Citations

ABSTRACT The TRUGENE HCV 5′NC genotyping kit (GeneLibrarian modules 3.1.1 and 3.1.2) and VERSANT HCV genotyping assay were compared by using 96 hepatitis C virus (HCV) RNA-positive patient specimens, including HCV genotypes 1, 2, 3, 4, 5, 6, and 10. The TRUGENE HCV 5 ′ NC genotyping kit (GeneLibrarian module 3.1.2) yielded the most accurate genotyping results.

Purpose: We sought to determine the maximum-tolerated dose (MTD) and evaluate the toxicities and clinical activity of two irinotecan (CPT-11), fluorouracil (FU), leucovorin (LV), and oxaliplatin schedules in patients with advanced solid tumors. Additionally, we investigated the effect of CPT-11 on oxaliplatin pharmacokinetics. Patients and Methods: Thirteen patients (cohort 1) received intravenous CPT-11 (infusion) and FU/LV (bolus) on days 1, 8, 15, and 22 and oxaliplatin (infusion) on days 1 and 15 every 6 weeks for a total 37 courses (median, three courses) at three dose levels. Twenty-two cohort 2 patients received intravenous CPT-11/oxaliplatin (infusion, day 1) and FU/LV (90-minute bolus infusion, days 2 to 5) every 3 weeks for a total of 122 courses (median, four courses) at three dose levels. Pharmacokinetic and neurotoxicity assessments were performed at the cohort 2 MTD. Results: Dose-limiting toxicity (DLT) seen in both cohorts at the starting dose required dose de-escalation. Cohort 1 DLT included diarrhea and neutropenia. In cohort 2, diarrhea, vomiting, dehydration, neutropenia, febrile neutropenia, and paresthesias were DLTs. Antitumor activity was seen in both cohorts. In cohort 2, the total platinum area under the curve of patients increased 17% in cycle 2 (P = .048), but objective neurotoxicity was not seen. Conclusion: The toxicities resulting from the addition of oxaliplatin to CPT-11/FU/LV are significant but manageable. The MTDs for the weekly schedule are CPT-11 (75 mg/m2), oxaliplatin (50 mg/m2), FU (320 mg/m2), and LV (20 mg/m2); and, for the 3-weekly schedule, the MTDs are CPT-11 (175 mg/m2), oxaliplatin (85 mg/m2), FU (240 mg/m2), and LV (20 mg/m2). Second-cycle platinum accumulation raises the possibility for enhanced cumulative neurotoxicity with CPT-11/oxaliplatin combinations.

ABSTRACT The COBAS AMPLICOR system has played a major role in the transition of molecular diagnostics from research to routine clinical laboratory use by automating the nucleic acid amplification and detection processes. However, sample preparation remains a labor-intensive portion of the procedure. In this study, we evaluated the performance of the COBAS AMPLICOR Hepatitis C Virus Test, version 2.0 (Roche Molecular Systems, Branchburg, N.J.) following manual hepatitis C virus (HCV) RNA extraction versus automated extraction with the MagNA Pure LC instrument (Roche Applied Science, Indianapolis, Ind.). Parallel replicate testing was performed with standard dilutions of 100, 75, 60, and 0 HCV IU/ml and 153 clinical specimens. An analytical sensitivity of 75 IU/ml was achieved with either the manual or the standard-volume (200 μl) automated extraction methodologies (25 of 26 [96.2%]; 95% confidence interval [95% CI], 80.4 to 99.9), whereas the clinical sensitivity and specificity were both 100% with either extraction method. A large-volume (1 ml) automated extraction method was also evaluated with standard dilutions of 40, 25, 10, and 0 IU/ml and the same 153 clinical specimens. The analytical sensitivity of the COBAS AMPLICOR assay with the large-volume extraction method was 25 HCV IU/ml (26 of 26 [100%]; 95% CI, 86.8 to 100), whereas the clinical sensitivity and specificity were both 100%. The MagNA Pure LC instrument is a versatile, labor-saving platform capable of integration with minimal modification of the existing assay procedure. The increased sensitivity of the COBAS AMPLICOR Hepatitis C Virus Test, version 2.0 performed in conjunction with large-volume HCV RNA extraction may be important in HCV diagnostic testing as new therapeutic strategies evolve.

ABSTRACT Activation of cyclic nucleotide‐dependent signaling pathways leads to phosphorylation of the small heat shock‐related protein, HSP20, on serine 16, and relaxation of vascular smooth muscle. In this study, we used an enhanced protein transduction domain (PTD) sequence to deliver HSP20 phosphopeptide analogs into porcine coronary artery. The transduction of phosphoHSP20 analogs led to dose‐dependent relaxation of coronary artery smooth muscle. Peptides containing the protein transduction domain coupled to a random orientation of the same amino acids did not. Direct fluorescence microscopy of arterial rings incubated with fluorescein isothiocyanate (FITC)‐PTD or FITC‐PTD‐HSP20 peptides showed a diffuse peptide uptake. Mass spectrometric immunoassays (MSIAs) of smooth muscle homogenates were used to determine whether the phosphopeptide analogs affected the phosphorylation of endogenous HSP20. Treatment with the phosphodiesterase inhibitor papaverine led to a mass shift of 80 Da. However, there was no mass shift of HSP20 in muscles treated with phosphoHSP20 analogs. This suggests that the PTD‐phosphoHSP20 peptide alone is sufficient to inhibit force maintenance and likely has a direct effect on the target of phosphorylated HSP20. These results suggest that transduction of phosphopeptide analogs of HSP20 directly alters physiological responses of intact muscles. The data also support a direct role for phosphorylated HSP20 in mediating vasorelaxation.

