Antigen clearing from porcine heart valves with preservation of structural integrity.

Bioprostheses currently used for replacement of diseased cardiovascular tissue are preserved and partially protected from immune rejection through chemical fixation. However, after implantation, chemically preserved (fixed) material has limited durability and lacks the ability to revitalize through cellular ingrowth and remodeling. As an alternative to fixation, we aimed at thoroughly removing antigens from tissue, leaving an intact scaffold, suitable for integration and revitalization in the host. Extensive washing of porcine heart valves with a mixture of two detergents (SDS and Triton X-100) yielded an intact matrix devoid of cells and depleted of soluble proteins that was minimally immunogenic in rabbits. A detailed characterization of the biomechanics and durability of the tissue is under way. If the lack of immunogenicity is confirmed in primates, our results would suggest that a detergent-washed, unfixed porcine heart valve can be an attractive non-inflammatory scaffold for heart valve regeneration in humans.

Improved local control of invasive bladder cancer by concurrent cisplatin and preoperative or definitive radiation. The National Cancer Institute of Canada Clinical Trials Group.

PURPOSE: A prospective randomized trial was conducted to determine whether the addition of concurrent cisplatin to preoperative or definitive radiation therapy in patients with muscle-invasive bladder cancer improved local control or survival. PATIENTS AND METHODS: Ninety-nine eligible patients with T2 to T4b transitional cell bladder cancer participated, 64% with cT3b or cT4. Patients and their physicians selected either definitive radiotherapy or precystectomy radiotherapy; patients were then randomly allocated to receive intravenous cisplatin 100 mg/m2 at 2-week intervals for three cycles concurrent with pelvic radiation, or to receive radiation without chemotherapy. Patients were stratified by clinical tumor stage and by radiation plan. The median follow-up duration is 6.5 years. RESULTS: The occurrence of distant metastases was the same in both study arms. However, 25 of 48 control patients have had a first recurrence in the pelvis, compared with 15 of 51 cisplatin-treated patients (P = .036). The pelvic relapse rate in the two groups was significantly reduced by concurrent cisplatin (P = .038, log-rank test) and this effect was preserved in a stepwise Cox regression model of prognostic factors (hazards ratio, 0.50; 90% confidence interval [CI], 0.29 to 0.86; P = .036). The hazard reduction was similar for both radiation plans. Pretreatment leukocytosis and high clinical stage were independent adverse factors in a Cox model of overall survival, but the effect of cisplatin was not significant. CONCLUSION: Concurrent cisplatin may improve pelvic control of locally advanced bladder cancer with preoperative or definitive radiation, but has not been shown to improve overall survival. The use of concurrent cisplatin had no detectable effect on distant metastases.

Chemotherapy with mitoxantrone plus prednisone or prednisone alone for symptomatic hormone-resistant prostate cancer: a Canadian randomized trial with palliative end points.

PURPOSE: To investigate the benefit of chemotherapy in patients with symptomatic hormone-resistant prostate cancer using relevant end points of palliation in a randomized controlled trial. PATIENTS AND METHODS: We randomized 161 hormone-refractory patients with pain to receive mitoxantrone plus prednisone or prednisone alone (10 mg daily). Nonresponding patients on prednisone could receive mitoxantrone subsequently. The primary end point was a palliative response defined as a 2-point decrease in pain as assessed by a 6-point pain scale completed by patients (or complete loss of pain if initially 1 +) without an increase in analgesic medication and maintained for two consecutive evaluations at least 3 weeks apart. Secondary end points were a decrease of > or = 50% in use of analgesic medication without an increase in pain, duration of response, and survival. Health-related quality of life was evaluated with a series of linear analog self-assessment scales (LASA and the Prostate Cancer-Specific Quality-of-Life Instrument [PROSQOLI]), the core questionnaire of the European Organization for Research and Treatment of Cancer (EORTC), and a disease-specific module. RESULTS: Palliative response was observed in 23 of 80 patients (29%; 95% confidence interval, 19% to 40%) who received mitoxantrone plus prednisone, and in 10 of 81 patients (12%; 95% confidence interval, 6% to 22%) who received prednisone alone (P = .01). An additional seven patients in each group reduced analgesic medication > or = 50% without an increase in pain. The duration of palliation was longer in patients who received chemotherapy (median, 43 and 18 weeks; P < .0001, log-rank). Eleven of 50 patients randomized to prednisone treatment responded after addition of mitoxantrone. There was no difference in overall survival. Treatment was well tolerated, except for five episodes of possible cardiac toxicity in 130 patients who received mitoxantrone. Most responding patients had an improvement in quality-of-life scales and a decrease in serum prostate-specific antigen (PSA) level. CONCLUSION: Chemotherapy with mitoxantrone and prednisone provides palliation for some patients with symptomatic hormone-resistant prostate cancer.

