Clozapine Suppresses the Gene Expression and the Production of Cytokines and Up-Regulates Cyclooxygenase 2 mRNA in Human Astroglial Cells.

Schizophrenia (SCZ) is a chronic neurodevelopmental psychotic disorder. The immune system and neuroinflammation seem to play a central role in the pathophysiology of SCZ. Clozapine is an effective atypical antipsychotic used for treatment-resistant SCZ. Life-threatening side effects, such as myocarditis, limit its use. We investigated the immunomodulatory effects of clozapine in an astroglial model of neuroinflammation. We thus assessed the effect of clozapine on the production of inflammatory mediators in human-derived astroglial (A172) cells, stimulated with a cytokine mix (TNFα, IL-1ß, IFNγ). RT-PCR and ELISA analyses demonstrated that clozapine suppressed gene expression and production of TNFα, IL-1ß and IL-8 and increased COX2 mRNA 24 h after stimulation. Clozapine inhibited Akt phosphorylation induced by the cytokine mix at 10 min and 40 min, as assessed by Western blot analysis with anti-pT308Akt antibody. Pretreatment with the Akt inhibitor MK-2206 increased COX2 gene expression in cytokine-stimulated cells, suggesting that Akt inhibition may be involved in COX2 gene expression upregulation. Clozapine may possess dual beneficial effects: inhibiting astroglial production of proinflammatory cytokines, thus attenuating neuroinflammation, and upregulating COX2 expression that may be relevant to improvement of neural functioning while accounting for some of its detrimental effects. Patients with TRS and neuroinflammatory markers may benefit particularly from clozapine treatment.

Differential Serum and Urine CRP, IP-10, and TRAIL Levels in Pediatric Urinary Tract Infection.

Background: It is estimated that clinical evaluation and urinalysis are unable to diagnose >10% of urinary tract infections (UTI) in young children. TNF-related apoptosis induced ligand (TRAIL), interferon gamma induced protein-10 (IP-10), and C-reactive protein (CRP) exhibit differential expression in the blood in response to bacterial vs. viral infection. We assessed if the urinary and serum levels of these host biomarkers discriminate UTI, nephronia, and response to antibiotic treatment. Methods: Hospitalized febrile children aged <18 years with suspected UTI based on abnormal urinalysis were recruited prospectively between 2016 and 2018; also, non-febrile controls were recruited. Following urine culture results and hospitalization course, participants were divided into three groups based on AAP criteria and expert adjudication: UTI, viral infection, and indeterminate. Results: Seventy-three children were enrolled, 61 with suspected UTI and 12 non-febrile controls. Of the 61 with suspected UTI, 40 were adjudicated as UTI, 10 viral infection, and 11 as indeterminate. Urinary CRP and IP-10 levels were significantly higher in the UTI group (p ≤ 0.05). Urinary CRP differentiated UTI from non-bacterial etiology in children under and over 3 months of age, with AUCs 0.98 (95% CI: 0.93-1.00) and 0.82 (0.68-0.95), respectively. Similarly, urinary IP-10 discriminated with AUCs of 0.80 (0.59-1.00) and 0.90 (0.80-1.00), respectively. Serum CRP and IP-10 levels were significantly higher in UTI cases with nephronia (p ≤ 0.03). UTI-induced changes in the levels of urinary and serum biomarkers resolved during recovery. Conclusions: CRP, IP-10, and TRAIL represent biomarkers with potential to aid the clinician in diagnosis and management of UTI.

Ketamine upregulates eNOS expression in human astroglial A172 cells: Possible role in its antidepressive properties.

Ketamine is a potent anti-depressive agent. Nitric oxide plays an essential role in neuronal transmission and cerebral blood flow and has been implicated in the pathophysiology of major depressive disorder as well as cardiovascular functioning. We investigated the effect of ketamine on eNOS expression in human A172 astroglial cells. Ketamine (50-500µM) increased eNOS expression at 4-24h in a concentration-dependent manner. This effect was mediated by NMDA receptor, Akt inhibition and ERK1/2 activation and was synergistically augmented by rapamycin. The combined effect on the vascular, immune and neuronal systems may be relevant to the rapid antidepressive effect of ketamine.

