In vitro bacterial adherence to ventriculoperitoneal shunts.

Bacterial adherence to medical devices has been recognized as an important initial step in the infectious process, but it has not been fully elucidated regarding ventriculoperitoneal (VP) shunts. The aim of the present study was to quantitatively determine the adherence in vitro of bacteria known to cause VP shunt infections and to identify factors affecting the process. Clinical isolates studied included Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus pneumoniae, and Escherichia coli. Adherence was examined quantitatively per square centimeter, visualized by electron microscopy and related to slime production and hydrophobicity. Although all four strains adhered to VP shunts, there were marked differences, with S. epidermidis and S. aureus showing the highest adherence (67.0 x 10(3) and 15.2 x 10(3) bacteria/cm(2), respectively). Factors affecting adherence included incubation time and temperature, bacterial concentration, device material (lower for silicone than Teflon), slime production and hydrophobicity. These data might be helpful for devising novel strategies to reduce VP shunt infections.

Nitric oxide levels in preterm and term infants and in premature infants with bacteremia.

OBJECTIVE: To determine the serum nitric oxide levels in healthy neonates and in infants with bacteremia. METHODS: We performed a prospective study in a tertiary neonatal intensive care unit. The serum nitric oxide levels were measured in all infants at birth (basal) and in the infected neonates also on the first 2 days of bacteremia. RESULTS: Thirty-three neonates (10 term, 23 preterm) were included. Eleven preterm infants (mean gestational age 27 weeks) had bacteremia. The main blood culture isolates included coagulase-negative staphylococci (n=4), Klebsiella pneumoniae (n=3), and Escherichia coli (n=3). The serum nitric oxide levels increased during infection in 10 infants (p <0.008). The mean nitric oxide level before infection was 44 microM and during infection 96 microM (p=0.008). In the healthy babies, the mean nitric oxide level was 26 microM in those with a gestational age <27 weeks, 44 microM in those born between 28 and 36 weeks of gestation, and 63 microM in term infants. CONCLUSIONS: Bacteremic preterm infants produce significantly higher amounts of nitric oxide. The basal nitric oxide levels at birth may be correlated with gestational age.

Retrospective analysis of the first clonal outbreak of nalidixic acid-resistant Shigella sonnei shigellosis in Israel.

Reported here is a retrospective molecular analysis of the isolates recovered from the first outbreak of nalidixic acid (NA)-resistant Shigella sonnei shigellosis to occur in Israel. The outbreak affected 94 children. In the retrospective analysis, a total of 13 NA-resistant isolates and five NA-susceptible isolates recovered during the outbreak period were examined. Restriction fragment length polymorphism profiles obtained by digestion with BamHI, PvuI, HinfI or SmaI yielded identical profiles for all 18 isolates. All NA-resistant strains had an identical plasmid profile, but this profile differed from that displayed by the susceptible strains. In all of the NA-resistant strains a 304 bp fragment in the gyrA gene coding for a region associated with NA resistance was sequenced and showed a single point mutation, Ser83-->Phe. In this outbreak, the isolates of NA-resistant Shigella sonnei belonged to a single clone and NA resistance was associated with a point mutation in the gyrA gene.

Interaction between paracrine tumor necrosis factor-alpha and paracrine angiotensin II during myocardial ischemia.

OBJECTIVES: The purpose of this study was to explore interactions between paracrine angiotensin II (Ang-II) and tumor necrosis factor-alpha (TNF-alpha) during myocardial ischemia. BACKGROUND: Ischemic myocardium releases significant amounts of TNF-alpha. This paracrine release correlated with postischemic myocardial injury. Other studies showed myocardial protection obtained by the use of angiotensin-converting enzyme inhibitors (i.e., captopril) and the Ang-II type 1 receptor antagonist losartan after ischemia. The possibility that these agents decrease TNF-alpha synthesis has not yet been investigated. METHODS: Using the modified Langendorff model, isolated rat hearts underwent either 90 min of nonischemic perfusion (control group) or 1 h of global cardioplegic ischemia. In both groups, either captopril (360 micromol/liter) or losartan (182.2 micromol/liter) was added before ischemia. The hearts were assayed for messenger ribonucleic acid (mRNA) expression and effluent TNF-alpha levels. In addition, cardiac myocytes were incubated in cell culture with Ang-II. RESULTS: After ischemia, TNF-alpha mRNA expression intensified from 0.63 +/- 0.06 (control group) to 0.92 +/- 0.12 (p < 0.03), and effluent TNF-alpha levels were 711 +/- 154 pg/ml. The TNF-alpha mRNA expression declined to 0.46 +/- 0.07 (p < 0.01) and 0.65 +/- 0.08 (p < 0.02) in captopril- and losartan-treated hearts, respectively. Effluent TNF-alpha was below detectable levels. Concentrations of TNF-alpha in supernatants of incubated cardiac myocytes treated with 10 and 50 nmol/liter of Ang-II were 206.0 +/- 47.0 pg/ml and 810 +/- 130 pg/ml, respectively (p < 0.004). When pretreated with 700 micromol/liter of losartan, TNF-alpha was below detectable levels. CONCLUSIONS: This study presents an original explanation for previously reported myocardial protection after ischemia, obtained by the use of captopril and losartan. These drugs reduce TNF-alpha synthesis, providing strong evidence of active interactions between paracrine TNF-alpha and Ang-II in the evolution of the ischemic cascade.

