Detection of multiple hypervirulent Klebsiella pneumoniae strains in a New York City hospital through screening of virulence genes.

OBJECTIVES: The 'hypervirulent' variant of Klebsiella pneumoniae (hvKp) is a predominant cause of community-acquired pyogenic liver abscess in Asia, and is an emerging pathogen in Western countries. hvKp infections have demonstrated 'metastatic' dissemination in immunocompetent hosts, an unusual mode of infection associated with severe complications. Two cases alerted us to the possible presence of hvKp at our hospital, both involving elderly Hispanic males who presented with recurrent fever, bacteraemia, epigastric pain and liver abscesses/phlegmon, thus prompting an assessment of hvKp prevalence. METHODS: A surveillance of K. pneumoniae blood, body fluid and wound isolates was conducted using real-time PCR to detect virulence-associated genes (uni-rmpA, iucA and peg344). Positive isolates were further characterized by wzi gene sequencing to determine capsular types (K-type) and by multilocus sequence typing and pulsed-field gel electrophoresis to determine strain relatedness. RESULTS: Four-hundred and sixty-three K. pneumoniae isolates, derived from 412 blood, 21 body fluids and 30 abdominal wound specimens, were screened over a 3-year period. Isolates included 98 multidrug-resistant strains. Eighteen isolates from 17 patients, including two from the index patient, screened positive for all three virulence genes. Sixteen of 18 positive isolates had K-types associated with hvKp, and isolates from different patients were unrelated strains, indicating likely community acquisition. Of 13 patients with significant morbidity, five died; eight patients had co-existing hepatobiliary disease, and six had diabetes mellitus. CONCLUSIONS: Multiple strains of hvKp are emerging in New York City and are associated with high mortality relative to multidrug-resistant and classical Klebsiella infections. Co-existing hepatobiliary disease appears to be a potential risk factor for these infections.

MR Angiographic-Guided Percutaneous Sclerotherapy for Venous Vascular Malformations: A Radiation Dose-Reduction Strategy.

We present a new technique using MRA instead of the usual DSA to provide guidance in the treatment of venous vascular malformations. When one performs this embolization procedure, appropriate needle positioning within the malformation must be confirmed before injection of the sclerosing agent to prevent untoward complications. Time-resolved imaging of contrast kinetics-MRA can accurately depict the angioarchitecture of the lesion, which substantially reduces the total radiation dose in these patients who are commonly in the pediatric age group and usually require numerous treatment episodes.

Risk stratification of pT1-3N0 patients after radical cystectomy for adjuvant chemotherapy counselling.

BACKGROUND: In pT1-T3N0 urothelial carcinoma of the bladder (UCB) patients, multi-modal therapy is inconsistently recommended. The aim of the study was to develop a prognostic tool to help decision-making regarding adjuvant therapy. METHODS: We included 2145 patients with pT1-3N0 UCB after radical cystectomy (RC), naive of neoadjuvant or adjuvant therapy. The cohort was randomly split into development cohort based on the US patients (n=1067) and validation cohort based on the Europe patients (n=1078). Predictive accuracy was quantified using the concordance index. RESULTS: With a median follow-up of 45 months, 5-year recurrence-free and cancer-specific survival estimates were 68% and 73%, respectively. pT-stage, ge, lymphovascular invasion, and positive margin were significantly associated with both disease recurrence and cancer-specific mortality (P-values ≤ 0.005). The accuracies of the multivariable models at 2, 5, and 7 years for predicting disease recurrence were 67.4%, 65%, and 64.4%, respectively. Accuracies at 2, 5, and 7 years for predicting cancer-specific mortality were 69.3%, 66.4%, and 65.5%, respectively. We developed competing-risk, conditional probability nomograms. External validation revealed minor overestimation. CONCLUSION: Despite RC, a significant number of patients with pT1-3N0 UCB experience disease recurrence and ultimately die of UCB. We developed and externally validated competing-risk, conditional probability post-RC nomograms for prediction of disease recurrence and cancer-specific mortality.

Inhibition of malignant cell growth by 311, a novel iron chelator of the pyridoxal isonicotinoyl hydrazone class: effect on the R2 subunit of ribonucleotide reductase.

