16a cells failed to induce significant platelet aggregation. Aggregation of homologous platelets correlated positively with lung-colonizing ability. Additionally, LD/LP cells demonstrated higher adhesion to endothelium than HD/HP B16a cells. Finally, LD/LP B16a cells expressed higher levels of alpha IIb beta 3 integrins than HD/HP tumor cells, as determined by flow cytometry and immunofluorescence.

Studies on the role of platelet eicosanoid metabolism and integrin alpha IIb beta 3 in tumor-cell-induced platelet aggregation.

Platelet eicosanoid metabolism resulting from tumor-cell-induced platelet aggregation (TCIPA) was examined in a homologous in vitro system. Rat Walker 256 carcinosarcoma cells induced the aggregation of rat platelets via a thrombin-dependent mechanism with concomitant production of eicosanoid metabolites (e.g., 12-HETE, TXA2). TCIPA was dependent on the concentration of tumor cells inducing aggregation, as well as cyclooxygenase and lipoxygenase products. Cyclooxygenase inhibitors, but not lipoxygenase inhibitors, blocked platelet aggregation induced in vitro by a low concentration of agonist. At a high agonist concentration, neither cyclooxygenase nor lipoxygenase inhibitors alone affected platelet aggregation; however, the combined inhibition of both the cyclooxygenase and lipoxygenase pathways resulted in subsequent inhibition of platelet aggregation regardless of agonist concentration. The extent of platelet TXA2 and 12-HETE biosynthesis was likewise dependent on and correlated with agonist concentration. The inhibitors used in this study did not significantly inhibit protein kinase C activity at the doses tested. Platelet surface glycoprotein alpha IIb beta 3 play an important role in platelet aggregation. The effect of platelet cyclooxygenase and lipoxygenase inhibition in regulating alpha IIb beta 3 surface expression was examined by flow cytometric analysis. Thrombin stimulation of washed rat platelets resulted in significantly increased surface expression of platelet alpha IIb beta 3 integrin complex. The enhanced surface expression was not inhibited by a cyclooxygenase inhibitor (aspirin), a thromboxane synthase inhibitor (CGS-14854) or a thromboxane receptor antagonist (SQ 29,548), nor was it stimulated by a thromboxane A2 mimic (pinane-thromboxane A2). However, alpha IIb beta 3 expression was blocked by lipoxygenase inhibition and stereospecifically increased by the platelet lipoxygenase metabolite 12(S)-HETE. These results suggest that both the platelet lipoxygenase and cyclooxygenase pathways are important for TCIPA but that different mechanisms of action are involved.

Alpha IIb beta 3 integrin expression and function in subpopulations of murine tumors.

Subpopulations of B16 amelanotic melanoma (B16a) cells, isolated by centrifugal elutriation from enzymatically dispersed solid tumors, demonstrated different abilities to form lung colonies when injected intravenously. In contrast, no differences in experimental metastasis were observed among subpopulations obtained from Lewis lung (3LL) tumors. Lung colonization by B16a and 3LL subpopulations correlated positively with observed differences (B16a) or lack of differences (3LL) in tumor cell ability to induce aggregation of homologous platelets, to adhere to subendothelial matrix or fibronectin, and with the percentage of cells in the G2/M phase of the cell cycle. Both B16a and 3LL cells express alpha IIb beta 3 integrin receptors; however, differences in the receptor expression level were found only among B16a subpopulations. Comparison of the amount of alpha IIb beta 3 receptor expressed on cell surface with tumor cell ability to induce platelet aggregation (TCIPA) and to adhere to fibronectin or subendothelial matrix revealed a positive correlation. Pretreatment of tumor cells with alpha IIb beta 3-specific antibodies inhibited tumor cell matrix adhesion, TCIPA, and lung colony formation. We propose that alpha IIb beta 3 integrin receptor expression, tumor cell matrix adhesion, and tumor cell-induced platelet aggregation can be important parameters to indicate the metastatic potential of some tumor cells and that the alpha IIb beta 3 is a multifunctional receptor involved in both tumor cell-matrix and tumor cell-platelet interactions. Further, the correlation among cell cycle phase, metastatic ability, and receptor expression suggests that metastatic propensity may be transiently expressed and/or increased in some tumor cell subpopulations.