The description of chemotherapy delivery: options and pitfalls.

Delivered and planned drug doses in chemotherapy trials are analyzed in relation to dose size, dose rate, and total drug delivery. Time-dose factors are presented in different ways, further exploring what may be misleading when comparing one treatment program with another. The time frame over which treatment intensity is to be calculated is recognized as arbitrary and not necessarily from start to end of treatment. Considerations for doses missed are presented. The cumulative-dose plot represents a new method of data presentation. This method graphically captures both intensity and total drug delivery per patient, and is helpful when analyzing trials designed to isolate treatment variables and improve the therapeutic index of available drugs.

Locoregional first recurrence after mastectomy: prospective cohort studies with and without immediate chemotherapy.

PURPOSE: To evaluate prospectively the impact of combination chemotherapy in the combined modality treatment of isolated first locoregional recurrence (LRR) following mastectomy for breast cancer. METHODS AND MATERIALS: Between 1979 and 1989, 120 chemotherapy-naive women with isolated LRR as first failure after mastectomy were prospectively identified, uniformly staged, and systematically followed. Treatment consisted of excision if feasible, radical locoregional radiotherapy, and a hormonal maneuver (unless estrogen receptor negative). The initial chemotherapy cohort also received 8 cycles of doxorubicin and cyclophosphamide. This was compared to a subsequent control cohort. RESULTS: For all patients, the 10-year actuarial relapse-free survival +/- 95% confidence interval was 42.1+/-9.2%, and overall survival was 56.8+/-9.1%. No difference was seen in locoregional control between cohorts. At 5 years, distant recurrence-free survival for chemotherapy and control cohort respectively was 75.4+/-10.8% and 60.7+/-12.5% (p = 0.33) and overall survival was 81.9%+/-9.6 and 74.3%+/-11.2 (p = 0.24). Univariate analysis showed no prognostic importance for any imbalance between cohorts. Cox modeling confirmed that complete resection was strongly associated with fewer LRR (hazard ratio [HR] 0.32, p = 0.001) and also with better overall survival (HR 1.82, p = 0.019). Chemotherapy produced a substantial reduction in risk of death (HR 0.72 CI 0.421-1.235, p = 0.23). CONCLUSIONS: In this prospective but nonrandomized study of treatment for first LRR, the risk of death in the later control cohort was 1.39 times the risk in the chemotherapy cohort but failed to reach statistical significance. The results justify further study.

Mitomycin is active against refractory germ-cell tumors. A phase II study.

Mitomycin was used as single-agent therapy in seven male patients with nonseminomatous germ-cell tumors resistant to platinum analogues. The toxicity was high, especially hematological and pulmonary, in this heavily pretreated group. Mitomycin was active in this situation and one complete and one partial response of 18 and 20 weeks' duration, respectively, were seen.

Quantitation of liquid-crystalline ordering in F-actin solutions.

Actin filaments (F-actin) are important determinants of cellular shape and motility. These functions depend on the collective organization of numerous filaments with respect to both position and orientation in the cytoplasm. Much of the orientational organization arises spontaneously through liquid crystal formation in concentrated F-actin solutions. In studying this phenomenon, we found that solutions of purified F-actin undergo a continuous phase transition, from the isotropic state to a liquid crystalline state, when either the mean filament length or the actin concentration is increased above its respective threshold value. The phase diagram representing the threshold filament lengths and concentrations at which the phase transition occurs is consistent with that predicted by Flory's theory on solutions of noninteracting, rigid cylinders (Flory, 1956b). However, in contrast to other predictions based on this model, we found no evidence for the coexistence of isotropic and anisotropic phases. Furthermore, the phase transition proved to be temperature dependent, which suggests the existence of orientation-dependent interfilament interactions or of a temperature-dependent filament flexibility. We developed a simple method for growing undistorted fluorescent acrylodan-labeled F-actin liquid crystals; and we derived a simple theoretical treatment by which polarization-of-fluorescence measurements could be used to quantitate, for the first time, the degree of spontaneous filament ordering (nematic order parameter) in these F-actin liquid crystals. This order parameter was found to increase monotonically with both filament length and concentration. Actin liquid crystals can readily become distorted by a process known as "texturing." Zigzaging and helicoidal liquid crystalline textures which persisted in the absence of ATP were observed through the polarizing microscope. Possible texturing mechanisms are discussed.