The key roles of iron and iron proteins in cell proliferation make them potential targets for cancer therapy. However, clinical trials directed toward perturbation of tumor iron homeostasis by iron chelation have been limited to the use of deferoxamine (DFO). There is thus a need to develop agents with greater efficacy. In the present study, we investigated the mechanism of cytotoxicity of 311 (2-hydroxy-1-naphthylaldehyde benzoyl hydrazone), a novel iron chelator of the pyridoxal isonicotinoyl class. We found that 311 inhibited the growth of CCRF-CEM cells in a time- and concentration-dependent fashion with an IC(50) that was approximately 20-fold lower than that of DFO. 311 also inhibited the growth of breast, bladder, and head and neck cancer cell lines. Using electron spin resonance (ESR) spectroscopy analysis, we found that a 12-h exposure of CCRF-CEM cells to 311 inhibited the tyrosyl radical ESR signal of the R2 subunit of ribonucleotide reductase. However, overproduction of the R2 subunit in hydroxyurea-resistant CCRF-CEM cells was associated with a decrease in sensitivity of cells to 311 but not to DFO. Our studies show that 311 is a more potent cytotoxic agent than DFO, with activity against both hematopoietic and nonhematopoietic cell lines regardless of their p53 status. Furthermore, the ESR studies suggest that inhibition of the R2 subunit of ribonucleotide reductase is at least one mechanism by which 311 blocks cell proliferation.

Dimercaptosuccinic acid distribution in renal tubular acidosis.

A 99Tcm dimercaptosuccinic acid (DMSA) scan performed after a urinary tract infection demonstrated an unusual pattern of isotope uptake, promoting further investigations leading to a diagnosis of renal tubular acidosis secondary to nephropathic cystinosis. This is known to affect isotope imaging but a unique feature in this undiagnosed case was the uncorrected metabolic acidosis, which had further altered the distribution of the DMSA. It is noteworthy because other patients referred for imaging with renal disease may also have abnormalities of acid base balance.

Estradiol-17 beta effects on lipid metabolism of adipose tissue in nutritionally induced lean and obese ovariectomized ewes.

Nutritionally manipulated lean (68 kg) and obese (87 kg) ovariectomized ewes were administered estradiol-17 beta (E2) or sham implants. Ewes individually had ad libitum access to corn silage. Rates of de novo lipogenesis, palmitate esterification, and glycerol and fatty acid release were determined with slices of subcutaneous adipose tissue at 0, 5, and 30 d after implantation. Condition and E2 interacted over time (linear, P less than .12; quadratic, P less than .05) to affect intake. Lean ewes implanted with E2 decreased intake initially after implantation, whereas obese ewes implanted with E2 decreased intake later after implantation. The linear effect of time x condition x E2 interacted (P less than .02) for lipogenesis. Lipogenesis was inhibited in both the lean and obese ewes implanted with E2. Lean compared with obese ewes without E2 had increased lipogenesis at a faster rate over time. Esterification increased (linear, P less than .01) in the lean ewes and decreased (quadratic, P less than .01) in the obese ewes over time. A time x E2 interaction occurred for esterification (P less than .02). Glycerol and fatty acid release were variable over time within condition and E2. A 48-h adipose tissue culture determined the effect of E2 on lipid metabolism. Estradiol-17 beta decreased (P less than .05) lipogenesis, decreased (P less than .08) esterification, and maintained fatty acid and glycerol release. Data in vivo and in culture indicated that E2 acted to decrease de novo lipogenesis and palmitate esterification and had little or no effect on lipolysis.

Herniated intervertebral discs associated with unstable spinal injuries.

To examine the occurrence of traumatic herniated intervertebral discs associated with unstable spinal injuries, the authors reviewed the records of all patients with spinal cord level unstable spine injuries managed at their institution over a 26-month period. Ninety-three patients were identified. All patients had roentgenographic and computed tomographic (CT) evaluation. Magnetic resonance imaging was performed in 48 patients, and revealed the presence of a herniated intervertebral disc in 16, with the highest incidence being in the cervical spine. In the patients who had only plain film and CT scans, no disc pathology was identified. Magnetic resonance imaging provides a noninvasive means of examining intervertebral disc damage in unstable spinal injuries that might otherwise be unidentified and result in spinal cord injury at the time of surgery.

Photoaffinity labeling of the beta-adrenergic receptor from cultured lymphoma cells with [125I]iodoazidobenzylpindolol: loss of the label with desensitization.