The significance of elevated scrotal temperature in an adolescent with a varicocele.

Simultaneous measurements of left and right scrotal, and axillary skin temperatures were recorded in 58 consecutive adolescents (mean age 14.4 years) with a grade II-III left sided varicocele, and nine control adolescents without genital pathology (mean age 15.7 years). Left and right testicular volumes were determined in both groups. The adolescents with a varicocele had a significant bilateral elevation of the scrotal temperatures compared to the control subjects. This relative hyperthermia was present in both supine and standing positions. The mean left scrotal temperature of varicocele patients was significantly higher in the standing position than in the supine position, which may reflect the dependent venous filling of the varicocele. Those varicocele patients who maintained a left scrotal temperature at least 1.4 degrees C cooler than axillary did not have significant left testicular volume loss, whereas those whose left temperature was approximately equal to axillary did have significant growth retardation of the left testis. Following successful varicocele surgery, left scrotal temperatures were significantly cooler, and statistically indistinguishable from controls. The left testicular volumes were also significantly improved with respect to corresponding right testicular volumes. These observations suggest that adolescents with a moderate to large left varicocele have a significant bilateral loss of testicular thermoregulation. In those individuals with a significantly warmer left hemiscrotum, there is a definite increased potential for left testicular volume loss. Varicocele surgery can reverse this process.

Role of platelet membrane in enhancement of tumor cell adhesion to endothelial cell extracellular matrix.

Tumor cell adhesion to subendothelial matrix in the presence of platelets and plasma has been examined in vitro using an entirely homologous system of rat Walker 256 carcinosarcoma cells, matrix laid down by rat aortic endothelial cells and rat platelets and plasma. In the presence of platelets or platelets plus plasma, tumor cell adhesion was significantly enhanced when compared to adhesion in the absence of platelets. In the presence of plasma alone (0.1%), we observed no significant increase in tumor cell adhesion. In order to determine which platelet factors contribute to the enhancement of tumor cell adhesion by platelets, we subjected washed rat platelets to mechanical lysis or thrombin stimulation followed by centrifugation. The membrane fractions and supernatant fractions containing platelet attachment proteins were compared for their abilities to support tumor cell adhesion to subendothelial matrix. Platelet membranes were also recombined with platelet supernatant fractions to determine if platelet attachment proteins or platelet membranes required the presence of the other to enhance tumor cell adhesion. Platelet supernatant fractions which contained release reaction proteins (confirmed by polyacrylamide gel electrophoresis) did not enhance tumor cell adhesion. Purified thrombospondin, fibronectin, beta-thromboglobulin, platelet derived growth factor, and serotonin had no effect on tumor cell adhesion. Platelet membrane containing fractions affected tumor cell adhesion to subendothelial matrix as follows: (a) platelets formed an adhesive bridge between tumor cells and the subendothelial matrix as demonstrated by scanning electron microscopy; (b) intact platelets and thrombin stimulated platelets were the most effective at facilitating tumor cell adhesion; (c) preparations containing partially lysed platelet ghosts were more effective in supporting tumor cell adhesion to subendothelial matrix than were preparations containing completely lysed platelet membrane fragments; (d) recombination of platelet supernatant fractions with mechanically lysed platelets did not enhance their ability to support adhesion; (e) fixed platelets, either alone or in combination with platelet supernatant fractions, failed to enhance adhesion. These data indicate that platelet enhanced tumor cell adhesion appears to be dependent on platelet membrane factors including receptor mobility, rather than intraplatelet components.

Platelet enhancement of tumor cell adhesion to subendothelial matrix: role of platelet cytoskeleton and platelet membrane.

