Induction of B cell hyperplasia in simian immunodeficiency virus-infected rhesus macaques with the simian homologue of Kaposi's sarcoma-associated herpesvirus.

A simian homologue of Kaposi's sarcoma-associated herpesvirus (KSHV), the eighth human herpesvirus (HHV8), was isolated from a simian immunodeficiency virus (SIV)-infected rhesus macaque (Macaca mulatta) that developed a multicentric lymphoproliferative disorder (LPD). This simian rhadinovirus is genetically similar to a recently described rhesus rhadinovirus (RRV) (Desrosiers, R.C., V.G. Sasseville, S.C. Czajak, X. Zhang, K.G. Mansfield, A. Kaur, R.P. Johnson, A.A. Lackner, and J.U. Jung. 1997. J. Virol. 71:9764-9769) and is designated RRV 17577. RRV 17577 was experimentally inoculated into rhesus macaques with and without SIV(mac239) infection to determine if RRV played a role in development of the LPD observed in the index case. In contrast to control animals inoculated with SIV(mac239) or RRV alone, two animals coinfected with SIV(mac239) and RRV 17577 developed hyperplastic LPD resembling the multicentric plasma cell variant of Castleman's disease, characterized by persistent angiofollicular lymphadenopathy, hepatomegaly, splenomegaly, and hypergammaglobulinemia. Hypergammaglobulinemia was associated with severe immune-mediated hemolytic anemia in one RRV/SIV-infected macaque. Both RRV/SIV-infected macaques exhibited persistent RRV viremia with little or no RRV-specific antibody response. The macaques inoculated with RRV alone displayed transient viremia followed by a vigorous anti-RRV antibody response and lacked evidence of LPD in peripheral blood and lymph nodes. Infectious RRV and RRV DNA were present in hyperplastic lymphoid tissues of the RRV/SIV-infected macaques, suggesting that lymphoid hyperplasia is associated with the high levels of replication. Thus, experimental RRV 17577 infection of SIV-infected rhesus macaques induces some of the hyperplastic B cell LPDs manifested in AIDS patients coinfected with KSHV.

A liquid-filled tunable double-focus microlens.

A novel microlens design with tunable double-focus is presented. It is fabricated by adding only one SU-8 photolithography step to the well-developed liquid-filled microlens fabrication process. The thickness of this layer determines the thickness difference between the central and peripheral region of the membrane, the deformation of which is used to define the surface profile of the microlens. The stepped thickness variation is finally manifested as the difference in deformation contour at two different regions of the membrane when subjected to uniform applied pressure, thereby causing two focal lengths to appear. Experimental and simulation results are presented, from which the tunability of the focal lengths of the double-focus microlens is demonstrated to be effective over a wide range through combining the structural design with pressure control. The successful demonstration of this unconventional microlens design concept will potentially extend t application of liquid-filled microlens technology.

Polymer-based disposable microneedle array with insertion assisted by vibrating motion.

This work presents a disposable polymer-based microneedle array that carries out insertions by mimicking the vibrating motion of a mosquito's proboscis. The proposed device, which comprises a 10:1 high-aspect-ratio parylene microneedle array and a chamber structure, was monolithically realized using a novel fabrication process. The vibrating motion of the microneedles was generated using a piezoelectric actuator. This device can be potentially applied to extract and collect blood by puncturing the dermis layer of human skin. The fabricated device is advantageous because of its biocompatibility, simple fabrication process, and low associated costs. Additionally, the graph of the measured extraction flow rate versus the pressure drop that is presented shows an agreement with the results predicted by analytical models. A 40% reduction of insertion force was demonstrated when the microneedle insertion was assisted by actuator-induced vibratory motions. Buckling analyses for estimating the maximum loads that the microneedle can sustain before failure occurs were also evaluated. Finally, the relationship between the insertion force and the vibration frequency was demonstrated in this study.

Whole-body autoradiography in drug discovery.

In the drug discovery process the pharmacokinetic screening, drug stability studies, evaluation of metabolites, CYP involvement, enzyme induction and inhibition, and excretion studies play a major role. The use of more sensitive and novel detection systems have made the discovery process less cumbersome than in previous years. In particular, the use of whole-body autoradiography (WBA) for tissue distribution, which was once considered an impractical tool, owing to the long turn around time (4-10 weeks), is coming to the forefront for rapidly resolving issues encountered in discovery. In today's research environment early lead compounds can be radio-labeled and whole-body sections imaged quickly (3-5 days) using new techniques, which has made (14)C- and (3)H-WBA a viable tool. The technique has been used in vivo in species from mice to monkeys, and ex vivo and/or in vitro in larger animals and humans. WBA has considerable merit in identifying "pharmacodeficient" compounds and providing insight on mechanistic questions. WBA data can provide information related to tissue pharmacokinetics, routes of elimination, CYP or Pgp mediated drug-drug interactions, tissue distribution, site specific drug localization and retention, metabolism, clearance, compound solubility issues, routes of administration, penetration into specific targets (e.g., tumors), tissue binding (e.g., melanin), and interspecies kinetics. Thus, WBA is quickly becoming part of the battery of studies conducted during the lead optimization process to select optimal drug candidates. Examples of the use of the WBA tool in early discovery are reviewed.

