Urinary tract infections caused by endemic multi-resistant Enterobacter cloacae in a dialysis and transplantation unit.

In the period 1994-1998, 42 multi-resistant Enterobacter cloacae isolates responsible for urinary tract infections (UTIs) were obtained from children hospitalized in our dialysis and transplantation unit. E. cloacae isolates obtained between 1994 and 1996 were susceptible to aminoglycosides, carbapenems and fluoroquinolones, whereas E. cloacae isolates obtained between 1997 and 1998 were susceptible to carbapenems and fluoroquinolones, only. All isolates were characterized by use of the polymerase chain reaction-based random amplification of polymorphic DNA and pulsed-field gel electrophoresis. It was found that the majority of multiresistant E. cloacae isolates obtained during 1997-1998, and all isolates obtained during 1994-1996, belonged to predominant clones A or B. These strains were responsible for gastrointestinal tract colonization and UTI of renal transplant recipients for several years, and persisted as endemic E. cloacae strains in the dialysis and transplantation unit from 1994 to 1998.

Clinical and laboratory evaluation of the safety of a bioartificial liver assist device for potential transmission of porcine endogenous retrovirus.

BACKGROUND: The potential risk of transmission of porcine endogenous retroviruses (PERV) from xenogeneic donors into humans has been widely debated. Because we were involved in a phase I/II clinical trial using a bioartificial liver support system (BLSS), we proceeded to evaluate the biosafety of this device. MATERIALS AND METHODS: The system being evaluated contains primary porcine hepatocytes freshly isolated from pathogen-free, purpose-raised herd. Isolated hepatocytes were installed in the shell, which is separated by a semipermeable membrane (100-kD nominal cutoff) from the lumen through which the patients' whole blood is circulated. Both before and at defined intervals posthemoperfusion, patients' blood was obtained for screening. Additionally, effluent collected from a clinical bioreactor was analyzed. The presence of viral particles was estimated by reverse transcriptase-polymerase chain reaction (RT-PCR) and RT assays. For the detection of pig genomic and mitochondrial DNA, sequence-specific PCR (SS-PCR) was used. Finally, the presence of infectious viral particles in the samples was ascertained by exposure to the PERV-susceptible human cell line HEK-293. RESULTS: PERV transcripts, RT activity, and infectious PERV particles were not detected in the luminal effluent of a bioreactor. Culture supernatant from untreated control or mitogen-treated porcine hepatocytes (cleared of cellular debris) also failed to infect HEK-293 cell lines. Finally, RT-PCR, SS-PCR, and PERV-specific RT assay detected no PERV infection in the blood samples obtained from five study patients both before and at various times post-hemoperfusion. CONCLUSION: Although longer patient follow-up is required and mandated to unequivocally establish the biosafety of this device and related bioartificial organ systems, these analyses support the conclusion that when used under standard operational conditions, the BLSS is safe.

Safety observations in phase I clinical evaluation of the Excorp Medical Bioartificial Liver Support System after the first four patients.

A Phase I clinical safety evaluation of the Excorp Medical, Inc, Bioartificial Liver Support System (BLSS) is in progress. Inclusion criteria are patients with acute liver failure of any etiology, presenting with encephalopathy deteriorating beyond Parson's Grade 2. The BLSS consists of a blood pump, heat exchanger to control blood temperature, oxygenator to control oxygenation and pH, bioreactor, and associated pressure and flow alarm systems. Patient liver support is provided by 70-100 g of porcine liver cells housed in the hollow fiber bioreactor. A single support period evaluation consists of 12 hour extracorporeal perfusion with the BLSS sandwiched between 12 hours of pre (baseline) and 12 hours of post support monitoring. Blood chemistries and hematologies are obtained every 6 hours during monitoring periods and every 4 hours during perfusion. Physiologic parameters are monitored continuously. The patient may receive a second treatment at the discretion of the clinical physician. Preliminary evaluation of safety considerations after enrollment of the first four patients (F, 41, acetaminophen induced, two support periods; M, 50, Wilson's disease, one support period; F, 53, acute alcoholic hepatitis, two support periods; F, 24, chemotherapy induced, one support period) is presented. All patients tolerated the extracorporeal perfusion well. All patients presented with hypoglycemia at the start of perfusion, treatable by IV dextrose. Transient hypotension at the start of perfusion responded to an IV fluid bolus. Only the second patient required heparin anticoagulation. No serious or unexpected adverse events were noted. Moderate biochemical response to support was noted in all patients. Completion of the Phase I safety evaluation is required to fully characterize the safety of the BLSS.