Bleomycin and subsequent anaesthesia: a retrospective study at Vancouver General Hospital.

A retrospective review was made of 20 surgical procedures in 14 patients with testicular carcinoma, previously treated with bleomycin chemotherapy, to evaluate the incidence of postoperative pulmonary complications. Other studies have suggested an increased rate of pulmonary complications, including fatal respiratory failure, when inspired oxygen fraction (FIO2) exceeds 0.3 during or after operation. There is a suggestion that bleomycin may sensitize the lungs to the effect of oxygen, leading to oxygen toxicity. This study involved patients whose inspired oxygen fraction ranged from 0.3 to 1.0 and only one non-fatal pulmonary complication occurred. The risks of anaesthesia following bleomycin are discussed with particular reference to possible enhanced oxygen toxicity.

Permanent suppression of phase separation cataract in calf lens using amine modification agents.

Low temperature induced opacification (cold cataract) of the nucleus of young mammalian lenses is associated with a phase separation of proteins in the lens cell cytoplasm. Calf lenses were treated with a variety of imido-esters and N-hydroxysuccinimide-esters, which react specifically with amino groups. Many potent inhibitors of phase separation cataract were identified which lower the opacification temperature by 6 degrees C or more. Lenses generally remain clear, colorless and soft. Furthermore, suppression of the cold cataract temperature is permanent upon removal of excess reagent.

Chronic hypomagnesemia caused by cisplatin: effect of calcitriol.

A group of six patients with hypomagnesemia (serum magnesium less than or equal to 0.5 mmol/L), previously given treatment with cisplatin for ovarian or testicular cancer, received calcitriol at a dose of 0.5 to 1.0 microgram/day for a period of 4 weeks to determine whether treatment with this vitamin D metabolite could improve their hypomagnesemia. In response to treatment, the serum magnesium concentration fell progressively in association with a rise in serum and urinary calcium levels and a decrease in parathyroid hormone level. In a single previous report, active vitamin D metabolites markedly improved renal magnesium wasting. However, in the present study, increases in serum and urinary calcium levels and suppression of parathyroid hormone, factors known to decrease magnesium reabsorption, presumably overwhelmed any direct effect calcitriol may have had to enhance magnesium reabsorption, so that the net effect was a marked exacerbation of the renal magnesium wasting.

The combination of cyproterone acetate and low dose diethylstilbestrol in the treatment of advanced prostatic carcinoma.

Cyproterone acetate is a steroidal antiandrogen with weak progestational activity that results in the partial suppression of pituitary gonadotropin. We demonstrate that the associated decrease in serum testosterone is more complete and prolonged if cyproterone acetate (200 mg. daily) is combined with a low dose of diethylstilbestrol (0.1 mg. daily). The effectiveness of the combination in the treatment of prostatic carcinoma was investigated in 51 patients with stage D2 malignancy. From an initial concentration of 360 +/- 23 ng. per dl. (mean +/- standard error), serum testosterone decreased to 56 +/- 5 ng. per dl. after 1 week and reached a plateau of approximately 30 ng. per dl. after 2 months. This was accompanied by a decrease in serum prostatic acid phosphatase from a mean starting concentration of 43 +/- 11 ng. per ml. to a low of 3 +/- 1 ng. per ml. at 12 months; the proportion of normal values increased from 10 to 80 per cent. A complete response was observed in 7 patients (13 per cent), partial response in 35 (69 per cent) and stable disease in 8 (16 per cent), yielding an over-all objective response rate of 98 per cent (1980 National Prostatic Cancer Project criteria). The actuarial median time to progression was 17 months and the median survival time was 23.5 months. In 26 patients who subsequently had signs of progressive carcinoma the relapse rate in bone (85 per cent) far exceeded that in nonskeletal sites (23 per cent). The incidence of cardiovascular toxicity was 12 per cent. These results indicate that cyproterone acetate and low dose diethylstilbestrol may be co-administered to achieve a synergistic and potent androgen withdrawal effect in the treatment of advanced prostatic carcinoma.

The load dependence of kinesin's mechanical cycle.

