Determinants of CPT-11 and SN-38 activities in human lung cancer cells

Irinotecan (CPT-11) is a semisynthetic camptothecin derivative with a broad spectrum of anti-tumour activity. Carboxylesterase (CE) catalyses the conversion of CPT-11 to SN-38 (7-ethyl-10-hydroxycamptothecin), the active form of CPT-11. The antiproliferative effects of CPT-11 and SN-38, CE-activity and topoisomerase I protein expression were investigated in five human small-cell lung cancer (SCLC) cell lines and four human non-small-cell lung cancer (NSCLC) cell lines. Antiproliferative activity, expressed as IC50 values, was determined using the MTT assay. CPT-11 was significantly more active in SCLC than in NSCLC cell lines (P = 0.0036), whereas no significant difference between histological types was observed with SN-38. A significant correlation (r2 = 0.52, P = 0.028) was observed between CE activity and chemosensitivity to CPT-11 but not to SN-38, and significantly higher CE activity was observed in SCLC compared with NSCLC cell lines (P = 0.025). Western blotting experiments showed topoisomerase I protein expressions within a factor of 2, and a granular nuclear staining was detectable in all cell lines by immunocytochemistry of cytospins. No correlation was observed between protein expression and sensitivity to CPT-11 or SN-38. Cellular and medium concentrations of CPT-11 and SN-38 were measured by high-performance liquid chromatography (HPLC) in one SCLC cell line with high CE activity and high sensitivity to CPT-11, and one NSCLC cell line with low sensitivity to CPT-11 and CE activity. Intracellular concentrations of CPT-11 and SN-38 were higher in the SCLC cell line, and this was associated with an increase in cellular uptake of CPT-11 compared with the medium, and an increased intracellular formation of SN-38. In conclusion, CE activity appears to be associated with higher sensitivity to CPT-11 in human lung cancer cell lines and may partly explain the difference in the in vitro sensitivity to CPT-11 between SCLC and NSCLC cells. The assessment of CE activity in clinical material of lung cancer patients undergoing treatment with CPT-11 may be warranted. However, other mechanisms may influence sensitivity to CPT-11, possibly including drug transport.     

The Mayo Clinic-Canadian Cooperative trial of sulfasalazine in active multiple sclerosis

OBJECTIVE: To determine whether sulfasalazine is better than placebo in slowing disability progression in MS. METHODS: In this randomized, double-blind, placebo-controlled phase III trial, 199 patients with active relapsing-remitting (n = 151) or progressive (n = 48) MS were evaluated at 3-month intervals for a minimum of 3 years (94% completed 3 years of follow-up; mean follow-up, 3.7 years). MRI studies were performed at 6-month intervals on a subset of 89 patients. RESULTS: Sulfasalazine failed to slow or prevent disability progression as measured by the primary outcome (confirmed worsening of the Expanded Disability Status Scale [EDSS] score by at least 1.0 point on two consecutive 3-month visits). Sulfasalazine influenced favorably a number of secondary outcomes during the first 18 months of the trial (e.g., annualized relapse rate, proportion of relapse-free patients; progressive subgroup only: rate of EDSS progression at 1 and 2 years, median time to EDSS progression) but these positive findings were not sustained into the second half of the trial. CONCLUSIONS: Sulfasalazine does not prevent EDSS score progression in the subset of MS patients studied by this protocol. Treatments may improve relapse-related outcomes in MS, at least temporarily, without providing sustained slowing of EDSS progression. Phase III MS trials should be of sufficient length to determine a meaningful impact on disease course.     

Trabeculectomy function after cataract extraction

OBJECTIVE: To examine the effect of cataract extraction (CE) after trabeculectomy on intraocular pressure (IOP) control. DESIGN: Retrospective noncomparative case series. PARTICIPANTS: A total of 115 consecutive patients who underwent extracapsular CE (N = 58) or phacoemulsification (N = 57) with intraocular lens (IOL) placement after trabeculectomy were studied. INTERVENTION: Cataract extraction with IOL after trabeculectomy was performed. MAIN OUTCOME MEASURES: Preoperative, intraoperative, and postoperative factors were evaluated for association with loss of IOP control requiring additional medications, bleb needling, or further glaucoma surgery, using Kaplan-Meier survival analysis and Cox multivariate proportional hazards survival regression. RESULTS: After mean postoperative follow-up of 21.1 +/- 14.3 months, additional glaucoma medication or needling of the filtering bleb to maintain IOP control was required in 35 eyes (30.4%) and was significantly associated with intraoperative iris manipulation and early postoperative peak IOP greater than 25 mmHg. Additional glaucoma surgery was eventually required in 11 eyes (9.6%) and was significantly associated with age of 50 years or younger, preoperative IOP greater than 10 mmHg, and early postoperative peak IOP greater than 25 mmHg. The cumulative proportion of patients who did not require reoperation for glaucoma was 93% and 90% at 1 and 2 years, respectively. The mean IOP at last visit had increased 1.6 mmHg above the pre-CE level and did not vary significantly after the first postoperative month. The median interval from CE to the addition of glaucoma medication or bleb needling was 1.6 months (within 3 months in 20 of 33 eyes) and that from nonsurgical intervention to further glaucoma surgery was 3.6 months (before the 7th postoperative month in 6 of 11 eyes). Of 19 eyes with hypotony (IOP < or = 6 mmHg) before CE, 11 eyes remained hypotonous after CE despite an increase in the mean IOP from 4.6 to 7.5 mmHg. CONCLUSIONS: When CE is performed after trabeculectomy, age of 50 years or younger, preoperative IOP greater than 10 mmHg, intraoperative iris manipulation, and early postoperative IOP greater than 25 mmHg are associated with worsened postoperative IOP control. Most bleb failures occur soon after CE. Resolution of pre-existing hypotony after CE is unpredictable.     