Psychopathological correlates of reduced dopamine receptor sensitivity in depression, schizophrenia, and opiate and alcohol dependence.

A dysfunction of central dopaminergic neurotransmission has been found in various neuropsychiatric diseases, and may be associated with a common psychopathological correlate. One hypothesis suggests that dopaminergic stimulation of the brain reward system reinforces behavior because it is experienced as pleasurable, and that dopaminergic dysfunction leads to anhedonia, the inability to experience pleasure. An alternative hypothesis assumes that dopaminergic stimulation does not promote pleasure or "liking" of a reward but rather mediates "wanting" of a reward, and suggests that dopaminergic dysfunction is associated with a failure to be motivated by stimuli that indicate reward. We measured negative symptoms, psychomotor slowing and dopamine receptor sensitivity in twelve drug-free patients with major depression, seventeen alcohol-dependent and sixteen opiate-dependent patients, ten schizophrenics with neuroleptic medication, and ten healthy controls. The sensitivity of central dopamine receptors was assessed with the growth hormone response to apomorphine application. Psychomotor slowing was measured in a reaction-time test and anhedonia and other negative symptoms were assessed with self-rating scales and the Scale for the Assessment of Negative Symptoms. Patients with major depression, alcohol dependence and neuroleptic medication displayed a reduced sensitivity of central dopamine receptors compared to control subjects. Anhedonia was not a common correlate of dopamine receptor dysfunction. Instead, affective flattening was associated with both dopamine receptor sensitivity and psychomotor slowing. Our findings thus do not support the anhedonia hypothesis of central dopaminergic dysfunction. Rather, affective flattening may result from the lack of an emotional response towards reward-indicating stimuli. These findings indicate that patients with dopaminergic dysfunction are not unable to experience pleasure, but may fail to be motivated by environmental stimuli to seek reward.

Advances in bioartificial liver assist devices.

Rapid advances in development of bioartificial liver assist devices (BLADs) are exciting clinical interest in the application of BLAD technology for support of patients with acute liver failure. Four devices (Circe Biomedical HepatAssist, Vitagen ELAD, Gerlach BELS, and Excorp Medical BLSS) that rely on hepatocytes cultured in hollow-fiber membrane technology are currently in various stages of clinical evaluation. Several alternative approaches for culture and perfusion of hepatocytes have been evaluated in preclinical, large animal models of liver failure, or at a laboratory scale. Engineering design issues with respect to xenotransplantation, BLAD perfusion, hepatocyte functionality and culture maintenance, and ultimate distribution of a BLAD to a clinical site are delineated.

Effect of Pseudomonas aeruginosa exotoxin A on IFN-gamma synthesis: expression of costimulatory molecules on monocytes and activity of NK cells.

