Postantibiotic effect in Escherichia coli determined with real-time metabolic monitoring.

Metabolic activity was used to quantify the delay in recovery of Escherichia coli after limited exposures to cefotaxime and piperacillin. This was measured with a microphysiometer, a device which measures changes in pH in the cellular environment, which in turn reflects the metabolic activity of the cells. The antibiotics were rapidly pumped into and flushed from each cellular environment. The length of time that the bacteria were exposed to either antibiotic was determined by programmed valve changes. Metabolic activity was measured during periods in which the instrument's pumps were off. Graphical analysis of the data was used to determine the postantibiotic effect. The lengths of the postantibiotic effects of both drugs (95 to 101 min) determined with the microphysiometer corresponded with the reappearance of short, highly motile cells in significant numbers.

Microfabrication in silicon microphysiometry.

Over the past 5 years, microphysiometry has proved an effective means for detecting physiological changes in cultured cells, particularly as a functional assay for the activation of many cellular receptors. To demonstrate the clinical relevance of this method, we have used it to detect bacterial antibiotic sensitivity and to discriminate between bacteriostatic and bacteriocidal concentrations. The light-addressable potentiometric sensor, upon which microphysiometry is based, is well suited for structural manipulations based on photolithography and micromachining, and we have begun to take advantage of this capability. We present results from a research instrument with eight separate assay channels on a 5-cm2 chip. We discuss the planned evolution of the technology toward high-through-put instruments and instruments capable of performing single-cell measurements.

A metabolic view of receptor activation in cultured cells following cryopreservation.

The effect of cryopreservation on agonist-induced receptor activation in mammalian cells was investigated with the Cytosensor microphysiometer, a biosensor that monitors cellular metabolic activity by measuring changes in extracellular pH. In this study, two different cell types--nonadherent TF-1 cells (from a human erythroleukemia patient) and adherent WT3 cells (CHO-K1 cells transfected with the m1 muscarinic acetylcholine receptor)--were cryopreserved by freezing in a disposable cell capsule used in the microphysiometer. The recovery of metabolic activity by TF-1 cells was observed over approximately 1 h following thawing. Responses of the TF-1 cells to granulocyte-macrophage colony-stimulating factor (GM-CSF) and platelet activating factor (PAF) were measured before cryopreservation and 90 min after thawing. The GM-CSF and PAF responses retained 71 +/- 14% and 73 +/- 10% of maximum stimulation, respectively. Post-thaw cholinergic stimulation of WT3 cells was 73 +/- 9% of its level in similarly treated but unfrozen cells. Cryopreservation caused no detectable difference in desensitization of the response due to repeated application of carbachol. These results demonstrate the feasibility of pharmacological studies with cryopreserved cells in the microphysiometer and further suggest that the microphysiometer may be useful in exploring the biological consequences of cryopreservation in the early post-thaw period.

Detection of Neisseria meningitidis and Yersinia pestis with a novel silicon-based sensor.

A light-addressable potentiometric (silicon) sensor was used in an immunofiltration procedure for the detection of pathogenic bacteria. Yersinia pestis was detected by filtering the cells onto nitrocellulose membranes and then filtering anti-Y. pestis mouse monoclonal antibody and anti-mouse immunoglobulin G-horseradish peroxidase conjugate. For Neisseria meningitidis detection, mouse monoclonal antibody to the major outer membrane protein of this bacterium was coupled directly to horseradish peroxidase. N. meningitidis cell suspensions were filtered onto polycarbonate membranes, and the enzyme conjugate was allowed to react with the filtered bacteria. The presence of both enzyme conjugates was determined potentiometrically with the silicon sensor. The sensitivity of this technique relative to that of an enzyme-linked immunosorbent assay for N. meningitidis was determined. Fewer than 1,000 bacterial cells could be detected with the silicon sensor in a 20-min assay, whereas a 2.5-h enzyme-linked immunosorbent assay with the same antigen and antibody preparations was significantly less sensitive.

The role of silicone ureteral stents in extracorporeal shock wave lithotripsy of large renal calculi.

Between November 1984 and December 1985 extracorporeal shock wave lithotripsy was used to treat 1,645 kidneys at our institution. A total of 646 kidneys with stone burden greater than 14 mm. was evaluated with regard to the impact of silicone ureteral stents in post-extracorporeal shock wave lithotripsy morbidity. Our results indicate that small stones were pulverized and eliminated with minimum morbidity. Larger stones frequently were associated with post-treatment ureteral obstruction by sand and fragments. Of 283 kidneys with stone burden exceeding 25 mm. pretreatment placement of silicone ureteral stents reduced complications from 26 to 7 per cent and auxiliary procedure rates from 15 to 6 per cent. Silicone ureteral stents protect the kidney from ureteral obstruction, and allow for safe and effective extracorporeal shock wave lithotripsy of large renal calculi.