Kinesin is a dimeric motor protein that transports organelles in a stepwise manner toward the plus-end of microtubules by converting the energy of ATP hydrolysis into mechanical work. External forces can influence the behavior of kinesin, and force-velocity curves have shown that the motor will slow down and eventually stall under opposing loads of approximately 5 pN. Using an in vitro motility assay in conjunction with a high-resolution optical trapping microscope, we have examined the behavior of individual kinesin molecules under two previously unexplored loading regimes: super-stall loads (>5 pN) and forward (plus-end directed) loads. Whereas some theories of kinesin function predict a reversal of directionality under high loads, we found that kinesin does not walk backwards under loads of up to 13 pN, probably because of an irreversible transition in the mechanical cycle. We also found that this cycle can be significantly accelerated by forward loads under a wide range of ATP concentrations. Finally, we noted an increase in kinesin's rate of dissociation from the microtubule with increasing load, which is consistent with a load dependent partitioning between two recently described kinetic pathways: a coordinated-head pathway (which leads to stepping) and an independent-head pathway (which is static).

Detection of sub-8-nm movements of kinesin by high-resolution optical-trap microscopy.

Kinesin is a molecular motor that transports organelles along microtubules. This enzyme has two identical 7-nm-long motor domains, which it uses to move between consecutive tubulin binding sites spaced 8 nm apart along a microtubular protofilament. The molecular mechanism of this movement, which remains to be elucidated, may be common to all families of motor proteins. In this study, a high-resolution optical-trap microscope was used to measure directly the magnitude of abrupt displacements produced by a single kinesin molecule transporting a microscopic bead. The distribution of magnitudes reveals that kinesin not only undergoes discrete 8-nm movements, in agreement with previous work [Svoboda, K., Schmidt, C. F., Schnapp, B. J. & Block, S.M. (1993) Nature (London) 365, 721-727], but also frequently exhibits smaller movements of about 5 nm. A possible explanation for these unexpected smaller movements is that kinesin's movement from one dimer to the next along a protofilament involves at least two distinct events in the mechanical cycle.

Tubulin GTP hydrolysis influences the structure, mechanical properties, and kinesin-driven transport of microtubules.

Tubulin is a GTPase that hydrolyzes its bound nucleotide triphosphate after it becomes incorporated into a microtubule. The only known consequence of nucleotide hydrolysis is that it increases the dissociation rate of tubulin from the end of the microtubule by 2 orders of magnitude. In this study, we investigated whether microtubules composed of tubulin-GMPCPP (guanylyl alpha,beta-methylenediphosphate) (a very slowly hydrolyzed GTP analog) or tubulin-GDP exhibit additional structural or functional differences. We show that tubulin-GMPCPP microtubules are significantly stiffer than tubulin-GDP microtubules and have a 21% shallower protofilament twist angle. We also find that kinesin, a microtubule-based motor protein, transports tubulin-GMPCPP microtubules at approximately 30% faster rates than tubulin-GDP microtubules. These findings suggest that growing microtubule ends, which are thought to be composed of tubulin-GTP, may have different structural and mechanical properties from the remainder of the microtubule polymer.

Teratoma in primary testis tumor reduces complete response rates in the retroperitoneum after primary chemotherapy. The case for primary retroperitoneal lymph node dissection of stage IIb germ cell tumors with teratomatous elements.

BACKGROUND: Recent advances in the therapy of advanced testicular nonseminomatous germ cell tumors (NSGCT) have resulted in increased attention to avoiding double therapy in cases where single modality therapy will suffice. METHODS: Over an 8-year period, 104 patients with Stage II and III testicular NSGCT received primary chemotherapy. Seventy-nine patients had retroperitoneal lymph nodal metastases, 33 of whom had a radiologic complete response, 43 a radiologic incomplete response, and 3 were not re-evaluated after induction chemotherapy. Thirty-nine patients underwent retroperitoneal lymph node dissection (RPLND). The radiologic and pathologic response of the nodes to primary chemotherapy was correlated with tumor burden (lymph node metastasis size < or = 2 cm, 2.1-5 cm, 5.1-10 cm, and >10 cm), primary tumor pathology, and prechemotherapy marker levels. RESULTS: Larger initial lymph node size, metastases size, the presence of teratoma in the primary tumor, and prechemotherapy alpha-fetoprotein (alpha-FP) > 80 mg/L and beta-HCG (bHCG) 10000 IU/L were found to correlate significantly with an incomplete radiologic response. Lymph node metastases size was the only independent prognostic factor on multivariable logistic regression analysis. Prechemotherapy alpha-FP > 80 mg/L and beta-HCG > 10000 IU/L were associated with the presence of teratoma or carcinoma in the retroperitoneal nodes. The presence of teratoma in the primary tumor is associated with a higher incomplete response rate and residual teratoma in the retroperitoneal lymph nodes after primary chemotherapy. CONCLUSIONS: Predictors of need for postchemotherapy RPLND include large lymph node metastasis size and presence of teratomatous elements in the primary tumor. To reduce the need for and morbidity of double therapy, patients with low volume clinical Stage II, NSGCT and teratomatous elements in the primary tumor are arguably better served by primary RPLND.

