Carcinomatous meningitis: the natural history of successfully treated metastatic bladder cancer.

Carcinomatous meningitis due to bladder cancer is a rare entity reported only in case reports. Optimal therapy is thus poorly defined with earlier cases reporting an unsuccessful outcome. Here we report a case of late carcinomatous meningitis secondary to transitional cell carcinoma (TCC) of the bladder occurring in a patient in complete remission. He was successfully treated with intrathecal methotrexate and whole brain irradiation and experienced prolonged survival after treatment. With modern chemotherapy increasing complete remissions and survival rates in patients with TCC, more and more patients are being reported with carcinomatous meningitis. We raise the question of whether central nervous system prophylaxis should be considered in patients with TCC achieving a complete remission to chemotherapy in the metastatic setting.

Fusobacterium necrophorum: a ruminal bacterium that invades liver to cause abscesses in cattle.

Fusobacterium necrophorum, a Gram-negative, rod-shaped, and an aerotolerant anaerobe, is a normal inhabitant of the rumen of cattle. The organism is in ruminal contents and adherent to the ruminal wall. Its role in ruminal fermentation is to metabolize lactic acid and degrade feed and epithelial proteins. The ruminal concentration is higher in grain-fed than forage-fed cattle. From the rumen, the organism gains entry into the portal circulation and is trapped in the liver to cause abscesses. The organism is an opportunistic pathogen and a primary causative agent of liver abscesses, an economically important disease of grain-fed cattle. Liver abscesses are often secondary to ruminal acidosis and rumenitis in grain-fed cattle. Two subspecies of F. necrophorum, subsp. necrophorum (biotype A) and subsp. funduliforme (biotype B), are recognized that can be differentiated based on morphological, biochemical, biological and molecular characteristics. The subsp. necrophorum is more virulent and is isolated more frequently from infections than the subsp. funduliforme. Several toxins or secreted products have been implicated as virulence factors. The major factors contributing to ruminal colonization and invasion into the liver are hemagglutinin, endotoxin and leukotoxin, of which leukotoxin is the protective antigen. In some conditions, the organism synergistically interacts with Arcanobacterium pyogenes, a facultative anaerobic organism and a secondary etiologic agent, to cause liver abscesses.

Chromatographic process identification for protein C purification using frequency response analysis.

Frequency response analysis is applied for the analysis of liquid chromatography output of protein separation. Reduced data from simple chromatograms suggest that various Bode plot parameters, magnitude ratios, phase shift, the steady state gain, break frequency, and system order in the frequency domain, can be used to gain phenomenological insights on the system. Such an approach is advantageous because the validity of the model can be checked for two plots, the magnitude ratio vs. frequency and the phase shift vs. frequency, as compared to a single plot in the time domain. This approach also provides a useful empirical-tool which can be quantifiably used for process validation and scale-up, especially for immunoaffinity and immobilized metal affinity chromatographic systems used for protein C purification.

Scintillation proximity radioimmunoassay for the measurement of acyclovir.

A homogeneous, single-tube scintillation proximity radioimmunoassay (SPRIA) to quantitate acyclovir (Zovirax), ACV, (9-[(2[hydroxyethoxy)]methylguanine)] in human plasma is described. The reagents for the SPRIA are an anti-ACV monoclonal antibody (WACO4 MAb), tritiated ACV, and scintillation proximity reagent (goat anti-mouse immunoglobulin G (IgG) coupled to fluoromicrospheres). The ACV standard curve range in the SPRIA is from 0.7 ng ml-1 (3.0 nmol l-1) to 90.0 ng ml-1 (0.4 mumol l-1) with a 50% inhibitory concentration of 5.0 ng ml-1 (22.2 nmol l-1). However, the lower limit of quantification is 7 ng ml-1 at 1:10 dilution of plasma. Analytical recovery of ACV in spiked human plasma controls ranges between 90-110%. Intra- and inter-assay relative standard deviations were < 8%. This high throughput homogeneous assay is a rapid, convenient and simple alternative to the current radioimmunoassay that uses ammonium sulfate precipitation as the separation method. This technique is particularly attractive because it requires neither separation of bound from free drug nor use of scintillation fluid. The procedure was applied to quantitate ACV in samples from pre-clinical and clinical studies after the administration of valaciclovir, a prodrug of ACV (256U87, Valtrex, L-valyl ester of ACV). Automation of this assay will further improve efficiency in processing a larger number of samples.

Differential assay of zidovudine and its glucuronide metabolite in serum and urine with a radioimmunoassay kit.

We developed an ancillary procedure for the ZDV-Trac RIA (Incstar) to allow simultaneous determination of both zidovudine (3'-azido-3'-deoxythymidine, ZDV, AZT, Retrovir) and its metabolite, the glucuronide of ZDV (3'-azido-3'-deoxy-5'-O-beta-D-glucopyranuronosylthymidine, ZDVG, GAZT), in human serum and urine. Using the ZDV-Trac RIA, we measured ZDV concentrations before and after ZDVG in samples was hydrolyzed to ZDV by beta-glucuronidase (EC 3.2.1.31); ZDVG concentration was calculated as the difference between the two results. This method enables rapid evaluation of a large number of samples with a total turn-around time of 6 h. The lower detection limit of the RIA was 0.27 micrograms/L; the measurements varied linearly with ZDV concentrations from 0.27 to 217 micrograms/L, with the 50% inhibitory concentration being approximately 10 micrograms/L. Analytical recoveries of inhouse serum and urine controls for both ZDV and ZDVG exceeded 90%. Coefficients of variation (CVs) of serum controls were less than 6% for ZDV and less than 11% for ZDVG; for urine controls, CVs for both ZDV and ZDVG were less than 6%. Results for ZDVG concentrations obtained by HPLC and by the ZDV-Trac RIA system compared well: r = 0.978, slope 1.0, for serum samples, and r = 0.993, slope 1.09, for urine samples.