The aim of the study was (1) to evaluate the effect of Pseudomonas aeruginosa Exotoxin A (P-ExA) on the production of IFN-gamma in anti-CD3 induced human peripheral blood mononuclear cells (PBMC) and (2) to establish the effect of P-ExA on the IFN-gamma dependent cellular activities such as the expression of costimulatory molecules on monocytes and cytotoxicity of NK cells. The toxin in a high dose (100 ng/ml) inhibited IFN-gamma synthesis. Inhibitory effect of P-ExA was abolished by IL-1alpha which in a combination with P-ExA exerted a strong synergistic effect on IFN-gamma synthesis. Other monokines such as IL-1beta, IL-6, TNF-alpha neither reversed the inhibitory effect of P-ExA nor induced production of IFN-gamma. P-ExA also inhibited IFN-gamma-induced cellular events: (1) expression of costimulatory molecules on monocytes (CD80, CD86, ICAM-1, HLA-DR); (2) cytotoxic activity of NK cells. Inhibition of NK cells activity by P-ExA was not reversed by cytokines such as IL-2, IFN-alpha and TNF-alpha, which are known to enhance effector functions of NK cells. From these results we conclude that: (1) inhibition of IFN-gamma synthesis, as well as IFN-gamma-induced expression of costimulatory molecules and NK-cell effector functions may lead to suppression of specific and non-specific defense mechanisms, respectively, which are necessary for elimination of PA bacteria; (2) enhancement of IFN-gamma synthesis induced by P-ExA in a combination with IL-1alpha may cause harmful, Th1 cells dependent, inflammatory reactions of the host (septic shock, tissue damage) during infection with Pseudomonas aeruginosa.

Novel bioartificial liver support system: preclinical evaluation.

Preclinical safety and efficacy evaluation of a novel bioartificial liver support system (BLSS) was conducted using a D-galactosamine canine liver failure model. The BLSS houses a suspension of porcine hepatocytes in a hollow fiber cartridge with the hepatocytes on one side of the membrane and whole blood flowing on the other. Porcine hepatocytes harvested by a collagenase digestion technique were infused into the hollow fiber cartridge and incubated for 16 to 24 hours prior to use. Fifteen purpose-bred male hounds, 1-3 years old, 25-30 kg, were administered a lethal dose, 1.5 g/kg, of D-galactosamine. The animals were divided into three treatment groups: (1b) no BLSS treatment (n = 6); (2b) BLSS treatment starting at 24-26 h post D-galactosamine (n = 5); and (2c) BLSS treatment starting at 16-18 h post D-galactosamine (n = 4). While maintained under isoflurane anesthesia, canine supportive care was guided by electrolyte and invasive physiologic monitoring consisting of arterial pressure, central venous pressure, extradural intracranial pressure (ICP), pulmonary artery pressure, urinary catheter, and end-tidal CO2. All animals were treated until death or death-equivalent (inability to sustain systolic blood pressure > 80 mmHg for 20 minutes despite massive fluid resuscitation and/or dopamine administration), or euthanized at 60 hours. All animals developed evidence of liver failure at 12-24 hours as evidenced by blood pressure lability, elevated ICP, marked hepatocellular enzyme elevation with microscopic massive hepatocyte necrosis and cerebral edema, elevated prothrombin time, and metabolic acidosis. Groups 2b and 2c marginally prolong survival compared with Group 1b (pairwise log rank censored survival time analysis, p = 0.096 and p = 0.064, respectively). Since survival times for Groups 2b and 2c are not significantly different (p = 0.694), the groups were combined for further statistical analysis. Survival times for the combined active treatment Groups 2b and 2c are significantly prolonged versus Group 1b (p = 0.047). These results suggest the novel BLSS reported here can have a significant impact on the course of liver failure in the D-galactosamine canine liver failure model. The BLSS is ready for Phase I safety evaluation in a clinical setting.

Effect of Pseudomonas aeruginosa exotoxin A on CD3-induced human T-cell activation.