Induction of mRNA for tumor necrosis factor-alpha and interleukin-1 beta in mice brain, spleen and liver in an animal model of Shigella-related seizures.

BACKGROUND: The pathogenesis of neurological symptoms, the most common extraintestinal complication of childhood shigellosis, is unclear. To elucidate the mechanisms involved, we developed an animal model and demonstrated that TNF alpha and IL-1 beta play a role. OBJECTIVES: To determine whether TNF alpha and IL-1 beta genes are expressed in the brain following peripheral administration of Shigella dysenteriae 60R. METHODS: Expression of mRNA for TNF alpha and IL-1 beta was examined in the brain structures (hypothalamus and hippocampus) and peripheral organs by reverse transcriptase polymerase chain reaction, at different time points after intraperitoneal injection of S. dysenteriae sonicate. RESULTS: In our animal model of Shigella-related seizures, TNF alpha and IL-1 beta mRNA were induced in the brain, spleen and liver already 1 hour after injection of S. dysenteriae sonicate. The expression of TNF alpha and IL-1 beta mRNA in spleen, hippocampus and hypothalamus decreased after 6 h and increased again at 18 h post-injection. CONCLUSIONS: Local production of TNF alpha and IL-1 beta in the brain may be involved in the enhanced seizure response of mice after administration of S. dysenteriae. It is possible that intracerebral production of TNF alpha and IL-1 beta plays a role in neurological disturbances of human shigellosis.

Role of nitric oxide in the enhancement of pentylenetetrazole-induced seizures caused by Shigella dysenteriae.

Convulsions and encephalopathy are frequent complications of childhood shigellosis. We studied the role of nitric oxide (NO) in Shigella-related seizures in an animal model. Pretreatment of mice with Shigella dysenteriae 60R sonicate elevated serum NO levels and enhanced the convulsive response to pentylenetetrazole (PTZ), as indicated by a higher mean convulsion score and a higher number of mice responding with seizures. Treatment of the mice with S-methylisothiourea sulfate (SMT), a potent inhibitor of inducible NO synthase (NOS), prevented the elevation of serum NO levels and concomitantly reduced the enhanced response to PTZ. The mean convulsion scores were 0.7, 0.7, 1.3, and 0.8 for mice treated with saline, saline and SMT, S. dysenteriae 60R sonicate, and S. dysenteriae 60R sonicate with SMT, respectively (P = 0.001 for 60R sonicate versus saline and P = 0.013 for 60R sonicate versus 60R sonicate with SMT). The corresponding seizure rates were 40, 44, 75, and 47% for saline, saline with SMT, S. dysenteriae 60R sonicate, and S. dysenteriae 60R sonicate with SMT, respectively (P = 0.0004 for 60R sonicate versus saline and P = 0.005 for 60R sonicate versus 60R sonicate with SMT). In contrast, injection of N-nitro-L-arginine, a selective inhibitor of constitutive NOS, neither abolished the elevation of serum NO nor attenuated the enhancement of seizures. These findings indicate that NO, induced by S. dysenteriae 60R sonicate, is involved in enhancing the susceptibility to seizures caused by S. dysenteriae.

Involvement of tumor necrosis factor alpha and interleukin-1beta in enhancement of pentylenetetrazole-induced seizures caused by Shigella dysenteriae.

