Detection of ecotropic replication-competent retroviruses: comparison of s(+)/l(-) and marker rescue assays.

Guidelines for testing gene therapy products for ecotropic replication-competent retrovirus (Eco-RCR) have not been delineated as they have for amphotropic viruses. To evaluate biologic assays that can detect these viruses, we compared an S(+)/L(-) assay and a marker rescue assay designed specifically for Eco-RCR detection. Moloney murine leukemia virus (Mo-MuLV) obtained from the American Type Culture Collection was used as the positive control. For marker rescue, NIH 3T3 cells were transduced with a retroviral vector expressing the neomycin phosphotransferase gene (3T3/Neo). Inoculation and passage of test material in 3T3/Neo cells for 3 weeks (amplification) and subsequent testing in the S(+)/L(-) assay or the marker rescue assay increased the level of sensitivity for virus detection greater than 10-fold compared with direct inoculation of D56 S(+)/L(-) cells. When serial dilutions of Mo-MuLV stock were evaluated, six of six cultures had detectable virus by the S(+)/L(-) and marker rescue assays at dilutions of 10(-5) and 10(-6). At the 10(-7) dilution, five of six assays had detectable virus in both assays. The ability to detect virus-infected cells was also evaluated in a modification that substituted cells for supernatant. Fifteen 3T3/Neo cultures inoculated with 10(6) 293 cells containing 100 or 10 Mo-MuLV/3T3 cells were all positive by marker rescue. For dilution with 1 virus-infected cell per 10(6) 293 cells, 10 of 15 cultures were positive. At the 0.1-cell dilution only 2 of 15 cultures were positive. If we hope to detect one infected cell in a test article, the probability of detecting virus if the assay is performed in triplicate is 96.3%. In summary, after 3 weeks of amplification the S(+)/L(-) and marker rescue assays can detect virus with similar sensitivities. We prefer the marker rescue assay because of the more reliable growth features of NIH 3T3 cells compared with the D56 cell line. For laboratories analyzing clinical materials, this report may prove useful in establishing detection assays for Eco-RCR.

A retrospective analysis comparing patients 70 years or older to patients younger than 70 years with non-small-cell lung cancer treated with surgery at Indiana university: 1989-1999.

Surgery remains the cornerstone of therapy for medically operable patients with early-stage non-small-cell lung cancer (NSCLC). However, there are few reports on the short-term morbidity and long-term survival following surgery in elderly patients with NSCLC. The surgical experience in 280 patients with NSCLC at Indiana University from 1989-1999 are reported with a comparison of patients who are >or= 70 years versus < 70 years of age. Preoperative characteristics, operative procedures, postoperative courses, and survival were compared between the age groups. Fifty percent of elderly patients had squamous cell carcinoma and 36.2% had adenocarcinoma, versus 41.3% and 44.4% in younger patients, respectively. In both groups, most patients had T1 or T2 tumors and N0 disease. The majority of patients in both age groups had a lobectomy. However, more patients younger than 70 years had chest wall resections and were more likely to undergo a pneumonectomy (19.5% vs. 6.9%). The median number of postoperative hospital days was shorter for younger patients (9 days vs. 11 days). Overall, more complications occurred in older patients, but no significant difference in cardiac or pulmonary complications was observed between the groups. There was no significant difference in survival between the age groups. This single-institution series demonstrates that surgical intervention for appropriately selected elderly patients with NSCLC results in similar complication rates and long-term survival when compared to their younger counterparts.

Naltrexone and brief counseling to reduce heavy drinking in hazardous drinkers.

The present study examined the utility of daily naltrexone for decreasing alcohol drinking in hazardous drinkers. Forty-one participants participated in a 10-week trial and received 30 min of brief counseling on the first and second week of treatment, as well as a daily dose of 50 mg of naltrexone throughout the trial. Overall, naltrexone-treated participants did not show the same degree of improvement on drinking outcomes as placebo-treated participants. The placebo group drank fewer drinks per drinking day and achieved more abstinence days than the naltrexone group. Craving was also lower for the placebo group. The groups were not balanced on gender or family history of alcoholism and this may explain the lack of effect of naltrexone on the drinking outcomes.

Botulinum toxin type B in upper-limb poststroke spasticity: a double-blind, placebo-controlled trial.

