Ribonuclease A variants with potent cytotoxic activity

Select members of the bovine pancreatic ribonuclease A (RNase A) superfamily are potent cytotoxins. These cytotoxic ribonucleases enter the cytosol, where they degrade cellular RNA and cause cell death. Ribonuclease inhibitor (RI), a cytosolic protein, binds to members of the RNase A superfamily with inhibition constants that span 10 orders of magnitude. Here, we show that the affinity of a ribonuclease for RI plays an integral role in defining the potency of a cytotoxic ribonuclease. RNase A is not cytotoxic and binds RI with high affinity. Onconase, a cytotoxic RNase A homolog, binds RI with low affinity. To disrupt the RI-RNase A interaction, three RNase A residues (Asp-38, Gly-88, and Ala-109) that form multiple contacts with RI were replaced with arginine. Replacing Asp-38 and Ala-109 with an arginine residue has no effect on the RI-RNase interaction. In addition, these variants are not cytotoxic. In contrast, replacing Gly-88 with an arginine residue yields a ribonuclease (G88R RNase A) that retains catalytic activity in the presence of RI and is cytotoxic to a transformed cell line. Replacing Gly-88 with aspartate also yields a ribonuclease (G88D RNase A) with a decreased affinity for RI and cytotoxic activity. The cytotoxic potency of onconase, G88R RNase A, and G88D RNase A correlate with RI evasion. We conclude that ribonucleases that retain catalytic activity in the presence of RI are cytotoxins. This finding portends the development of a class of chemotherapeutic agents based on pancreatic ribonucleases.     

Scoliosis circa 2000: radiologic imaging perspective. II. Treatment and follow-up

Plain film imaging remains important for the diagnosis and surveillance of scoliosis, as well as for the detection of complications after surgery. New means of treating scoliosis have become established and should be understood by the radiologist. To the well-known postoperative complications, including pneumothorax, pneumonia, and gastrointestinal obstruction, are added new specific potential problems with the new surgical methodology.     

Immunogenicity of Plasmodium falciparum circumsporozoite protein multiple antigen peptide vaccine formulated with different adjuvants

Only low antibody levels were obtained from vaccinating human volunteers with single-chain peptide from the Plasmodium falciparum circumsporozoite protein (PfCSP). This resulted in modest protection against sporozoite challenge. In addition, HLA restriction limits the probability of synthesis of a vaccine effective for a diverse population. We report immunization studies with a multiple antigen peptide (MAP) system consisting of multiple copies of a B-cell epitope from the central repeat region of the PfCSP in combination with a universal T-cell epitope, the P2P30 portion of tetanus toxin. This MAP4(NANP)6P2P30 vaccine was highly immunogenic in four different strains of mice when used with various safe and nontoxic adjuvants. When this MAP vaccine was encapsulated in liposomes with lipid A and adsorbed to aluminium hydroxide and given three times at 4-week intervals, the resultant antibody prevented 100% of sporozoites from invading and developing into liver stage infection. This high degree of immunogenicity of MAP4(NANP)6P2P30 vaccine formulated in liposomes, lipid A and aluminum hydroxide provides the foundation for consideration of human trials with this formulation.     

Sulfasalazine in the treatment of juvenile chronic arthritis: a randomized, double-blind, placebo-controlled, multicenter study. Dutch Juvenile Chronic Arthritis Study Group

AIMS: To determine whether left ventricular volumes and ejection fractions calculated from single plane two-dimensional echocardiograms using the algorithm (0.85A2L) correlate with those calculated using the biplane Simpson's method, and whether small changes in volumes and ejection fraction occurring post-infarction could be detected from single-plane as well as from biplane two-dimensional echocardiograms. METHODS AND RESULTS: Serial two-dimensional echocardiograms were obtained in 371 patients from the DEFIANT II trial a mean of 2 days, 1 week and 6 months post-infarction. Single plane volumes from the apical four chamber and apical long axis correlated closely with biplane Simpson's left ventricular volumes. Both single-plane left ventricular volumes significantly over-estimated biplane Simpson's volumes. Biplane Simpson's ejection fractions were consistently slightly under-estimated from the single-plane images. Differences between biplane Simpson's and single-plane volumes increased independently with increasing left ventricular size and distortion. The small changes in left ventricular volumes and ejection fraction over time were as reliably detected from single plane as from biplane images. CONCLUSION: Single-plane left ventricular volumes over-estimate biplane Simpson's volumes and under-estimate ejection fraction, and these discrepancies are amplified in dilated hearts with abnormal shape.     