Condyloma acuminatum of the bladder and associated urothelial malignancy.

The extension of condylomata acuminata into the bladder is rare. Several reports have indicated an association of this disease with carcinoma of the penis and of the female genital tract. We report the first association of condyloma acuminatum with urothelial carcinoma in a man.

Clostridium difficile toxin A in infants.

C difficile produces two toxins, each of which is cytotoxic and lethal to animals [1]. Whether one or both of the toxins is responsible for pseudomembranous colitis in humans is unknown. C difficile is rarely found in the intestinal tracts of healthy adults, and then only in relatively low numbers. The same numbers of C difficile found in adults with pseudomembranous colitis may be present in infants with no obvious adverse effects [3]. The primary cytotoxin of C difficile, toxin B, may also be found in these infants, but the presence of toxin A, which causes intestinal fluid accumulation and mucosal necrosis in hamsters, has not been demonstrated [3]. However, an ELISA developed in our laboratory has been used to detect toxin A in fecal specimens from adults with pseudomembranous colitis [1]. Using this assay, we attempted to determine whether this toxin might be found in hospitalized infants with no obvious gastrointestinal distress. Ten of the 21 infants tested had toxin A in their feces. No infant had toxin A in the absence of toxin B. This is not surprising, because both toxins are produced in about the same ratio in vitro by all strains of C difficile we have tested [1]. In general, those specimens with high titers of toxin B had correspondingly high concentrations of toxin A. The infant with the highest titers of both toxin A and B was the eight-month-old infant with a clinical diagnosis of antibiotic-associated colitis. The other nine infants with toxin A had no enteric symptoms.(ABSTRACT TRUNCATED AT 250 WORDS)

Human chorionic gonadotropin detected in urinary concentrates from patients with malignant tumors of the testis, prostate, bladder, ureter and kidney.

Human chorionic gonadotropin levels in concentrated 24-hour urine and serum were determined by a specific radioimmunoassay of beta-human chorionic gonadotropin in 47 patients with genitourinary carcinoma. Elevated human chorionic gonadotropin levels in urine were observed in 6 of 8 patients with active testicular tumor, 3 of 14 with prostatic cancer, 2 of 10 with bladder cancer, 1 of 2 with ureteral or renal pelvic tumors and 2 of 6 with renal cancer. Of the 14 patients with positive urinary human chorionic gonadotropin 4 had undetectable human chorionic gonadotropin levels in the serum. The tumors of all 8 patients with nontesticular cancer who had increased urinary human chorionic gonadotropin levels were in an advanced clinical stage or of poor histological differentiation. Our results suggest that some nontrophoblastic carcinomas of the genitourinary system are capable of ectopic production of human chorionic gonadotropin, particularly if the malignancy is advanced or poorly differentiated. The radioimmunoassay of beta-human chorionic gonadotropin in concentrated 24-hour urine specimens enhances the sensitivity of detection of ectopic human chorionic gonadotropin production.

Effects of the two toxins of Clostridium difficile in antibiotic-associated cecitis in hamsters.

Hamsters were vaccinated with toxoids containing toxin A, toxin B, both toxins, or a preparation containing neither toxin of Clostridium difficile, the causative agent of antibiotic-associated cecitis in hamsters and pseudomembranous colitis in humans. To determine whether these vaccines would reduce the severity of antibiotic-associated cecitis, the hamsters were injected subcutaneously with clindamycin. Nearly all of the hamsters protected against neither toxin or only one toxin died. These animals developed enlarged hemorrhagic ceca and diarrhea, although the ceca from the animals immunized against toxin B were less hemorrhagic. The hamsters immunized against both toxins survived clindamycin treatment and had ceca of normal size and appearance. Concentrations of both toxins were lower in the ceca of the latter animals than in the unprotected animals. To determine the effects of either toxin alone on the animals, nonimmunized hamsters were injected with either purified toxin A, which produced enlarged ceca with moderate hemorrhaging, or partially purified toxin B, which produced hemorrhagic ceca of normal size. All of the hamsters injected with either toxin at concentrations found in the ceca after clindamycin treatment died. These results suggest that toxin A causes the water influx, that both toxins cause hemorrhaging to different extents in the ceca of hamsters with antibiotic-associated cecitis and that either toxin alone can cause death. These studies may help explain the etiology of pseudomembranous colitis in humans.

Production of antitoxins to two toxins of Clostridium difficile and immunological comparison of the toxins by cross-neutralization studies.

We prepared antitoxins specific for each of two toxins of Clostridium difficile and used these to demonstrate that the toxins are immunologically distinct.

Production of Clostridium difficile antitoxin.

We have produced antitoxin to the toxin of Clostridium difficile in rabbits and in goats. Antitoxin dilutions of 1/8,000 and 1/5,120 were capable of neutralizing lethal doses of the toxin in mice and in tissue culture, respectively.