Abstract Glucocorticoids (GCs) are commonly used to treat inflammatory disease; unfortunately, the long-term use of these steroids leads to a large number of debilitating side effects. The antiinflammatory effects of GCs are a result of GC receptor (GR)-mediated inhibition of expression of proinflammatory genes as well as GR-mediated activation of antiinflammatory genes. Similarly, side effects are most likely due to both activated and repressed GR target genes in affected tissues. An as yet unachieved pharmaceutical goal is the development of a compound capable of separating detrimental side effects from antiinflammatory activity. We describe the discovery and characterization of AL-438, a GR ligand that exhibits an altered gene regulation profile, able to repress and activate only a subset of the genes normally regulated by GCs. When tested in vivo, AL-438 retains full antiinflammatory efficacy and potency comparable to steroids but its negative effects on bone metabolism and glucose control are reduced at equivalently antiinflammatory doses. The mechanism underlying this selective in vitro and in vivo activity may be the result of differential cofactor recruitment in response to ligand. AL-438 reduces the interaction between GR and peroxisomal proliferator-activated receptor γ coactivator-1, a cofactor critical for steroid-mediated glucose up-regulation, while maintaining normal interactions with GR-interacting protein 1. This compound serves as a prototype for a unique, nonsteroidal alternative to conventional GCs in treating inflammatory disease.

Abstract We tested the hypothesis that cells of ventilator-injured lungs are subject to reversible plasma membrane stress failure. Rat lungs were perfused with the membrane impermeable fluorescent marker propidium iodide and randomized to one of four ventilation strategies. Subpleural lung regions were imaged with confocal microscopy, and cell injury was quantified as the number of propidium iodide–positive cells per alveolus. The number of injured cells was significantly greater in lungs ventilated with large tidal volumes and zero end-expiratory pressure than in lungs ventilated with small tidal volumes and positive end-expiratory pressure (p < 0.01). Cell injury correlated with lung weight gain, change in dynamic compliance, and histologic injury scores. In a second set of experiments, lungs were mechanically ventilated for 30 minutes at high tidal volume settings, whereas propidium iodide was perfused either during or after injurious ventilation. Labeling after removal of injurious stress revealed significantly fewer injured cells (0.25 ± 0.09 to 0.08 ± 0.08, p < 0.01). We conclude that cells of ventilator-injured lungs are subject to reversible plasma membrane stress failure.

Renal artery stenosis (RAS) may cause hypertension, azotemia, episodes of flash pulmonary edema and congestive heart failure. Renal artery angioplasty and stenting was performed in 207 patients from 1991 to 1997. Thirty-nine of these patients (19%) underwent renal artery stenting for the control of recurrent episodes of congestive heart failure and flash pulmonary edema. All patients had angiographic evidence of severe (>70%) bilateral RAS (n = 18) or severe RAS to a solitary functioning kidney (n = 21). Sixteen patients (41%) were male and 23 (59%) were female, mean age 69.9 years (range 50- 85 years). Of the 18 patients with bilateral RAS, 12 (66.6%) underwent bilateral stenting. Mean blood pressure decreased from 174/85 6 32/23 mmHg to 148/72 6 24/14 mmHg (p < 0.001). Mean number of blood pressure medications decreased from 3 6 1 to 2.5 6 1 (p = 0.006). Twenty-eight patients (71.8%) had improvement in blood pressure control. The mean serum creatinine decreased from 3.16 6 1.61 to 2.65 6 1.87 (p = 0.06). Six of 39 patients (15.4%) used angiotensin converting enzyme (ACE) inhibitors prior to stenting whereas 19 of 39 patients (48.7%) used ACE inhibitors poststenting (p = 0.004). Twenty of 39 patients (51.4%) demonstrated improvement in serum creatinine, 10 of 39 patients (25.6%) had stabilization of serum creatinine and nine of 39 patients (23%) demonstrated worsening. The number of hospitalizations due to congestive heart failure in the year preceding renal artery stenting was 2.4 6 1.4 and poststenting was 0.3 6 0.7 (p < 0.001). The New York Heart Association Functional Class decreased from 2.9 6 0.9 prestenting to 1.6 6 0.9 poststenting (p < 0.001). Thirty of 39 patients (77%) had no hospitalizations for congestive heart failure during a mean follow-up period of 21.3 months. Nine patients expired during the course of follow up; eight of the nine patients died within the first year after renal artery stenting. Renal artery stenting decreased the frequency of congestive heart failure, flash pulmonary edema, and the need for hospitalization in most patients. Blood pressure was markedly improved in the majority of patients with improved or stabilized renal function. Evaluation for RAS is important in hypertensive patients who present with recurrent congestive heart failure or flash pulmonary edema.

Acute vascular or humoral rejection, a vexing outcome of organ transplantation, has been attributed by some to activation and by others to apoptosis of endothelial cells in the graft. We asked which of these processes causes acute vascular rejection by tracing the processes during the development of acute vascular rejection in porcine cardiac xenografts performed in baboons. Apoptosis, assayed by terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL), expression of activated caspase-3, and proapoptotic genes Bax and Bcl-x L , was not detected until acute vascular rejection was well advanced, and even then, apoptosis was largely confined to myocytes. Activation of the endothelium, as evidenced by expansion of rough endoplasmic reticulum and increased ribosomal antigen and phospho-p70 S6 kinase, occurred early in the course of acute vascular rejection and progressed through the disease process. These findings suggest that acute vascular rejection is caused by an active metabolic process and not by apoptosis in the endothelium.