Immunomodulatory activity of ketamine in human astroglial A172 cells: Possible relevance to its rapid antidepressant activity.

To determine if the immunomodulatory effect of ketamine is relevant to its rapid antidepressant activity, cultured human astroglial cells were incubated with ketamine, cytokine mix, or both. At 24h, ketamine dose-dependently (100-500 µM) decreased IL-6 and TNFα production and gene expression and, at clinically relevant concentration (100 µM), augmented IL-ß release and gene expression in both unstimulated and cytokine-stimulated cells. In unstimulated cells, ketamine also increased IL-8 production and mRNA expression. The reduction in IL-6 mRNA was significant within 1h in unstimulated cells and at 4h after stimulation. Ketamine suppressed the production of the only established depression-relevant proinflammatory cytokines, IL-6 and TNFα.

Apnea induced by respiratory syncytial virus infection is not associated with viral invasion of the central nervous system.

We aimed to study whether direct central nervous system invasion is responsible for the neurologic manifestations seen in hospitalized infants with respiratory syncytial virus (RSV) infection. Cerebrospinal fluid from infants with RSV infection was tested for the detection of the following respiratory RNA viruses: RSV, influenza A and B, pandemic influenza H1N1, Parainfluenza-3, human metapneumovirus, adenovirus, parechovirus and enterovirus. All children tested negative for the presence of viral material in the cerebrospinal fluid. Our results support the notion that the mechanism of RSV-induced neurologic manifestations, including apnea, is not direct central nervous system invasion.

Effect of nitric oxide on microRNA-155 expression in human hepatic epithelial cells.

OBJECTIVE: Nitric oxide (NO) is a signaling molecule and regulator of immunity and inflammation. MicroRNAs (miRNAs) regulate gene transcription and are involved in inflammatory processes and cancer. This study sought to determine if NO activity affects miRNA expression. METHODS: Human liver epithelial (HepG2) cells were treated with the NO-releasing S-nitroso-N-acetylpenicillamine (SNAP) 100 µM for 4 h and subjected to microarray analysis. To examine the underlying mechanisms, cells were exposed to cGMP analog 8-bromo-cGMP, protein kinase inhibitor Rp-*-Br-PET-cGMPS (Rp-PET), or nitric synthase inhibitor L-NAME and evaluated with RT-PCR. RESULTS: MiR-155 was the only miRNA of the 887 arrayed that showed a change in expression after SNAP treatment. Incubation of the cells with 8-bromo-cGMP increased miR-155 expression 4.0 ± 0.7-fold (p < 0.05); Rp-PET before SNAP had a dual, concentration-dependent effect. SNAP treatment induced a 3.1 ± 0.7-fold change in miRNA-155 expression, Rp-PET 25 µM, a 7.3 ± 2.2-fold change, and Rp-PET 100 µM, a 0.79 ± 0.09-fold change (SNAP vs SNAP + Rp-PET, p < 0.05). In unstimulated cells, Rp-PET or L-NAME treatment increased miR-155 expression by 3.5 ± 0.7-fold and 5.6 ± 2.2-fold, respectively (p < 0.05). CONCLUSION: In HepG2 cells, exogenous NO increases miR-155 expression, but endogenous basal NO inhibits it. Both effects are mediated via cGMP/PKG signaling. The upregulation of miR-155 by NO provides a new link between NO, inflammation, and cancer.

Effect of rifampin on production of inflammatory mediators in HepG2 liver epithelial cells.

Rifampin, a potent antibacterial agent, is one of the main drugs used in the treatment of mycobacterial infections. Hepatotoxicity is a well-documented adverse event. The aim of this study was to investigate the effect of rifampin on the production of inflammatory mediators in human epithelial HepG2 liver cells in the absence or presence of proinflammatory cytokines. Incubation of HepG2 cells with a cytokine mix plus rifampin was associated with a significant dose-dependent increase in the production of nitric oxide compared to incubation with the cytokine mix alone (P < 0.05) as well as with an increase in inducible nitric oxide synthase protein and mRNA expression. Rifampin significantly increased the secretion of interleukin 8 (IL-8) in both untreated cells (P < 0.001) and cytokine-treated cells (P < 0.006). An array screening assay revealed that rifampin stimulated the production of IL-1ß and gamma interferon-induced protein-10 (IP-10) in untreated cells and increased the secretion of RANTES in cytokine-treated cells. Together, these results indicate that rifampin may exert proinflammatory effects on liver cells.