Neurologic manifestations, mainly convulsions, are the most frequent extraintestinal complications of shigellosis. We used an animal model to study the roles of tumor necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1beta) in Shigella-related seizures. Administration of Shigella dysenteriae 60R sonicate enhanced the sensitivity of mice to the proconvulsant pentylenetetrazole (PTZ) within 7 h. This was indicated by a significantly higher mean convulsion score and an increased number of mice responding with clonic-tonic seizures in the Shigella-pretreated group. Preinjection of mice with anti-murine TNF-alpha (anti-mTNF-alpha) or anti-murine IL-1beta (anti-mIL-1beta) 30 min prior to administration of Shigella sonicate abolished their enhanced response to PTZ at 7 h. Mean convulsion scores were reduced by anti-mTNF-alpha from 1.2 to 0.8 (P = 0.017) and by anti-mIL-1beta from 1.3 to 0.7 (P = 0.008). Preinjection of anti-mTNF-alpha also reduced the percentage of mice responding with clonic-tonic seizures, from 48 to 29% (P = 0.002), and preinjection of anti-mIL-1beta reduced it from 53 to 21% (P = 0. 012). Neutralization of TNF-alpha or IL-1beta did not protect the mice from death due to S. dysenteriae 60R. These findings indicate that TNF-alpha and IL-1beta play a role in the very early sensitization of the central nervous system to convulsive activity after S. dysenteriae administration. Similar mechanisms may trigger neurologic disturbances in other infectious diseases.

Anti-tumor necrosis factor-alpha improves myocardial recovery after ischemia and reperfusion.

OBJECTIVES: This study sought to assess the importance of locally released or paracrine myocardial tumor necrosis factor-alpha (TNF-alpha) in the evolution of postischemic myocardial dysfunction and to use immunohistochemical studies to localize TNF-alpha within the myocardium. BACKGROUND: TNF-alpha is implicated as a systemic mediator in the development of myocardial ischemia-reperfusion injury by promoting leukocyte myocardial infiltration, and it has been shown to originate from noncardiac peripheral mononuclear cells. We have recently documented in a blood-free environment the release of TNF-alpha from the ischemic-reperfused myocardium. METHODS: Isolated rat hearts undergoing 1 h of global cardioplegia-induced ischemia and 30 min of reperfusion were investigated with use of the modified Langendorff model. Hearts were randomly divided into three subgroups: group A, control group; and groups B and C, isolated hearts receiving cardioplegic solution containing monoclonal hamster antimurine TNF-alpha antibodies (group B) or hamster IgG (group C). RESULTS: Significant amounts of TNF-alpha were detected in group A and group C effluent on 1 min of reperfusion (752 +/- 212 and 958 +/- 409 pmol/ml, respectively). However, in group B, TNF-alpha was below detectable levels. In this group, postischemic left ventricular peak systolic pressures, first derivative of the rise in left ventricular pressure (dP/dtmax), pressure-time integral, coronary flow and O2 consumption improved (analysis of variance [ANOVA] p < 0.0001 for all variables) compared with values in groups A and C; creatine kinase levels decreased (p < 0.005); and myocardial structure was preserved. Immunohistochemical staining localized TNF-alpha to cardiac myocytes and to endothelial cells. CONCLUSIONS: Anti-TNF-alpha neutralizes local TNF-alpha release from cardiac myocytes after ischemia and improves myocardial recovery during reperfusion, indicating that postischemic paracrine TNF-alpha release plays an active role in myocardial dysfunction.

Induction of tumor necrosis factor and nitric oxide by Shigella strains isolated from patients with or without neurologic manifestations.

The pathogenesis of the Shigella-associated neurological symptoms is unclear. We examined the potential role of host factors. Sonicates of Shigella strains isolated from children with and without neurologic disturbances were compared regarding their ability to induce tumor necrosis factor (TNF) and nitric oxide (NO) in vitro, in mouse macrophage J744 cell line. The mean concentrations of TNF (14.6 vs. 4.4 ng/ml) and NO (7.4 vs. 3.7 microM) induced were higher in response to strains isolated from children with neurologic complications; the differences were not statistically significant. TNF was also measured in plasma of children with shigellosis, and was found to be elevated in all patients. The mean concentration of TNF in plasma of children with neurologic manifestations was higher than that of children with no neurologic symptoms (450 vs. 138 pg/ml, P <0.05). It is concluded that TNF and NO may play a role in the development of neurologic manifestations of shigellosis.

Induction of nitric oxide production in mouse macrophages by Shiga toxin.