Platelet involvement during tumor cell adhesion to subendothelial matrix was examined in vitro. Platelets were subjected to thrombin stimulation and mechanical lysis and examined for their effects on tumor cell adhesion. These treatments altered the platelet ultrastructure and cytoskeletal integrity. Untreated washed rat platelets (WRP) exhibited extensive adhesion to and spreading on substrates and substantially enhanced tumor cell adhesion to the same substrates (i.e., 250% greater than tumor cells without platelets). Thrombin prestimulation of platelets limited platelet adhesion and spreading and platelet facilitation of tumor cell adhesion. Complete mechanical lysis disrupted both the platelet membrane and the cytoskeleton and eliminated the ability of platelets to adhere or to enhance tumor cell adhesion. Partially lysed platelets resembled membrane ghosts and facilitated tumor cell adhesion by a mechanism independent of spreading and cytoskeletal rearrangement. Fractionation studies indicated that platelet cytoskeletal components played a role in the adhesion process. Pretreatment of WRP with cytochalasin A or B dose dependently inhibited microfilament-mediated platelet spreading and platelet-enhanced tumor cell adhesion. Colchicine and vinblastine induced microtubule depolymerization, but they had no observable effect on platelet spreading or platelet-enhanced tumor cell adhesion. It was concluded that platelet-enhanced tumor cell adhesion to subendothelial matrix depends on an intact platelet cytoskeleton and on a platelet membrane component(s) and is mediated by surface contact between platelets and tumor cells. Furthermore, platelet-mediated tumor cell adhesion to subendothelial matrix may involve two mechanisms: one dependent on, and one independent of, platelet spreading and cytoskeletal rearrangement.

A new in vitro model for investigation of tumor cell-platelet-endothelial cell interactions and concomitant eicosanoid biosynthesis.

We have developed a new in vitro model system to examine tumor cell-platelet-endothelial cell interactions under dynamic conditions. Using the same model, we can determine endogenous eicosanoid metabolism and alterations in the prostacyclin-thromboxane A2 balance associated with interactions among tumor cells, platelets, and endothelial cells. The model consisted of cloned rat aortic endothelial cells grown on gelatin microcarrier beads under dynamic conditions (i.e., spinner culture). Interactions of these endothelial cells with platelets (heparinized rat platelet rich plasma) and/or tumor cells (rat Walker 256 carcinosarcoma) were assessed in an aggregometer. Gelatin beads alone or microcarrier grown endothelial cells did not elicit spontaneous aggregation of platelet rich plasma over a time period of 30 min. Microcarrier grown endothelial cells inhibited tumor cell induced platelet aggregation in a dose dependent fashion (i.e., depending on endothelial cell number). The ability of microcarrier grown endothelial cells to inhibit tumor cell induced platelet aggregation depended on endogenous production of prostacyclin. This conclusion is based on the following results: an increased number of microcarrier grown endothelial cells caused a prolongation of the aggregation lag time; an increased number of microcarrier grown endothelial cells caused a proportionate increase in 6-keto-prostaglandin F1 alpha concentration; an increased number of microcarrier grown endothelial cells was inversely correlated with thromboxane A2 production by platelets; indomethacin pretreatment of microcarrier grown endothelial cells caused a decrease in prostacyclin production and therefore overcame the associated inhibition of tumor cell induced platelet aggregation; and the inhibition of tumor cell induced platelet aggregation in the presence of endogenous prostacyclin produced by microcarrier grown endothelial cells was the same as that observed in the presence of exogenous prostacyclin. Scanning electron microscopy of aggregometry samples revealed: little or no platelet or tumor cell adhesion to gelatin beads alone, a low basal adhesion of tumor cells to microcarrier grown endothelial cells, and large aggregates of platelets and tumor cells located primarily at gaps in the monolayer of indomethacin treated microcarrier grown endothelial cells. This new in vitro model provides a method for examining the effects of eicosanoid metabolism by endothelial cells on tumor cell-platelet-endothelial cell interactions under dynamic conditions.