The effect of Pseudomonas aeruginosa (PA) exotoxin A (P-ExA) on CD3-induced T-cell activation was studied on the level of T-cells (proliferation, synthesis of interleukin (IL)-2, expression of IL-2R complex, ICAM-1,2 and LFA-1 molecules), and on the level of monocytes (expression of ICAM-1,2, LFA-1 molecules, as well as FcRI and CD14 receptors). We found that: (1) P-ExA blocked T-cell proliferation and this effect was totally reversed by intact monocytes, and partially by IL-2 or TPA but not by costimulatory cytokines (IL-1alpha, IL-1beta, TNF-alpha or IL-6); (2) P-ExA transiently, in short-term cultures (48 h), inhibited synthesis of IL-2; (3) prolonged stimulation (96 h) of peripheral blood mononuclear cells (PBMC) or CD4 + T-cells with P-ExA in high or low doses (100 and 10 ng/ml, respectively), enhanced the level of IL-2 in the cultures; (4) P-ExA at low dose, combined with IL-1beta, TNF-alpha or IL-6, up-regulated synthesis of IL-2; and (5) stimulation of T-cells with anti-CD3 monoclonal antibody (mAb) and P-ExA at high dose diminished the expression of the p55 chain but not of the p75 chain of IL-2R complex and slightly affected the expression of CD3 complex, ICAM-1,2 and LFA-1 molecules. Hence, P-ExA can regulate the level of IL-2 in cultures of CD3-induced T-cells either by inhibition of IL-2 consumption (when P-ExA is applied in high dose), or by induction of IL-2 production (a costimulatory effect exerted by P-ExA in low dose in combination with monokines). Action of P-ExA on monocytes resulted in: (1) inhibition of the expression of ICAM-1,2 molecules and their ligand LFA-1 molecule; (2) low expression of FcRI receptor (a ligand for Fc part of CD3 mAb); and (3) inhibition (over 90%) of the expression of CD14 molecule. In conclusion, P-ExA-induced anergy of T-cells depends on: (a) decrease in the affinity of IL-2R complex on activated T-cells; and (b) inhibition of the accessory activities of monocytes.

A microchip glucose sensor.

A major problem in development of a glucose sensor for use in an implantable artificial pancreas is the lack of reproducibility in signals from sensor to sensor. Each glucose sensor fabricated with currently used methods has a unique response to varying levels of glucose concentration and thus needs to be individually calibrated before use. We have adapted microchip manufacturing techniques for the fabrication of electrochemically based glucose sensors with standardized and reproducible function. Scanning electron microscopic study of the resulting electrode surfaces shows them to be smooth and featureless at all levels of magnification. X-ray diffraction analysis of the electrodes indicates preferential exposure of the [1,1,1] crystal interface. Cyclic voltammetry evaluation of initial sensor response to varying glucose concentrations shows excellent sensor to sensor reproducibility for all sensors made with the same underlayment. Sensors made with titanium underlayment appear to be more differentiated and thus more sensitive to variations in glucose concentration than are sensors with chromium underlayment. Although the initial response of microchip glucose sensors appears to be standardized and reproducible, additional development of an appropriate electrical insulation material is required before long-term study of signal stability is feasible.

Antibiotic resistance of bacterial strains isolated from children in Child Health Center, Warsaw.

This study examines the antibiotic susceptibility of 1792 bacterial strains isolated from hospitalized children between January and December 1993. A total of 1015 Gram-negative rods represented by members of Enterobacteriaceae family (770) and nonfermenters (245) were isolated. The most resistant strains were noticed among Klebsiella pneumoniae and Enterobacter cloacae. From 38% to 46% of K. pneumoniae strains were resistant to third-generation cephalosporins, but all of them were sensitive to imipenem. From 60% to 80% of E. cloacae isolates were resistant to all beta-lactams, but sensitive to imipenem. Resistance of P. aeruginosa to aminoglycosides varied from 30% for gentamicin to 5% for amikacin. About 40% of P. aeruginosa strains were resistant to carbenicillin, and 25% to azlocillin and piperacillin, but only two strains were resistant to ceftazidime and imipenem. Among Gram-positive cocci the most frequently encountered were coagulase-negative staphylococci, followed by Staphylococcus aureus and Enterococcus faecalis. The methicillin-resistant strains of coagulase-negative staphylococci and S. aureus consisted 74.8% and 34%, respectively. All strains of methicillin-resistant coagulase-negative staphylococci and S. aureus were sensitive to vancomycin.

Zeolitic ammonium ion exchange for portable hemodialysis dialysate regeneration.