Determinants of early and late results of combined valve operations and coronary artery bypass grafting

BACKGROUND: Factors determining the outcome of operative correction of valvular abnormalities combined with coronary artery bypass grafting are still incompletely defined. METHODS: Determinants of early and late (more than 90 days) deaths and event-free survival were studied for combined valve operations and coronary artery bypass grafting in 741 patients using multivariate analysis. RESULTS: Ninety-day survival probability was 89% (95% confidence interval, 87% to 92%). Preoperative risk factors for early death were age, female sex, renal failure, New York Heart Association class IV or V, and mitral insufficiency. The operative risk factor was the duration of aortic cross-clamping. Five- and 10-year survival probabilities were 74% (95% confidence interval, 71% to 78%) and 43% (95% confidence interval, 36% to 50%), respectively. Preoperative risk factors for late death were age, preoperative renal failure, New York Heart Association class IV or V, vessel disease, and nonsinus rhythm. Five- and 10-year event-free survival probabilities were 57% (95% confidence interval, 53% to 61%) and 23% (95% confidence interval, 17% to 28%), respectively. Preoperative risk factors for non-event-free survival were age, female sex, reduced left ventricular function, mitral regurgitation, and pacemaker rhythm. CONCLUSION: The demographic factors of age and female sex; the comorbid condition of renal failure; the cardiac conditions of advanced New York Heart Association class, left ventricular function, mitral regurgitation, vessel disease, and cardiac rhythm; and the operative condition of ischemia time are the most important predictors of clinical outcome after combined valve operations and coronary artery bypass grafting.     

Synthesis and comparative molecular field analysis (CoMFA) of antitumor 3-arylisoquinoline derivatives

In this study a series of 3-arylisoquinoline derivatives were synthesized and cytotoxicity against human melanoma tumor cell evaluated, and a three dimensional quantitative structure-activity relationship was investigated using the comparative molecular field analysis (CoMFA). The results suggested that the electrostatic, steric and hydrophobic factors of 3-arylisoquinolines were strongly correlated with the antitumor activity. Considerable predictive ability (cross-validated r2 as high as 0.721) was obtained through CoMFA.     

Suppression of in vitro IgM rheumatoid factor production by diphtheria toxin interleukin 2 recombinant fusion protein (DAB 486IL-2) in patients with refractory rheumatoid arthritis

OBJECTIVE: To investigate the effect of diphtheria toxin interleukin 2 recombinant fusion protein (DAB 486IL-2) on in vitro synthesis of immunoglobulin and rheumatoid factor (RF) in patients with severe refractory rheumatoid arthritis (RA) enrolled in a phase II, double blind, placebo controlled study. METHODS: Anticoagulated venous blood samples were obtained before (Day 1) and after (Day 28) intravenous infusion of either DAB 486IL-2 at 0.075 mg/kg/day (12 patients) or saline placebo (10 patients) on Days 1-5. Peripheral blood leukocytes (PBL) were prepared by density gradient centrifugation, cultured in the presence and absence of pokeweed mitogen (PWM) for one week, and culture supernatants assayed for immunoglobulins and IgM RF by ELISA. RESULTS: Compared to placebo treated patients, PWM induced IgM RF synthesis by PBL decreased after treatment with DAB 486IL-2 (p = 0.043). However, there was no apparent correlation with clinical improvement. PWM induced IgM, IgA, and IgG synthesis also tended to decrease, although the changes did not attain statistical significance. In contrast, PWM induced IgM RF, IgM, IgA, and IgG synthesis by PBL from patients treated with placebo tended to increase during the observation period. Spontaneous immunoglobulin and IgM RF production by PBL from either the DAB 486IL-2 or placebo patients remained stable. CONCLUSION: These observations raise the possibility that DAB 486IL-2 may diminish B cell function either directly or indirectly through effects on T cell function, but the change may not correspond to clinical response.     