OBJECTIVE: To determine whether botulinum toxin type B (BTX-B) is effective in controlling upper-limb spasticity. DESIGN: A single-site, double-blind, placebo-controlled, randomized trial and open-label study. SETTING: Outpatient. PARTICIPANTS: Subjects with an Ashworth Scale score of 2 or more at the elbow, wrist, and fingers. INTERVENTIONS: Subjects were injected with 10000 U of BTX-B or placebo at the elbow, wrist, and finger flexors. Main outcome measures Measures recorded at weeks 0, 2, 4, 8, 12, and 16, with a 12-week open-label study. Ashworth Scale score, a global assessment of change (GAC), adverse events and mouse neutralization antibody testing. RESULTS: BTX-B did not decrease muscle tone in the elbow, wrist, or finger flexors at 10000 U over the 16-week period. A decrease in Ashworth Scale score for the BTX-B patient group was present at the wrist at week 2 of the double-blind study (P=.003) but was not statistically significant at other visits. In the open-label study, improvement was noted at week 4 for the elbow (P=.039), wrist (P=.002), finger (P=.001), and thumb flexors (P=.002). In the double-blind study, the Physician GAC did not reach significance. Dry mouth was reported by 8 of 9 BTX-B subjects in the double-blind study. Mouse neutralization antibodies were negative. CONCLUSIONS: Our study does not show a significant decrease in tone from 10000 U of BTX-B. Dry mouth was common.

Total colpocleisis with high levator plication for the treatment of advanced pelvic organ prolapse.

OBJECTIVES: Our purpose was (1). to report long-term objective and subjective outcome data after total colpocleisis with high levator plication and (2). to compare operative morbidity of total colpocleisis in patients with prior versus concurrent hysterectomy. STUDY DESIGN: The medical records of patients who underwent total colpocleisis with high levator plication between August 1, 1988, and December 31, 2000, were retrospectively reviewed. Patient characteristics, operative data, and objective outcome measures including pelvic organ prolapse staging measurements were obtained from subject records. Subjective outcome measures were obtained by a standardized telephone survey. A t test was used to compare continuous variables between patients who underwent prior versus concurrent hysterectomy. The Fisher exact test was used to test for association between patient group and each categorical variable. RESULTS: During the 12-year study period, 92 subjects underwent total colpocleisis with high levator plication. Of the 92 subjects, 90 (97.8%) underwent concurrent operations and 37 (40.2%) underwent simultaneous hysterectomy. With objective cure defined as absence of prolapse to the hymen, 90 subjects (97.8%) were objectively cured after a median follow-up of 12 months (range, 0-64 months). Of 62 subjects available for telephone follow-up, 56 (90.3%) reported being satisfied or very satisfied with how the surgery cured their prolapse after a median follow-up of 24 months (range, 13-161 months). Concurrent hysterectomy was associated with statistically significant increases in absolute change in hematocrit (9.5% without vs 11.9% with hysterectomy) and transfusion requirement (12.7% without vs 35.1% with hysterectomy). There was no significant difference in surgical complications between groups. CONCLUSION: Total colpocleisis is an effective operation for the treatment of advanced pelvic organ prolapse. Concurrent hysterectomy is associated with higher blood loss and transfusion requirements.

Human immunodeficiency virus type 1-derived lentivirus vectors pseudotyped with envelope glycoproteins derived from Ross River virus and Semliki Forest virus.

Ross River virus (RRV) and Semliki Forest virus (SFV) are two alphaviruses that have a high degree of amino acid homology, as well as a very broad host range. We show here that envelope glycoproteins derived from both viruses can pseudotype human immunodeficiency virus type 1 (HIV-1)-derived lentivirus vectors. Both RRV and SFV glycoproteins considerably expand the host range of the lentivirus vector, and vectors can be efficiently concentrated by ultracentrifugation. A systematic analysis comparing the alphaviral glycoproteins to the vesicular stomatitis virus glycoprotein (VSV-G) revealed that lentivirus vectors incorporate RRV glycoproteins with an efficiency comparable to that of VSV-G. Both pseudotypes have comparable physical titers, but infectious titers with the RRV pseudotype are lower than with VSV-G. Incorporation of SFV glycoproteins into lentivirus vector is less efficient, leading to decreased physical and infectious titers. The transduction rates with VSV-G-, RRV-, and SFV-pseudotyped lentivirus vectors into adherent cell lines can be significantly increased by using a combination of Polybrene and plates coated with CH-296 recombinant fibronectin fragments. Together, our data suggest that RRV and SFV glycoproteins might be suitable as alternatives to VSV-G for pseudotyping lentivirus vectors.