Ammonia removal from a recirculating dialysate stream is a major challenge in developing a truly portable, regenerable hemodialysis system. Three zeolites, type F, type W, and clinoptilolite, were found to have good ammonia ion exchange capacity with linear equilibrium ion exchange coefficients of 0.908, 0.488, and 0.075 L/g, respectively. The linear equilibrium ion exchange coefficient relates dialysate ammonia concentration (mumol/L) to the amount of ammonia absorbed by zeolite (mumol/g) at equilibrium. Ammonia uptake by zeolite powders was fast, with equilibrium reached within 15 sec. Zeolite ammonia ion exchange and regeneration through multiple cycles was studied using an ion exchange column containing clinoptilolite pellets. Zeolite ion exchange capability was regenerated by flushing the column with 2 mol/L sodium chloride after an ion exchange run. The column maintained ammonia ion exchange capacity through six ion exchange/regeneration cycles, demonstrating multiple dialysis use possibilities. Atomic absorption spectroscopy of the column effluent showed no detectible (< 1 part per million) Si or Al leached from the zeolite.

Ammonia transport across hydrophobic membranes. Application to dialysate regeneration.

Removal of ammonia from a recirculating dialysate buffer in a portable hemodialysis application can be achieved by countercurrent, gas phase ammonia transfer across a hydrophobic membrane into an acid solution. Ammonia transfer fluxes as high as 0.076 mumol/sec/m2 have been achieved using a Sarns Turbo Membrane Oxygenator (Sarns-3M, Ann Arbor, MI) with a 1.9 m2 membrane surface area (0.145 mumol/sec actual rate). A simple physical model based upon ammonia desorption at the gas-dialysate buffer interface in a membrane pore, ammonia diffusion through the gas filled pore, and subsequent ammonia absorption at the gas/acid interface side of the pore quantitatively describes the experimental data. The ammonia transfer rate is most dependent upon dialysate buffer pH (higher pH promoting transfer rate) and ammonia concentration in the dialysate buffer (higher concentrations promoting transfer rate). A 500 fold improvement in transfer rate, however, will be required for clinical application.

Interference of glucose sensing by amino acids.

Interference by membrane permeable substances on nonspecific electrodes is a major problem in glucose sensing. Alanine, lysine, phenylalanine, and cystine were chosen for study to gain insight into this problem. These compounds represent the classes of mono-amino aliphatic, di-amino aliphatic, aromatic, and sulfur containing amino acids, respectively. Cyclic voltammetry experiments were performed using a Pt electrode (1.77 mm2). The reductive current of glucose at -0.750 V versus Ag/AgCl was measured with increasing concentrations of interfering substances in Krebs-Ringer phosphate buffer (pH 7.4) at 37 degrees C. Experimental results have shown that these amino acids have an inhibitory effect on the glucose signal. An important finding was that the interferences from phenylalanine and cystine were more pronounced than those of lysine and alanine. An initial drop in the glucose signal was seen at less than 2.0 mg/dl of alanine or lysine and at less than 0.5 mg/dl of phenylalanine or cystine. Additional increase in the concentrations of interfering substance did not cause further appreciable signal reduction. The results confirm that glucose sensing using a non-specific electrode is possible in fluids containing interfering substances such as amino acids.

Pseudomonas aeruginosa exotoxin A enhances automaticity and potentiates hypoxic depression of isolated rat hearts.