Soluble triggering receptor expressed on myeloid cells-1 for diagnosing empyema.

BACKGROUND: Studies have shown that soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) is upregulated by microbial products in the bronchoalveolar lavage fluid, and cerebrospinal fluid of patients with pneumonia and bacterial meningitis, respectively. Our goal was to evaluate whether sTREM-1 in pleural fluid can distinguish pleural empyema from postthoracotomy-related pleural effusion and effusions of other etiologies. METHODS: Patients who presented with pleural effusion were identified through laboratory records. In addition to routine biochemical markers, differential white blood cells, cytology, Gram stain, and pleural fluid culture, pleural fluid sTREM-1 was measured by enzyme-linked immunosorbent assay using a commercial kit (R&D Systems, Minneapolis, MN). RESULTS: Eighty-nine patients were included in the study: 17 with empyema, 7 simple parapneumonic effusion, 18 transudate, 12 postthoracotomy pleural effusion, 22 malignancy, 1 connective tissue disease, and 12 with undetermined effusion. Mean levels of sTREM-1 were significantly higher in empyema than in postthoracotomy pleural effusion (687 +/- 479 pg/mL vs 34 +/- 81 pg/mL, p < 0.0001, respectively) and in effusions of other etiologies (15 +/- 54 pg/mL, p < 0.0001). A cutoff value of 114 pg/mL for pleural sTREM-1 achieved a sensitivity of 94% and a specificity of 93% in differentiating empyema from pleural effusions of other etiologies. The area under the receiver operating characteristic curve for pleural effusion sTREM-1 as a predictor for empyema was 0.966. CONCLUSIONS: Our findings suggest that sTREM-1 in the pleural fluid can potentially assist clinicians in the differentiation of bacterial from nonbacterial pleural effusion, particularly in postthoracotomy pleural effusion.

Roles of NF-kappaB activation and peroxisome proliferator-activated receptor gamma inhibition in the effect of rifampin on inducible nitric oxide synthase transcription in human lung epithelial cells.

Rifampin (rifampicin), an important antibiotic agent and a major drug used for the treatment of tuberculosis, exerts immunomodulatory effects. Previous studies have found that rifampin increases inducible nitric oxide (NO) synthase (iNOS) expression and NO production. The present study investigated the potential mechanism(s) underlying these actions. The incubation of human lung epithelial A549 cells with a cytokine mix (interleukin-1beta, tumor necrosis factor alpha, and gamma interferon) induced the expression of iNOS mRNA. The addition of rifampin increased the iNOS level by 1.9 +/- 0.3-fold at a dose of 10 microg/ml (P < 0.01) and by 4.0 +/- 0.3-fold at a dose of 50 microg/ml (P < 0.001). Rifampin treatment also affected the transcription factors that regulate iNOS mRNA: there was an increased and prolonged degradation of the inhibitory subunit of NF-kappaB, a corresponding increase in the level of cytokine-induced DNA binding of NF-kappaB (2.1 +/- 0.2-fold), and a decrease in the level of expression of peroxisome proliferator-activated receptor gamma (PPARgamma). Specifically, the level of PPARgamma expression dropped by 15% in response to cytokine stimulation and by an additional 40% when rifampin was added (P < 0.001). Rifampin had no effect on the activation of mitogen-activated protein kinases or the signal transducer and transcription activator (STAT-1). In conclusion, rifampin augments NO production by upregulating iNOS mRNA. It also increases the level of NF-kappaB activation and decreases the level of PPARgamma expression. The increases in the levels of NF-kappaB activation and NO production probably contribute to the therapeutic effects of rifampin. However, given the role of NF-kappaB in upregulating many inflammatory genes and the roles of PPARgamma in downregulating inflammatory genes and in lipid and glucose metabolism, these findings have implications for potential adverse effects of rifampin in patients with chronic inflammatory diseases and glucose or lipid disorders.