Prognostic importance of lymph node tumor burden in melanoma patients staged by sentinel node biopsy.

BACKGROUND: The objective of this study was to investigate the relationship between nodal tumor burden and the outcomes of recurrence and survival in sentinel node-positive melanoma patients. METHODS: We reviewed a series of sentinel node-positive patients with primary cutaneous melanoma treated with completion lymph node dissection (CLND). Microscopic nodal tumor deposits were counted and measured with an ocular micrometer. Various measures of tumor burden and traditional melanoma prognostic indicators were studied in multivariate Cox regression models. RESULTS: Sentinel lymph node and CLND specimens were evaluated in 90 node-positive patients. The diameter of the largest lymph node tumor nodule and the total lymph node tumor volume were significant predictors of recurrence (two-sided P <.0001 for both) and survival (two-sided P =.0018 and P =.0002, respectively). A tumor deposit diameter of 3 mm was identified as the most significant cut point predictive of recurrence (P <.0001; hazard ratio, 5.18) and survival (P <.0001; hazard ratio, 5.43). The 3-year survival probability was.86 for patients with largest tumor deposit diameters of 3 mm (P <.0001). CONCLUSIONS: Microstaging of melanoma sentinel lymph node/CLND specimens by using the diameter of the largest tumor deposit is a highly significant predictor of early relapse and survival.

Anatomic distribution of periprostatic adipose tissue: a mapping study of 100 radical prostatectomy specimens.

BACKGROUND: Because the prostatic capsule is often indistinct, recognition of extraprostatic extension by carcinoma depends heavily on the identification of carcinoma cells in the periprostatic adipose tissue. However, the distribution of adipose tissue along the prostatic surfaces has not been studied extensively. METHODS: The authors analyzed the periprostatic adipose tissue in specimens from 100 patients treated with radical retropubic prostatectomy and bilateral pelvic lymphadenectomy for prostatecarcinoma. Each specimen was totally embedded and examined by the whole mount method. The presence or absence of adipose tissue on the anterior, posterior, right, and left surfaces of the prostate was determined and analyzed. RESULTS: Periprostatic adipose tissue was present on 48% of all prostatic surfaces examined. The distribution of periprostatic adipose tissue varied among the different surfaces of the prostate, with the anterior, posterior, right, and left surfaces showing 44%, 36%, 59%, and 57% adipose tissue, respectively. The amount of periprostatic adipose tissue was similar among specimens from operations performed by different surgeons (P = 0.72). However, nerve-sparing procedures resulted in less adipose tissue (46%) than non-nerve-sparing procedures (54%) (P = 0.01). CONCLUSIONS: Less than half of the prostatic surfaces examined were covered by adipose tissue. The absence of adipose tissue over large areas of the prostatic surface, especially the posterior surface, makes the evaluation of extraprostatic extension difficult and unreliable. Further refinement of the prostate carcinoma staging system is needed.

Clinicians' and patients' experiences and satisfaction with unscheduled, nighttime, Internet-based video conferencing for assessing acute medical problems in a nursing facility.

Videoconferencing between patients and their physicians can increase patients' access to healthcare. Unscheduled videoconferencing can benefit patients with acute medical problems but has not been studied extensively. We conducted a clinical trial of unscheduled, nighttime videoconferencing in a nursing home, where on-call physicians usually provide care by telephone from remote locations. Although most calls for medical problems did not lead to videoconferencing, physicians and nursing-home residents were satisfied with videoconferencing when it did occur, and physicians reported that making medical decisions was easier with videoconferencing. Videoconferencing was most often conducted to assess residents with changes in mental status, abnormal laboratory values, or falls. Physicians often lacked immediate access to videoconferencing equipment when medical problems with residents occurred. This application could benefit from improved access and portability of equipment.