Limb salvage surgery for widely infiltrating bony sarcomas

OBJECTIVE: To determine whether neoadjuvant chemotherapy and radiotherapy for bony sarcomas extending into soft tissues would allow limb salvage yet maintain local disease control. DESIGN: A prospective cohort study. SETTING: A university-affiliated cancer centre in Alberta. PATIENTS: All patients with potentially curable, widely infiltrating bony sarcomas of the extremity without neurologic deficit, referred to the centre in the 6 years from January 1984 to December 1990. There were 11 patients; 1 did not complete the protocol. The mean follow-up was 24 months. INTERVENTIONS: Adriamycin (doxorubicin) was infused for 3 days at a rate of 30 mg/d. A few days later radiotherapy was given 5 days a week for 10 doses at a rate of 3.0 Gy per dose. Four to 5 weeks later the tumour was excised surgically, with placement of a bone allograft or prosthesis, allowing a 1-cm margin of healthy soft tissue and a 5-cm margin of healthy bone and marrow cavity whenever possible. MAIN OUTCOME MEASURES: Need for limb amputation, infectious complications, recurrence of local or regional disease. RESULTS: One patient underwent amputation after fracture through the tumour site. There were two postoperative infections, one acute and one chronic. All patients had full neurologic function of the distal limb. There was no local or regional recurrence of disease. CONCLUSION: Neoadjuvant chemotherapy followed by radiotherapy and tumour excision provides control of aggressive local bone sarcomas while maintaining limb integrity.     

FRC measurement in intensive care patients. A definition of standards

Determination of Functional Residual Capacity (FRC) can be performed through washout methods, indicator gas dilution or bodyplethysmography. Some of these techniques have been adapted for use in intensive care patients whilst being mechanically ventilated. However, most measurement setups are bulky, cumbersome to use and their running costs are high. Hence FRC measurement has not become a routine method in intensive care although it offers considerable advantages in the management of ventilated patients such as the determination of "best PEEP", the detection of progressive alveolar collapse in the course of acute lung injury and during weaning from mechanical ventilation. Up to now most efforts to improve and simplify FRC measurement were made at the expense of accuracy. An ideal method ought to be accurate, easy to handle and cost-effective. It should supply not only FRC data but also information about intrapulmonary gas distribution and dead space. These demands can be met using modern data acquisition software. The pros and cons of all methods available for FRC measurement are discussed in view of their suitability for intensive care patients. A conventional nitrogen washout using emission spectroscopy for measurement of nitrogen concentration gives satisfying exact values for the determination of the parameters mentioned above. The measurement error can be lowered under 5% by special corrections for flow and nitrogen signal (delay and rise times, changes of gas viscosity). For flow measurement a normal pneumotachograph can be used. Using a laptop computer for data acquisition the bed-side monitor fulfills most of the demands in intensive care. It is then also possible to measure indices of intrapulmonary gas distribution such as Alveolar Mixing Efficiency and Lung Clearance Index.     

Episodic gonadotropin-releasing hormone gene expression revealed by dynamic monitoring of luciferase reporter activity in single, living neurons

The existence of an intrinsic oscillator for pulsatile gonadotropin-releasing hormone (GnRH) secretion in normal and transformed GnRH neurons raises the question of whether the corresponding gene also is expressed in an episodic manner. To resolve this question, we used a modification of conventional luciferase technology, which enabled continuous monitoring of GnRH gene activity in single, living neurons. With this method, the relative rate of endogenous gene expression is estimated by quantification of photons emitted by individual neurons microinjected with a GnRH promoter-driven luciferase reporter construct. Immortalized GT1-1 neurons, which secrete the decapeptide GnRH in a pulsatile manner conceptually identical to that of their nontransformed counterparts in vivo, were chosen as the model for these studies. First, we injected individual cells with purified luciferase protein and established that the reporter half-life was sufficiently short (50 min) to enable detection of transient changes in gene expression. Next, we subjected transfected GT1-1 cells to continuous monitoring of reporter activity for 16 h and found that the majority of them exhibited spontaneous fluctuations of photonic activity over time. Finally, we established that photonic activity accurately reflected endogenous GnRH gene expression by treating transfected GT1-1 cells with phorbol 12-myristate 13 acetate (a consensus inhibitor of GnRH gene expression) and observing a dramatic suppression of photonic emissions from continuously monitored cells. Taken together, these results demonstrate the validity of our "real-time" strategy for dynamically monitoring GnRH gene activity in living neurons. Moreover, our findings indicate that GnRH gene expression as well as neuropeptide release can occur in an intermittent manner.