Rifampin inhibits prostaglandin E2 production and arachidonic acid release in human alveolar epithelial cells.

Rifampin, a potent antimicrobial agent, is a major drug in the treatment of tuberculosis. There is evidence that rifampin also serves as an immunomodulator. Based on findings that arachidonic acid and its metabolites are involved in the pathogeneses of Mycobacterium tuberculosis infections, we investigated whether rifampin affects prostaglandin E(2) (PGE(2)) production in human alveolar epithelial cells stimulated with interleukin-1beta. Rifampin caused a dose-dependent inhibition of PGE(2) production. At doses of 100, 50, and 25 microg/ml, it inhibited PGE(2) production by 75%, 59%, and 45%, respectively (P < 0.001). Regarding the mechanism involved, rifampin caused a time- and dose-dependent inhibition of arachidonic acid release from the alveolar cells. At doses of 100, 50, 25, and 10 mug/ml, it significantly inhibited the release of arachidonic acid by 93%, 64%, 58%, and 35%, respectively (P < 0.001). Rifampin did not affect the phosphorylation of cytosolic phospholipase A(2) or the expression of cyclooxygenase-2. The inhibition of PGE(2), and presumably other arachidonic acid products, probably contributes to the efficacy of rifampin in the treatment of tuberculosis and may explain some of its adverse effects.

Rifampin augments cytokine-induced nitric oxide production in human alveolar epithelial cells.

Rifampin increased nitric oxide production and inducible nitric oxide synthase expression in alveolar cells stimulated with cytokines. Nitric oxide concentrations after induction with cytokines, cytokines with 10 microg/ml rifampin, and cytokines with 50 microg/ml rifampin were 3.2, 4.5, and 8.8 microM, respectively (P < 0.02 versus cytokines alone). This indicates that rifampin modulates the immune response.

Involvement of prostaglandins in an animal model of Shigella-related seizures.

We investigated whether prostaglandins (PGs), proinflammatory mediators implicated in excitatory activity, are involved in Shigella-related seizures. Pretreatment with S. dysenteriae sonicate (2LD(50)) enhanced mice response to pentylenetetrazole-induced seizures, without increase of brain concentrations of PGE(2), PGD(2) or PGF(2alpha). Preinjection of NS-398, an inhibitor of cyclooxygenase-2, before treatment with Shigella sonicate, had no effect on seizures. The anticonvulsive PGD(2) increased after injection of 8 LD(50) of Shigella sonicate, which did not enhance seizures (32 pg/mg vs 26 pg/ml, p=0.0063). The findings indicate that PGs are not involved in the enhanced seizure response after exposure to Shigella. However, induction of PGD(2) may play an inhibitory role.

Heparin added to cardioplegic solution inhibits tumor necrosis factor-alpha production and attenuates myocardial ischemic-reperfusion injury.

OBJECTIVES: Tumor necrosis factor (TNF)-alpha is known to be a proinflammatory cytokine that has a pronounced negative inotropic effect and plays an important role in ischemic-reperfusion injury. METHODS: Twenty isolated rat hearts were randomly divided equally into two groups (heparin and non-heparin) and were perfused with a Krebs-Henseleit solution using a modified Langendorff model. The influence of heparin on the synthesis and release of TNF-alpha by isolated rat hearts after 1 h of global cardioplegic ischemia and on left ventricular (LV) performances during 30 min of postischemic reperfusion was investigated. RESULTS: Significant mean (+/- SEM) amounts of TNF-alpha in myocardial tissue (1,149 +/- 33.7 pg/g) and effluent (951.8 +/- 27.3 pg/mL) from the coronary sinus were detected after global cardioplegic ischemia. The addition of heparin to the cardioplegic solution significantly improved the recovery of LV function in the postischemic heart (p < 0.0001 for all measurements). TNF-alpha protein production in the heparin-treated hearts was below detectable levels despite a postischemic increase of TNF-alpha messenger RNA expression in both heparin-treated hearts and nontreated hearts (0.71 +/- 0.06 and 0.8 +/- 0.12 relative optical density, respectively). CONCLUSION: This study shows, for the first time, that heparin causes the inhibition of TNF-alpha protein synthesis and release from the isolated ischemic rat heart within the posttranscriptional stage, and that it prevents the depression of LV function caused by ischemic-reperfusion injury.