Pharmacokinetic parameters and mechanisms of inhibition of rat type 1 and 2 steroid 5alpha-reductases: determinants for different in vivo activities of GI198745 and finasteride in the rat.

The interaction of baculovirus expressed rat steroid 5alpha-reductase types 1 and 2 (r5AR1 and r5AR2) with 17beta-N-(2,5-bis(trifluoromethyl)phenyl)carbamoyl-4-aza-5alpha-androst-1-en-3-one (GI198745) was investigated at pH 7 and 37 degrees. This 5alpha-reductase inhibitor was found previously to be a time-dependent inhibitor of the two human 5alpha-reductase isozymes. In contrast, we demonstrate in the present study that although GI198745 is a potent time-dependent inhibitor of r5AR2, it is a classical rapid-equilibrium inhibitor of r5AR1. This type of behavior with human and rat 5alpha-reductases has been shown for the inhibitor 17beta-(N-tert-butylcarbamoyl)-4-aza-5alpha-androst-1-en-3-one (finasteride), a current therapy for benign prostatic hyperplasia. Inhibition of r5AR1 by GI198745 was competitive with testosterone and followed Michaelis-Menten kinetics with a K(i) value of 0.3 +/- 0.02 nM. Data for the inhibition of r5AR2 by GI198745 were consistent with a two-step mechanism, where K(i) is the dissociation constant for an initial enzyme-inhibitor complex and k(3) is the rate constant for the second slow step. The pseudo-bimolecular rate constant (k(3)/K(i)) for the association of GI198745 with r5AR2 was (2.0 +/- 0.4) x 10(7) M(-1) sec(-1). The high affinity of this inhibitor for r5AR2 was further demonstrated by the inability of the enzyme-inhibitor complex to dissociate after approximately 7 days of dialysis at 4 degrees. Both GI198745 and finasteride appear to inactivate r5AR2 by apparent irreversible modification, but are classical, reversible inhibitors of r5AR1. Therefore, we hypothesize that because of its pharmacokinetic parameters and increased potency against r5AR1, GI198745 is more effective than finasteride in preventing the growth of the rat prostate.

Further isolations of non-flagellate Pseudomonas aeruginosa.

Three strains of non-flagellate Ps. aeruginosa isolated from pathological specimens have already been described. The purpose of this paper is to report further isolates. All strains have been shown to be different types isolated from a variety of infections.

Non-flagellate Pseudomonas aeruginosa in pathological material.

Three isolates of non-flagellate Pseudomonas aeruginosa obtained from pathological material of human cases are described. Two were found in urinary tract infections and the third in a respiratory infection. They were shown by typing methods to be distinct strains.

Sequential delta-integration for the regulated insertion of cloned genes in Saccharomyces cerevisiae.

A novel delta-integration vector was developed to allow the sequential insertion of multiple cloned genes in the yeast Saccharomyces cerevisiae. To allow repetitive integrations, the reusable URA3 Blaster selection cassette was employed; the insertions (of CUP1p-lacZ in this study) were selected using the URA3 marker which was subsequently "popped" out by recombination between flanking direct repeats. Transformants contained only one new integrated copy after the loss of the URA3 marker, and subsequent transformations were effective for the sequential insertion of a series of genes (one at a time) into dispersed chromosomal delta sequences. The structural stability of the integrations was location-dependent (ranging from 75% to 100% after 50 generations in complex medium with or without gene expression), and the integrations (at least up to five) had no significant effects on the growth of the cells. In addition, beta-galactosidase specific activity levels varied linearly with integrated copy number. The repetitive, regulated nature of integration with this vector is not possible with traditional delta-integration or other homologous recombination methods, and is promising for fine-tuning cloned gene copy number and for the insertion of metabolic pathway genes.

Ty1-mediated integration of expression cassettes: host strain effects, stability, and product synthesis.

The yeast retrotransposon Ty1 has been used to insert multiple copies of heterologous genes into the genome of Saccharomyces cerevisiae. Amplification using a GAL1-regulated Ty1 element carrying a 4.6 kilobase pair expression cassette (Escherichia coli lacZ structural gene under the control of the yeast CUP1 promoter, and the bacterial neo gene) was compared with that of a GAL1-regulated Ty1 element carrying only the neo gene. Mobilization of Ty1 was induced from a chromosomal element and a 2 mu-plasmid-based element; similar results were obtained for both locations. The two marked Ty1 cassettes were successfully integrated into the genomes of three different S. cerevisiae strains. Efficiencies were found to vary significantly between strains. The size of the inserted cassette was also important; the efficiency for the CUP1p-lacZ-neo cassette was much lower than that for the neo cassette in the same host. All amplified copies were found to be quite stable with or without expression for at least 50 generation in nonselective medium; moreover, there were no significant effects on the growth of the cells. After integration, beta-galactosidase specific activity from the lacZ construct in the three hosts was found to correlate well with the copy number of the CUP1p-lacZ expression cassette amplified by the Ty1 retrotransposon.