The beta-adrenergic antagonist [125I]iodoazidobenzylpindolol ( [125I]IABP) specifically photolabeled two polypeptides in membrane preparations from wild-type (WT) and coupling protein-deficient cyc- cultured lymphoma cells. The molecular weights of the two polypeptides determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis were 65,000 and 55,000. They were labeled in a ratio of approximately 1:1. Pretreatment of intact WT or cyc- cells with 1.0 microM epinephrine for 15 min (desensitization) resulted in a greater loss of the 55,000 Mr polypeptide (40-60%) relative to the 65,000 Mr peptide (10-30% loss). An 18- to 24-hr pretreatment of WT cells with terbutaline (down-regulation) led to a greater than 90% reduction of the photolabeling of both polypeptides, whereas a similar pretreatment of cyc- cells resulted in no further loss of labeled receptor than that observed after only a 15-min pretreatment with epinephrine. There was no indication of a change in the electrophoretic mobility of the [125I]IABP-labeled receptors after either short- or long-term agonist pretreatment. These data provide direct evidence for heterogeneity of the beta-adrenergic receptor in lymphoma cells. The differential loss of the [125I]IABP labeling in the two polypeptides suggests a functional heterogeneity as well.

Repair of alkylated DNA: Drosophila have DNA methyltransferases but not DNA glycosylases.

DNA methyltransferase activity has been identified in crude extracts of Drosophila melanogaster pupae for the removal of methyl groups from O-6 methylguanine appearing in alkylated DNA. Additionally, N-7 methylguanine and 3 methyladenine appear to be uniquely susceptible to methyltransferase activity that resides in Drosophila pupae. Consistent with this, tests to detect DNA glycosylase activity for the repair of the latter two modified bases was unsuccessful, even though a substantial loss of methyl groups from these bases was observed. Conversely, the repair of methylated purines was not detected in extracts of Drosophila embryos. The removal of methyl groups from methylated purines was dependent upon incubation temperature and was proportional to the amount of protein added to reaction mixtures. Results indicate that the methyl group is attached to protein during the repair of methylated DNA, suggesting that it is similar to the O6-methylguanine-DNA methyltransferase identified in other organisms. Although other explanations are possible, the inability to detect DNA glycosylase activity suggests that Drosophila may not rely on base excision repair for the removal of modified or nonconventional basis in DNA.

Effect of dithiothreitol on the beta-adrenergic receptor of S49 wild type and cyc- lymphoma cells: decreased affinity of the ligand-receptor interaction.

Dithiothreitol (DTT) treatment of WT or cyc- lymphoma membranes resulted in the simultaneous loss of epinephrine-stimulated adenylate cyclase activity and beta-adrenergic antagonist binding. The treatment produced no significant decrease in NaF-stimulated activity and only a partial loss of the PGE1 stimulation. Epinephrine partially protected against the loss of epinephrine-stimulated cyclase activity. The decrease in beta-adrenergic stimulation of adenylate cyclase was characterized by over a 100-fold increase in Kact for epinephrine stimulation of adenylate cyclase (10 mM DTT for 30 min) with no effect on the Vmax. We have previously shown that two polypeptides (Mr = 55,000 and 65,000 daltons) are specifically labeled by [125I]iodoazidobenzylpindolol (IABP) in WT and cyc-. The IABP photolabeling of the 55,000 dalton beta-receptor polypeptide was preferentially reduced by 1.0 mM DTT, whereas 10 mM DTT eliminated the photolabeling of both polypeptides. The effects of DTT were not due to either scavenging of the nitrene or reduction of the azide. A reduction in epinephrine stimulation of adenylate cyclase was also observed after treatment of intact WT, and the effects were readily reversed in the cells by washout of the DTT. The DTT effects on membranes were not reversed by washout. Our results demonstrate that the oxidized state of the lymphoma beta-receptor is necessary for maximum sensitivity to agonist stimulation of adenylate cyclase and that low concentrations of reducing agent selectively decrease specific photo-labeling of the 55,000 dalton beta-receptor polypeptide.

Differences in the forskolin activation of adenylate cyclases in wild-type and variant lymphoma cells.