Host mediators play an important role in the pathogenesis of shigellosis and Shiga toxin toxicity. Nitric oxide (NO) production in mouse peritoneal macrophages and in the macrophage J744 cell line in response to purified Shiga toxin and lipopolysaccharide (LPS) from Shigella flexneri were studied. Shiga toxin induced NO production in a dose-dependent manner up to 800 ng/ml. Detectable levels of NO were present as early as 4 h after induction and continued to increase during 72 h; Shiga toxin induced greater NO production with time than did LPS. Pre-treatment of Shiga toxin (400 ng/ml) or LPS (10 ng/ml) with polymyxin B, which inactivates LPS, reduced their ability to induce NO by 28% and 96%, respectively. Induction in the presence of anti-TNF alpha antibodies did not reduce the amount of NO in the supernate. These studies showed that Shiga toxin induces NO production in murine macrophages.

Tumor necrosis factor-alpha is released from the isolated heart undergoing ischemia and reperfusion.

OBJECTIVES: The purpose of this study was to examine whether tumor necrosis factor-alpha (TNF-alpha) is released directly from the ischemic myocardium undergoing reperfusion. BACKGROUND: Tumor necrosis factor-alpha is a protein hormone produced by systemic leukocytes (primarily by activated macrophages). It has been implicated as a systemic mediator in the development of septic shock and other pathologic conditions. Serum TNF-alpha has also been detected in a variety of cardiac disease states and after myocardial ischemia-reperfusion injury. METHODS: Nine isolated rat hearts undergoing 30 min of perfusion, followed by warm cardioplegic arrest, 1 h of global ischemia and 30 min of reperfusion, were investigated using the modified Langendorff model. RESULTS: Significant amounts of TNF-alpha (752 +/- 212 pmol/ml) were detected in the effluent during the first minute of reperfusion. Tumor necrosis factor-alpha levels correlated with postischemic deterioration in peak systolic pressures (r = 0.7882, p = 0.012), dP/dt max (r = 0.6795, p = 0.044), time-pressure integral (r = 0.7661, p = 0.0016) and postischemic creatine kinase levels (r = 0.8367, p = 0.005). The deterioration in coronary flow, however, was inversely correlated with TNF-alpha levels (r = -0.7581, p = 0.018). CONCLUSIONS: To our knowledge, this study is the first to suggest that the isolated rat myocardium synthesizes and releases TNF-alpha in response to ischemia and reperfusion, which directly correlates with the postischemic deterioration in myocardial mechanical performance and the amount of cellular necrosis.

An animal model for the study of neurotoxicity of bacterial products and application of the model to demonstrate that Shiga toxin and lipopolysaccharide cooperate in inducing neurologic disorders.

An approach for studying neurotoxicity of bacterial products is presented. Pentylenetetrazol, a convulsant drug, was injected into mice, and increased sensitivity to pentylenetetrazol was used as an indicator of neurotoxicity. The preinjection of sonicates of Shigella dysenteriae 60R or Escherichia coli H30 (producing Shiga toxin or Shiga-like toxin I, respectively) enhanced the response of mice to pentylenetetrazol within 6 h. This was indicated by a higher mean convulsion score, increased number of mice responding with convulsions, and induction of seizures in animals pretreated with a subepileptic dose of pentylenetetrazol. Preinjection of purified Shiga toxin significantly changed the response to pentylenetetrazol only when coadministered with bacterial lipopolysaccharide (LPS); mean convulsion scores were 1.6 and 0.9 for the Shiga toxin-LPS group and controls, respectively. LPS alone did not affect sensitivity to pentylenetetrazol. These results suggest that Shiga toxin and LPS together induce neurologic disorders early in the course of infection.

The effect of shiga toxin and sonicates of Shigella isolates from children with neurologic manifestation on neuroblastoma cell lines.

Although neurologic manifestations are frequent during childhood shigellosis, their pathogenesis is unclear and controversial. Shiga toxin and other cytotoxins are often implicated, but their effect on neuronal cells has not been determined. We examined the effect of purified Shiga toxin and sonicates of Shigella isolates from children with neurologic symptoms on well-characterized human neuroblastoma cells in vitro. Quantitative determinations showed high cytotoxicity of Shiga toxin on HeLa cells (1.2 x 10(6) CD50/mg purified toxin), but no effect on LA-N-1, LA-N-5 and IMR neuroblastoma cell lines. Pretreatment with tumor necrosis factor, which increases expression of the Shiga toxin receptor, globotriosyl ceramide, in endothelial cells and enhanced Shiga toxin cytotoxicity, did not affect the susceptibility of neuroblastoma cells to the toxin. Low dilutions (up to 1:16-1:64) of sonicates of Shigella isolates from children with neurologic symptoms caused agglutination of neuroblastoma cells, but no cell killing was observed morphologically. This study shows that Shiga toxin does not exhibit cytotoxic activity on the human neuroblastoma cell lines examined, neither do sonicates of relevant Shigella strains. The mechanism and significance of the agglutination activity on neuroblastoma cells should be further studied.