Oxidative modifications to crystallins induced in calf lenses in vitro by hydrogen peroxide.

Calf lenses which are incubated in solutions of 1-150 mM H2O2 for 24 hr remain clear at 20 degrees C. While insoluble lens protein increases by at most 2-3%, we find extensive oxidation of exposed protein thiols, major shifts in the size distribution of crystallins, and progressive generation of more acidic polypeptides. Some of these oxidative modifications are reversible with reducing agent. beta H-Crystallins are particularly susceptible to oxidation: disulfide-bonded soluble aggregates form at low H2O2 levels, while irreversible dissociation to beta L-crystallins occurs at high H2O2 concentration. The gamma-crystallins are particularly prone to charge modification. In contrast, the size and charge distributions of alpha-crystallins appear to be virtually unaffected by H2O2.

Phase II study of lonidamine in patients with metastatic renal cell carcinoma: a National Cancer Institute of Canada Clinical Trials Group Study.

The National Cancer Institute of Canada Clinical Trials Group conducted a phase II study of lonidamine, given in an escalating oral daily schedule in patients with measurable advanced renal cell carcinoma. Two responses were seen in 25 evaluable patients. Toxicity was mild or moderate in most patients and included myalgia, nausea, vomiting, somnolence, and testicular pain. Lonidamine was not myelosuppressive. This agent had only minimal activity against renal cell carcinoma when given in this oral schedule.

Phase II study of lonidamine in patients with metastatic breast cancer: a National Cancer Institute of Canada Clinical Trials Group Study.

The National Cancer Institute of Canada Clinical Trials Group conducted a phase II study of lonidamine, given in an escalating oral daily schedule to a maximum dose of 450 mg/m2 in patients with previously treated advanced breast cancer. Five responses were seen in 30 evaluable patients (17%). Treatment was discontinued because of toxicity in seven patients. Toxicity generally consisted of myalgia, nausea, vomiting, skin hyperesthesia, somnolence, and ototoxicity. All side effects were reversible and no hematologic toxicity was observed. The absence of myelosuppression and the suggestive lack of cross-resistance between lonidamine and standard chemotherapeutic drugs warrant further studies of lonidamine in breast cancer, particularly in combination with other agents.

Renal magnesium wasting and hypocalciuria in chronic cis-platinum nephropathy in man.

1. The renal handling of calcium and magnesium was studied in six patients with persistent hypomagnesaemia after cis-platinum treatment for testicular tumours. 2. In comparison with normal subjects, the patients showed hypomagnesaemia (mean 0.54 mmol/l), which was associated with a normal urinary magnesium excretion (mean 4.83 mmol/24 h). Urinary calcium excretion was significantly lower in the patients than in the normal subjects (mean 2.05 vs 5.15 mmol/24 h, respectively; P less than 0.01), despite slightly higher total serum calcium levels (2.53 vs 2.38 mmol/l, respectively; P less than 0.05). During magnesium chloride infusion, when serum magnesium levels were comparable in patients and controls, urinary calcium excretion remained lower in the patients, indicating that hypomagnesaemia was not the cause of the hypocalciuria. 3. Dietary magnesium supplementation resulted in a significant increase in the serum magnesium levels in the patients, while dietary magnesium deprivation resulted in a comparable decrease in urinary magnesium excretion in patients and controls (to 1.46 and 2.00 mmol/day, respectively), although the serum magnesium level fell further (to 0.46 mmol/l) in the patients. 4. The dissociation of renal calcium and magnesium excretion appears to be part of the intrinsic tubular defect caused by cis-platinum. This dissociation of urinary calcium and magnesium excretion, which resembles that seen in Bartter's syndrome, may result from a lesion in the distal convoluted tubule.