The ability of the diterpene forskolin to stimulate cyclic AMP accumulation in intact cell and membrane preparations of wild-type S49 lymphoma cells (WT) and a number of variants has been confirmed. Additionally, a number of salient new findings have emerged: (a) A time delay in forskolin stimulation of cyclic AMP accumulation and adenylate cyclase (t 1/2 approximately equal to 1.5 min) occurred in all hormone-sensitive WT and variant cell and membrane preparations tested. (b) The time delay was missing in the adenylate cyclase-deficient variant (cyc-) of the S49 lymphoma cell, which also lacks functional adenylate cyclase-coupling proteins. (c) The simultaneous addition of epinephrine and forskolin to WT cells or to membrane preparations eliminated the time delay. (d) Forskolin stimulation of intact WT cells did not appear to desensitize adenylate cyclase. (e) The activation of WT adenylate cyclase by forskolin was biphasic with respect to concentration, with both high- and low-affinity components being apparent. In cyc-, only the low-affinity component was detected.

Specific muscarinic-cholinergic desensitization in the neuroblastoma-glioma hybrid NG108-15.

The cholinergic agonist carbachol, epinephrine, and the opiate morphine all inhibit prostaglandin E1 (PGE1)-stimulated adenylate cyclase in homogenates from the neuroblastoma-glioma hybrid NG108-15. Pretreatment of the hybrid with 100 microM carbachol resulted in the rapid loss (desensitization) of the carbachol inhibition of adenylate cyclase (t1/2 less than 3 min). The desensitization of the carbachol inhibition was blocked by 0.1 microM atropine. Pretreatment with carbachol (1-24 h) did not significantly affect the inhibition of adenylate cyclase by either epinephrine or morphine, nor did it alter the PGE1-stimulated activity, that is, no supersensitization was observed. Cholate extracts of the particulate fraction from either carbachol-desensitized or of control NG108-15 were able to reconstitute adenylate cyclase activities of the coupling proteins (G/F)-deficient cyc- lymphoma cell membranes with equal efficacy. These results suggested that the coupling proteins of the adenylate cyclase were not altered by the carbachol pretreatment and that desensitization occurs at the receptor or at a receptor-associated level. However, the possibility remained that specific domains of the G/F, which interact only with muscarinic receptors, were altered.

Direct evidence for the role of the coupling proteins in forskolin activation of adenylate cyclase.

Forskolin stimulation of adenylate cyclase in wild type (WT) S49 lymphoma membrane preparations exhibited a lag and biphasic kinetics as a function of the forskolin concentration (i.e., both a high and low affinity component were observed). In contrast to WT, forskolin stimulation of cyc- adenylate cyclase in membranes demonstrated no observable lag and only the low affinity component. Both the lag and the high affinity component characteristic of forskolin activation of WT were observed in cyc- reconstituted with cholate extracts of WT which contained the G/F protein. The potency of forskolin stimulation of reconstituted adenylate cyclase was increased still further if the reconstitution was carried out with epinephrine and Gpp(NH)p. The Vmax of the forskolin stimulation of adenylate cyclase was approximately the same in the reconstituted and unreconstituted cyc-. In addition to the experiments with reconstituted cyc-, we have demonstrated that forskolin increased the apparent affinity of epinephrine for activation of adenylate cyclase in WT, and reciprocally, epinephrine increased the apparent affinity of forskolin for activation. We conclude that the lag, biphasic kinetics of forskolin activation and the synergism of hormone and forskolin activation of WT are attributable to functional G/F and are consistent with the forskolin stabilization of the activated catalytic unit of adenylate cyclase.

beta-Adrenergic receptors and cyclic AMP responses to epinephrine in cultured human fibroblasts at various population densities.

The beta-adrenergic receptors of the intact human lung diploid fibroblast line Wl-38 and an SV-40 transformed clone of Wl-38, Wl-38-VA-13-2RA (VA13), were estimated in experiments utilizing the beta-adrenergic ligand, 125l-hydroxybenzylpindolol (125IHYP). When specific 125IHYP binding was measured in cells grown to relatively low population densities (0.15x10(6)cells/35mm dish), both Wl-38 and VA13 cells had approximately 40,000 beta-adrenergic receptors per cell. Wl-38 cells, when cultured to a high population density (0.5x10(6) cells/35/mm dish) had clearly diminished numbers of beta-adrenergic receptors and greatly decreased cAMP responses to epinephrine stimulation. On the other hand, in VA13 cells, neither the receptor number nor the beta-adrenergic response was affected by cell population density. In Wl-38 cells, the diminished cAMP response to epinephrine paralleled the decrease in number of beta-adrenergic receptors. Prostaglandin E1 (PGE1) stimulation of cAMP levels was unaffected by cell population density in either Wl-38 or VA13 cells. Thus, increased cell population density, perhaps related to density dependent inhibition of growth, caused a specific diminution in 125IHYP binding concomitant with decreased cAMP responses to epinephrine.