Glycerol induced ARF in rats is mediated by tumor necrosis factor-alpha.

Glycerol-induced acute renal failure (ARF) in rats is a model of acute trauma in which intra-muscular injection of 50% glycerol causes rapid myoglobinuria, oliguria, and a rapid reduction in glomerular filtration rate. We found that plasma tumor necrosis factor-alpha (TNF-alpha) is rapidly induced in glycerol injected rats. It can be detected in some animals as early as 30 minutes post-injection, peaks at one hour (range: 4 to 32 U/ml) with no significant difference between blood from renal vein and vena cava, and decreases by three hours. None was detected in control saline injected rats (P < 0.001). Four out of five rats infused with neutralizing anti-TNF-alpha antiserum (200 microliters/300 g body wt) immediately prior to glycerol injection had significantly protected kidney function (P = 0.001). In these rats, plasma urea (104.8 +/- 58.9 mg%) and creatinine (1.16 +/- 0.38 mg%) were lower and creatinine clearance higher (0.34 +/- 011 ml/min) than in glycerol injected animals pretreated with normal serum (291.8 +/- 41.8 mg%, 3.15 +/- 0.74 mg%, and 0.03 +/- 0.03 ml/min, respectively) or animals injected with glycerol alone (302.6 +/- 76.8 mg%, 3.45 +/- 0.97 mg%, and 0.03 +/- 0.03 ml/min, respectively). These results imply a direct role for TNF-alpha in pathogenesis of glycerol induced ARF in rats.

Inhibition of tumor necrosis factor-induced cell killing by tryptophan and indole.

Cells sensitive to the cytocidal effect of tumor necrosis factor (TNF) were protected against this effect when growth in the presence of elevated concentrations of tryptophan. Several other indole derivatives also provided protection against TNF cytotoxicity. Most effective were indole itself and its monomethyl derivatives, providing a degree of protection greatly exceeding that observed with tryptophan. Protection was also observed against the cytocidal effect of TNF applied in the presence of a protein synthesis inhibitor. The protective effect of tryptophan was largely dependent on preexposure of the cells, for several hours, to a high concentration of this amino acid. On the other hand, indole was protective also when applied to cells together with TNF, or even two hours after TNF application. The inhibition of the cytotoxicity of TNF by tryptophan and other indole derivatives may serve as a useful experimental tool in exploring the mechanisms and the physiological implications of TNF cytotoxicity.

Presence of an anti-viral factor in peritoneal dialysis effluent.

Viral peritonitis is an exceptionally rare occurrence in peritoneal dialysis. In fact, up to now, only one case report has been documented in the literature. In a prospective study, peritoneal dialysis effluent (PDE) was specifically cultured for the following viruses: the herpes group of viruses, including herpes simplex types I (HSV) and II, cytomegalovirus (CMV) and varicella-zoster (V-Z), and the enteroviruses group including coxsackie B-5 (Cox B), echo, enterovirus and polio. Cultures were performed under both basal conditions and in the presence of peritonitis. No viral growth was demonstrated. The possible existence of an anti-viral factor in the PDE was therefore raised. In order to investigate this hypothesis, the PDE of 16 patients undergoing intermittent peritoneal dialysis and of 24 patients on continuous ambulatory peritoneal dialysis were examined for anti-viral activity. The method used was analogous to that employed for testing the anti-viral effect of interferon (IFN). The inhibition of the cytopathic effect (CPE) of various viruses was examined in the following tissue cultures: Vero cells (a line of monkey kidney cells) incubated with HSV, vesicular stomatitis virus (VSV) and Cox B; human kidney cells incubated with parainfluenza 3 (Para-3); human foreskin fibroblasts incubated with CMV, HSV and VSV and L-929 (a line of mouse cells) incubated with VSV. As control, unused Dianeal (Travenol, Ashdod, Israel) 1.5 and 4.25 g/dl, normal saline and 5 g/dl dextrose solutions were tested under the same conditions using VSV on Vero. The PDE was also examined for the presence of specific anti-viral antibodies by microneutralization and ELISA tests.(ABSTRACT TRUNCATED AT 250 WORDS)