Efficient directional cloning of recombinant adenovirus vectors using DNA-protein complex

We describe an efficient cloning system utilizing adenoviral DNA-protein complexes which allows the directional cloning of genes into adenoviral expression vectors in a single step. DNA-protein complexes derived from a recombinant adenovirus (AVC2.null) were isolated by sequential use of CsCl step gradients followed by isopycnic centrifugation in a mixture of CsCl and guanidine HCl. AVC2.null is an adenoviral expression vector containing unique restriction sites between the human CMV-IE promoter and the SV40 intron/polyadenylation site. Transgenes were prepared for cloning into this vector by introduction of compatible restriction sites by PCR. A vector expressing rat granulocyte-macrophage colony-stimulating factor (GM-CSF) was constructed using DNA-protein complex as well as by traditional recombination techniques. The efficacy of our adenoviral cloning system utilizing DNA-protein complex was two logs higher than that seen using homologous recombination. All viruses generated by directional ligation of the insert into the vector DNA-protein complexes contained the desired transgene in the correct orientation. This technique greatly simplifies and accelerates the generation of recombinant adenoviral vectors.     

1998 William J. Stickel Bronze Award. Antifungal activity of Melaleuca alternifolia (tea-tree) oil against various pathogenic organisms

Tea-tree oil (oil of Melaleuca alternifolia) has recently received much attention as a natural remedy for bacterial and fungal infections of the skin and mucosa. As with most naturally occurring agents, claims of effectiveness have been only anecdotal; however, several published studies have recently demonstrated tea-tree oil's antibacterial activity. This study was conducted to determine the activity of tea-tree oil against 58 clinical isolates: Candida albicans (n = 10), Trichophyton rubrum (n = 8), Trichophyton mentagrophytes (n = 9), Trichophyton tonsurans (n = 10), Aspergillus niger (n = 9), Penicillium species (n = 9), Epidermophyton floccosum (n = 2), and Microsporum gypsum (n = 1). Tea-tree oil showed inhibitory activity against all isolates tested except one strain of E floccosum. These in vitro results suggest that tea-tree oil may be useful in the treatment of yeast and fungal mucosal and skin infections.     

One-stage sternal stenting with homograft bone after cardiac operation in pediatric patients

Low cardiac output after open heart operations in neonates and infants carries a high mortality. Delayed sternal closure may be life-saving but may prolong hospital stay and increase costs. To circumvent these issues, we shaped homograft bone and interposed it between the sternal edges to allow primary wound closure in 2 pediatric patients. Midterm results are satisfactory.     

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.     

Peripheral O2 diffusion does not affect V(O2)on-kinetics in isolated insitu canine muscle

To test the hypothesis that muscle O2 uptake (V(O2)) on-kinetics is limited, at least in part, by peripheral O2 diffusion, we determined the V(O2) on-kinetics in 1) normoxia (Control); 2) hyperoxic gas breathing (Hyperoxia); and 3) hyperoxia and the administration of a drug (RSR-13, Allos Therapeutics), which right-shifts the Hb-O2 dissociation curve (Hyperoxia+RSR-13). The study was conducted in isolated canine gastrocnemius muscles (n = 5) during transitions from rest to 3 min of electrically stimulated isometric tetanic contractions (200-ms trains, 50 Hz; 1 contraction/2 s; 60-70% peak V(O2)). In all conditions, before and during contractions, muscle was pump perfused with constantly elevated blood flow (Q), at a level measured at steady state during contractions in preliminary trials with spontaneous Q x Adenosine was infused intra-arterially to prevent inordinate pressure increases with the elevated Q x Q was measured continuously, arterial and popliteal venous O2 concentrations were determined at rest and at 5- to 7-s intervals during contractions, and V(O2) was calculated as Q x arteriovenous O2 content difference. PO2 at 50% HbO2 saturation (P50) was calculated. Mean capillary PO2 (Pc(O2)) was estimated by numerical integration. P50 was higher in Hyperoxia+RSR-13 [40 +/- 1 (SE) Torr] than in Control and in Hyperoxia (31 +/- 1 Torr). After 15 s of contractions, Pc(O2) was higher in Hyperoxia (97 +/- 9 Torr) vs. Control (53 +/- 3 Torr) and in Hyperoxia+RSR-13 (197 +/- 39 Torr) vs. Hyperoxia. The time to reach 63% of the difference between baseline and steady-state V(O2) during contractions was 24.7 +/- 2.7 s in Control, 26.3 +/- 0.8 s in Hyperoxia, and 24.7 +/- 1.1 s in Hyperoxia+RSR-13 (not significant). Enhancement of peripheral O2 diffusion (obtained by increased PcO2 at constant O2 delivery) during the rest-to-contraction (60-70% of peak V(O2)) transition did not affect muscle V(O2) on- kinetics.