Testing paradigm for prediction of development-limiting barriers and human drug toxicity.

The financial investment grows exponentially as a new chemical entity advances through each stage of discovery and development. The opportunity exists for the modern toxicologist to significantly impact expenditures by the early prediction of potential toxicity/side effect barriers to development by aggressive evaluation of development-limiting liabilities early in drug discovery. Improved efficiency in pharmaceutical research and development lies both in leveraging "best in class" technology and integration with pharmacologic activities during hit-to-lead and early lead optimization stages. To meet this challenge, a discovery assay by stage (DABS) paradigm should be adopted. The DABS clearly delineates to discovery project teams the timing and type of assay required for advancement of compounds to each subsequent level of discovery and development. An integrative core pathology function unifying Drug Safety Evaluation, Molecular Technologies and Clinical Research groups that effectively spans all phases of drug discovery and development is encouraged to drive the DABS. The ultimate goal of such improved efficiency being the accurate prediction of toxicity and side effects that would occur in development before commitment of the large prerequisite resource. Good justification of this approach is that every reduction of development attrition by 10% results in an estimated increase in net present value by $100 million.

Clinics go electronic: two stories from the field.

We hear a lot about computerized record system implementations in hospitals, but other settings are making the transition as well. How does the move to an electronic record system affect day-to-day operations in clinics? Our writers tell how a student health center and a family clinic did it--and how they're working now.

Adoption of electronic medical records as a technology innovation for ambulatory care at the Medical University of South Carolina.

The study described in this article measured and compared the attitudes of groups of Medical University of South Carolina (MUSC) ambulatory care staff and physicians toward adopting an electronic medical record (EMR) system, using the Perceived Characteristics of Innovating (PCI) scales developed by Moore and Benbasat (1991). The PCI scale scores were compared by professional group and by clinic location. The overall findings of this study were that potential users of the ambulatory care EMR at MUSC had generally positive or neutral attitudes toward the system. There were a number of significant differences noted among the professional groups, particularly between the physician groups and other groups. The physician groups had less positive feelings than the other groups did. There were few significant differences noted for comparisons by clinic location.

Can computer-aided systems engineering tools enhance the development of health care information systems? A critical analysis.

Computer-aided systems engineering (CASE) is a software technology that helps systems developers write complex application programs. CASE has been identified as having the potential to help health information systems developers complete large-scale projects such as the computer-based patient record or community health information network. The article evaluates the current use of CASE in health care and other settings through a review of the literature. There is no consensus on the value of CASE. Many experts believe that it is an emerging technology that will become widespread, whereas others see it as expensive and cumbersome to use.

Evolution of computer-based information systems and networks to support integrated health care delivery systems.

As health care delivery systems become more integrated with an emphasis on community wellness and prevention, well-developed information infrastructures will be needed to support their activities. The article introduces several previously published independent models for the evolution of integrated delivery systems, computer-based patient records (CPRs), and health information networks as well as a summary model that illustrates the relationship among these entities and their somewhat parallel development.

Tiopinac absorption, distribution, excretion, and pharmacokinetics in man and animals.

[14C]Tiopinac disposition was evaluated in man, monkey, rabbit, mouse, minipig, and rat. The absorption of tiopinac was rapid and essentially complete in these species. Peak plasma levels occurred in less than 1 hr to 2 hr in all species. Following iv and po doses, tiopinac was recovered predominantly in the urine. In man, 93.2 and 2.6% of the administered radioactivity was recovered in the urine and feces, respectively. In the rat, 61.3% of the radioactivity was accounted for in urine and 32.7% in feces. In the mouse, radioactivity was concentrated in the kidney and liver, with tissue/plasma ratios ranging from 1.3 to 7.0 for kidney and 0.5 to 1.7 for liver. Radioactivity in all other tissues was generally lower than in plasma. Tiopinac was shown to be the major circulating entity in plasma, accounting for 90% of total radioactivity levels in the mouse and 61% in the rat. The half-life of tiopinac was 2.3 +/- 0.3 hr in man, 0.8 hr in the minipig, and 2.6 hr in the rabbit. The volume of distribution was 0.29 +/- 0.05 liter/kg in man, 0.16 liter/kg in the rabbit and minipig, and 0.42 liter/g in the mouse. Tiopinac was highly bound (99.5%) in human serum.