Adenylate cyclase coupling proteins are not essential for agonist-specific desensitization of lymphoma cells.

Epinephrine or prostaglandin E1 pretreatment of intact S-49 lymphoma cells specifically desensitized adenylate cyclase activity. Cholate extracts of membranes from these desensitized cells showed no difference from controls in their reconstitution of hormone-stimulated adenylate cyclase activity in the adenylate cyclase-deficient variant (cyc-) of the lymphoma cell line. Adenylate cyclase in the cyc- variant was also specifically desensitized by epinephrine in prostaglandin E1. The desensitization was assessed by reconstituting the cyc- adenylate cyclase with cholate extracts of the wild type lymphoma cells. Since the cyc- has been shown to lack the coupling or G/F proteins of the adenylate cyclase complex, and these proteins are restored by cholate extracts from wild type cells, we conclude that the G/F proteins are not altered or necessary for specific desensitization of the lymphoma cell lines.

Hydrolysis of soybean trypsin inhibitor by the gamma subunit of 7SNGF and EGF-BP.

Although soybean trypsin inhibitor (STI) does not inhibit the esterase activity of either epidermal growth factor binding protein (EGF BP) or the gamma subunit of 7SNGF, it does behave as a substrate for proteolysis. Cleavage of the active site peptide bond of STI does occur when incubated in the presence of either EGF-BP or the gamma subunit of 7SNGF. The hydrolysis id pH dependent with maximum proteolysis at pH 6.0-7.0. the newly formed C-terminal arginine residue in modified STI can be released by carboxypeptidase B digestion. Both enzymes are inhibited by low concentrations (2-4 microgram/ml) of the microbial protease inhibitors leupeptin and antipain. These inhibitors are specific for trypsin-like proteases. Since both enzymes can be found as part of high molecular weight complexes with growth factors these results confirm the hypothesis that they are involved during a postranslational modification event.

Epinephrine desensitization of adenylate cyclase from cyc- and S49 cultured lymphoma cells.

The characteristics of the specific beta-adrenergic desensitization of adenylate cyclase from the adenylate cyclase deficient lymphoma cell line (cyc-) and the wild type S49 (WT) are presented in detail in this report. We have previously shown that the cyc- adenylate cyclase desensitized with 1-3 hr pretreatment of the cells with the beta-adrenergic agonist epinephrine. Adenylate cyclase of cyc- was measured after reconstitution with cholate extracts of the coupling proteins (G/F) from WT. We have now demonstrated that: (i) the initial epinephrine-induced desensitization of adenylate cyclase from either cyc- or WT was similar and occurred rapidly, with a half-life of approximately 2 min, although WT desensitized to a greater extent with prolonged hormone treatment (18 hr pretreatment of cyc- or WT with 0.1 mM terbutaline resulted in a 92% desensitization of the WT adenylate cyclase and only a 48% desensitization of cyc-); (ii) the 60 min epinephrine desensitization of cyc- was reversed after addition of propranolol and continued (40-60 min) incubation, while that of WT was only partially reversed; (iii) similar concentrations of epinephrine (0.2-0.4 muM) were required for half-maximal desensitization of both cell lines; and (iv) the Kact for epinephrine stimulation of reconstituted adenylate cyclase from cyc- or WT was increased after a 1 hr desensitization with 10 muM epinephrine. Kact was approximately 5-fold less than the half-maximal concentration required for desensitization. The data support the conclusion that the mechanisms of the rapid, reversible specific beta-adrenergic desensitization of adenylate cyclase in cyc- and WT cells are similar and occur independently of the G/F proteins of the adenylate cyclase complex, whereas the slower, apparently irreversible phase of desensitization occurs only in WT.