The potent virulence factor exotoxin A, produced by Pseudomonas aeruginosa, has been reported to suppress the synthesis of the alpha-subunit of cardiac Gi protein and may have general effects upon synthesis of other myocardial proteins. To determine whether such exotoxin A actions influence specific functional properties of the intact heart, characteristics of isolated perfused hearts obtained from rats receiving injections of exotoxin A 48 hr before sacrifice were compared with those of rats receiving no exotoxin A. Exotoxin A treatment increased the spontaneous beating rates and potentiated the suppressive effects of hypoxia upon heart rate, left ventricular systolic pressure, and rates of ventricular contraction and relaxation. On the other hand, exotoxin A treatment did not influence the magnitude or rate of pressure development under control conditions, the positive chronotropic and inotropic responses to isoproterenol, or the negative chronotropic responses to adenosine. Since a specific exotoxin A-induced suppression of myocardial alpha-subunit of the Gi protein should confer hypersensitivity to isoproterenol and reduced sensitivity to adenosine, the absence of alterations in responses to these interventions suggests that exotoxin A's effect was not confined to specific suppression of this protein. However, net effects of exotoxin A exposure included a pronounced increase in excitability of the hearts and enhanced vulnerability to hypoxic insults.

The resistance patterns and serotypes of Pseudomonas aeruginosa strains isolated from children.

Among 916 Pseudomonas aeruginosa strains isolated from children in a Warsaw hospital during the period 1985-1988 the most frequently encountered serotypes were O6, O11, O12, and O16. The majority of isolates resistant to aminoglycosides were characterized by simultaneous resistance to gentamicin and tobramycin and belonged to serotype O11. Among isolates resistant to beta-lactam antibiotics the most frequently encountered serotype was O6, followed by serotype O3 and O11. Most strains of serotype O12 were resistant to aminoglycosides and beta-lactam antibiotics as has been reported from other countries.

Voltage polarity relay--optimal control of electrochemical urea oxidation.

Voltage polarity relay (VPR) is shown to optimize the urea oxidation rate and urea current utilization under constant current conditions in direct electrochemical urea oxidation. Direct electrochemical urea oxidation is characterized by reversible deactivation of the working electrode due to oxidation products remaining on the surface and the requirement that the working electrode potential remain below about 1.1 V relative to Ag/AgCl in order to prevent undesirable secondary electrochemical oxidations. The VPR method monitors the potential of the working electrode relative to a suitable reference and changes system polarity when the upper potential set limit is reached. Thus, what was the working electrode becomes the counter electrode and vice versa. Since urea oxidation products are desorbed from the counter electrode when its potential drops below about -0.6 V relative to Ag/AgCl, alternating electrode functions between working and counter provides cyclic electrode regeneration and continuous urea oxidation. VPR is believed to optimize constant current control for any electrochemical system that exhibits behavior similar to direct electrochemical urea oxidation.

Characterization of antibody response to Pseudomonas aeruginosa in patients with wound infections.

ELISA was used to measure the amount and avidity of IgG antibodies to exotoxin A (ExA) and 7 Fisher's immunotypes of lipopolysaccharide (LPS) in the sera of 13 patients with mild or moderate Pseudomonas aeruginosa infections. Changes in the specificity of tested sera during the course of infection were demonstrated. A statistically significant increase was seen in the amount and avidity of the antibodies to ExA in a majority of the sera, and an increase was seen in amount of antibodies to LPS immunotype 4 in the sera of patients with moderate infections.

Pseudomonas aeruginosa exotoxin A primes human monocyte oxidative burst response in vitro.

Modulation of the oxidative burst responsiveness of human blood monocytes and neutrophils after incubation with purified exotoxin A from Pseudomonas aeruginosa was studied in a lucigenin- and luminol-enhanced chemiluminescence system. Exotoxin A alone caused a dose-dependent stimulation of monocyte chemiluminescence responses, whereas neutrophil responses were inconsistent. Preincubation of monocytes with exotoxin A primed the cells for a significantly higher oxidative burst response when N-f-methionyl-leucyl-phenylalanine (fMLP) was used as a secondary stimulus, especially in the lucigenin-enhanced system. Heat-treatment at 100 degrees C for 15 min completely abolished the priming activity of the exotoxin A preparation. These findings demonstrate that exotoxin A modulates monocyte responsiveness in the chemiluminescence assay and suggest that increased release of toxic oxygen radicals from mononuclear phagocytes may contribute to the tissue damage in lungs with chronic P. aeruginosa infections.