Development of in vitro performance tests and evaluation of nonabsorbable monofilament sutures for cardiovascular surgery

There have been reports suggesting that polypropylene (PP) monofilament sutures are associated with mechanical failure. To overcome this problem, a new monofilament suture made from polyvinylidene fluoride, under the trade name of Teflene, has been developed. Few studies have measured the in vitro properties of Teflene sutures, and those that have, have been limited to a few tensile properties of the straight suture such as tensile strength, elongation, and creep behavior. The in vitro performance properties of Teflene sutures were evaluated and compared with those of commercial sutures made from PP such as Prolene and Surgilene in four sizes, 2-0, 3-0, 4-0, and 5-0. The performance properties of sutures included both the physical properties of straight sutures, such as suture diameter, tensile strength, elongation, surface roughness, coefficient of friction, bending stiffness, and tissue drag, and the knot characteristics, such as knot pull strength, knot run-down, and knot security. Existing standard test methods and testing instruments were used if available to measure certain suture properties such as diameter, tensile strength, knot pull strength, and some physical properties. The other novel test methods and unique accessory devices needed to perform the tests for measuring tissue drag, knot run-down, coefficient of friction, and knot security were developed in the authors' laboratories, and the comparative results are reported for the first time. From the test results, Teflene sutures were found in general to possess equivalent characteristics to those of existing PP commercial sutures, but some differences also were observed, such as greater elongation and less knot run-down. These differences may give them a unique feel and handling performance, especially in terms of making a knot, sliding it into position, and causing less damage to adjacent tissue.     

Multicenter study of case finding in elderly emergency department patients

OBJECTIVES: To assess the feasibility of a brief comprehensive case-finding program for detecting functional, cognitive, and social impairments among elderly ED patients and to estimate the prevalence of unknown, undetected, or untreated impairments elderly patients may have. METHODS: A multicenter prospective study conducted at five private and public hospital EDs in five different communities across the country. Patients aged 60 years and older released to their homes during 52 randomly selected evening and weekend shifts between February 1 and April 30, 1993, were eligible for the case-finding program. They were evaluated by medical students who received special training (instructional videotape, supervised examinations, and conference calls) in the administration of a standardized 17-item protocol that included an interview and simple tests of function. The patients' physicians were notified of the screening results and were asked to return a one-month follow-up questionnaire. The physicians answered whether the presumed problem had been confirmed and whether a treatment plan for a new problem had been developed. RESULTS: Patient acceptance of the case-finding program was good; 252 of 338 eligible patients (75%) agreed to participate, and 281 conditions were detected for 242 screened patients (96%). The most frequently reported problems were with: performing the activities of daily living (79%); vision (55%); lack of influenza vaccination (54%); home environment (49%); mental status (46%); general health (41%); falls (40%); and depression (36%). The physicians returned questionnaires for 153 patients (63%); 76 patients (50%) were evaluated at follow-up visits, during which 47 newly identified problems (62%) were confirmed and treatment plans were developed for 25 problems (53%) among 21 patients. A mean time of 17.7 +/- 10.2 minutes was required to complete the screen. CONCLUSIONS: A brief comprehensive case-finding program for functional, cognitive, and social impairment among elderly ED patients is feasible. The screening uncovered a significant amount of morbidity among older patients visiting EDs.     

Pharmacokinetics, oral bioavailability, and metabolism in mice and cynomolgus monkeys of (2'R,5'S-)-cis-5-fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl] cytosine, an agent active against human immunodeficiency virus and human hepatitis B virus

(2'R,5'S-)-cis-5-Fluoro-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl] cytosine (524W91) is a nucleoside analog with potent anti-human immunodeficiency virus and anti-human hepatitis B virus activities in vitro. The pharmacokinetics and bioavailability of 524W91 after oral dosing were studied in mice dosed with 10, 100, and 600 mg of 524W91 per kg of body weight by the oral and intravenous routes. Cynomolgus monkeys were dosed with 10 and 80 mg of 524W91 per kg. In both species, the clearance of 524W91 was rapid, via the kidney, and was independent of dose. In monkeys, the total body clearance of 10 mg of 524W91 per kg was 0.7 +/- 0.1 liter/h/kg, and the volume of distribution at steady state was 0.8 +/- 0.02 liter/kg. The terminal elimination half-life was 1.0 +/- 0.2 h. The absolute bioavailability after oral dosing was 63% +/- 4% at 10 mg/kg. Concentrations of 524W91 in the cerebrospinal fluid were 4% +/- 0.7% of the corresponding levels in plasma. In mice, the total clearance of 10 mg of 524W91 per kg was 2.3 liters/kg/h, and the volume of distribution at steady state was 0.9 liter/kg. Absolute bioavailability in mice after oral dosing was 96% at a dose of 10 mg/kg. The metabolism of orally administered [6-3H]524W91 was studied in cynomolgus monkeys at a dose of 80 mg/kg and in mice at a dose of 120 mg/kg. Monkeys excreted 41% +/- 6% of the radioactive dose in the 0- to 72-h urine, 33% +/- 10% in the feces, and 10% +/- 7% in the cage wash. Unchanged 524W91 was 64% of the total radiolabeled drug recovered in the urine. The glucuronide was a minor urinary metabolite. 5-Fluorouracil was not detected (less than 0.02% of the dose). Mice dosed orally with 120 mg of [6-3H]524W91 per kg excreted 67% +/- 7% of the radiolable in the )- to 48-h urine. Small amounts of the 3' -sulfoxide and glucuronide metabolites were observed in the urine, but 5-fluorouracil was not detected. Good bioavailability after oral dosing and resistance to metabolism recommend 524W91 for further preclinical evaluation.