Determination of zidovudine concentration in serum by enzyme-linked immunosorbent assay and by time-resolved fluoroimmunoassay.

Two solid phase, nonradioactive immunoassays were developed and evaluated for the determination of zidovudine (Retrovir, ZDV, AZT) concentrations in serum. The first, an enzyme-liked immunosorbent assay (ELISA), used an anti-AZT monoclonal antibody (MAb) and subsequent alkaline phosphatase second antibody system for the detection method. The second, a time-resolved fluoroimmunoassay (TR-FIA), used a polyclonal anti-AZT antibody followed by a europium-labeled goat anti-rabbit immunoglobulin (IgG) as the fluorescent probe. The ELISA had a detection range from 125 to 4,000 nM and a 50% inhibitory concentration (IC50) of about 500 nM. Development of the TR-FIA was based on a very sensitive detection system of metal chelate chemistry and time-resolved fluorometry. The standard curve in the TR-FIA was from 5 to 4,000 nM with an IC50 of 200 nM. Intra- and interassay precision of the ELISA was good, with coefficients of variation from 3.9 to 7.3% and 3 to 17%, respectively, while the same values for the TR-FIA were 6.2 to 10.9% and 5.4 to 19.9%, respectively. The results obtained from each method were compared individually with those of high-performance liquid chromatography (HPLC) and radioimmunoassay (RIA). There was good agreement among the results obtained by each method. These two methods enable laboratories not licensed for radioisotopes to analyze potentially infectious samples without aerosol-forming centrifugation.

Zidovudine-induced fever.

Zidovudine (3'-azido-3'deoxythymidine, AZT, Retrovir) is the first antiretroviral drug to demonstrate clinical efficacy for symptomatic human immunodeficiency virus infection. In a large, placebo-controlled trial, nausea and hematologic toxicity, but not fever, occurred more frequently in zidovudine- than in placebo-treated patients. However, in an open label study, fever severe enough to halt zidovudine administration occurred in 10% of 70 acquired immune deficiency syndrome (AIDS) patients receiving the drug. We now describe three AIDS patients with severe zidovudine-induced fever in whom other causes of fever were excluded. Zidovudine-induced fever was confirmed in each case by drug rechallenge. Using an enzyme immunoassay, we detected IgM antibodies directed against a zidovudine-serum protein conformational determinant in one of these three patients. Neither IgG nor IgM anti-zidovudine antibodies were present in sera from the other two patients with zidovudine fever, from four AIDS patients who discontinued zidovudine for reasons other than fever, or from five AIDS patients who never received zidovudine. Zidovudine may cause fever as a severe adverse effect in patients with AIDS. Either type III or type IV hypersensitivity may mediate this reaction.

Radioimmunoassay for Retrovir, an anti-human immunodeficiency virus drug.

A direct radioimmunoassay (RIA) for the quantitation of Retrovir (zidovudine, azidothymidine, AZT) in biological fluids has been developed. The assay is sensitive with an I50 value of about 30 nM and with a lower limit of detection of about 3 nM. Intra-assay precision gave sample coefficients of variation that ranged from 1.77 to 8.65% for the standard curve with human plasma. Inter-assay precision and accuracy were within acceptable limits. The RIA was validated by comparing results obtained form the analysis of rat plasma samples by both this RIA and a high-performance liquid chromatography method. None of the crossreactivities recorded should interfere with the assay system. The affinity constant of the antibody chosen for use was 1.4 z 10(9) L/mol.

Radioimmunoassay for desciclovir, 2-[(2-amino-9H-purin-9-yl)methoxy]ethanol, a prodrug for the antiviral acyclovir.

A direct radioimmunoassay for the detection of desciclovir (DCV)(2) in biological fluids has been developed. The radioimmunoassay was validated by comparing results obtained from human plasma samples analyzed by both this RIA and a gas chromatographic method. None of the crossreactivities noted interfere with the assay system. Although the succinylated antigen has a slightly higher affinity constant, the non-succinylated tritiated antigen was chosen for routine use. The assay is sensitive with an I50 value of 20 nM and with a lower limit of detection of about 3 nM. Intra-assay precision gave sample coefficients of variation which ranged from 2.2 to 9.6% for the standard curve with human plasma and from 2.0 to 8.2% for the standard curve with human urine. Inter-assay precision and accuracy were within acceptable limits.

A competitive enzyme-linked immunosorbent assay to quantitate acyclovir and BW B759U in human plasma and urine.

A simple and sensitive enzyme-linked immunosorbent assay for the detection and quantitation of acyclovir in human plasma and urine was developed. Acyclovir immobilized on a solid phase and free acyclovir in the sample solution were allowed to compete for a limited amount of anti-acyclovir monoclonal antibody. The specific antibody bound to the immobilized acyclovir was detected by the use of alkaline phosphatase-conjugated anti-mouse immunoglobulin. The resulting enzyme activity was inversely related to acyclovir concentration in the sample. The Hill plot of standard acyclovir concentrations was linear over a 100-fold concentration range, with a lower detection limit of 0.2 nM and a concentration of soluble ligand displacing 50% of available antibody of approximately 1 nM. The metabolites of acyclovir cross-reacted minimally, and there was no detectable interference by various unrelated compounds tested in the assay. However, BW B759U [9-(2-hydroxy-1-hydroxymethylethoxy)methylguanine], a congener of acyclovir, cross-reacted significantly. As a consequence, the assay was found useful in measuring the concentrations of BW B759U in clinical samples devoid of acyclovir.