Involvement of the neuropeptide corticotropin-releasing hormone in an animal model of Shigella-related seizures.

Convulsions are common complications of shigellosis in children. Corticotropin-releasing hormone (CRH), a stress neurohormone, has been implicated in the susceptibility of young children to seizures. We investigated the role of CRH in Shigella-related seizures. Pretreatment with Shigella dysenteriae sonicate enhanced mice response to pentylenetetrazole (PTZ)-induced seizures. Preinjection of antalarmin, a CRH-receptor 1 antagonist, decreased both the mean convulsion score (MCS: 1.6 vs. 1.1, p<0.05) and the percent of mice having seizures (48% vs. 28%, p=0.03). This indicates that CRH plays a role in the increased susceptibility to seizures following exposure to S. dysenteriae.

Effect of tumor necrosis factor-alpha on endothelial and inducible nitric oxide synthase messenger ribonucleic acid expression and nitric oxide synthesis in ischemic and nonischemic isolated rat heart.

OBJECTIVES: The present study aimed to investigate the influence of endogenous tumor necrosis factor-alpha (TNF-alpha) that was synthesized during ischemia and exogenous TNF-alpha on endothelial and inducible nitric oxide synthase (eNOS and iNOS) messenger ribonucleic acid (mRNA) expression and nitric oxide (NO) production in the isolated rat heart. BACKGROUND: Tumor necrosis factor-alpha is recognized as being a proinflammatory cytokine with a significant cardiodepressant effect. One of the proposed mechanisms for TNF-alpha-induced cardiac contractile dysfunction is increased NO production via iNOS mRNA upregulation, but the role of NO in TNF-alpha-induced myocardial dysfunction is highly controversial. METHODS: Isolated rat hearts studied by a modified Langendorff model were randomly divided into subgroups to investigate the effect of 1-h global cardioplegic ischemia or the effect of 1-h perfusion with exogenous TNF-alpha on the expression of eNOS mRNA and iNOS mRNA and on NO production. RESULTS: After 1 h of ischemia, there were significant increases in TNF levels in the effluent (from hearts), and eNOS mRNA expression had declined (from 0.91 +/- 0.08 to 0.68 +/- 0.19, p < 0.001); but there were no changes in iNOS mRNA expression, and NO was below detectable levels. Perfusion of isolated hearts with TNF-alpha had a cardiodepressant effect and decreased eNOS mRNA expression to 0.67 +/- 0.04 (p < 0.002). Inducible nitric oxide synthase mRNA was unchanged, and NO was below detectable levels. CONCLUSIONS: We believe this is the first study to directly show that TNF-alpha does not increase NO synthesis and release but does downregulate eNOS mRNA in the ischemic and nonischemic isolated rat heart.

Bidirectional concentration-dependent effects of tumor necrosis factor alpha in Shigella dysenteriae-related seizures.

We have previously demonstrated that pretreatment of mice with Shigella dysenteriae sonicate enhanced their susceptibility to pentylenetetrazole-induced seizures and that tumor necrosis factor alpha (TNF-alpha) was proconvulsive in this respect. The present study shows that TNF-alpha, at high concentrations, may also exert a suppressive effect on Shigella-mediated seizures. This implies that high levels of TNF-alpha may play a protective role in neurologic complications of S. dysenteriae infection.

Seroepidemiology of hepatitis A antibodies among children's hospital staff.

BACKGROUND: With improved socioeconomic conditions, adults are more frequently seronegative for hepatitis A virus (HAV) and therefore susceptible to infection. A safe and efficacious active HAV vaccine has been developed and licensed. The general recommendation is to vaccinate populations at increased occupational exposure to HAV. AIM: To determine the seroprevalence of HAV antibodies among children's hospital employees and to correlate seropositivity with demographic and occupational variables as a basis for formulating vaccine recommendations. METHODS: The staff of a tertiary pediatric medical center participated by answering a structured questionnaire on demographic and occupational data and by donating venous blood for determining HAV antibodies by an enzyme immunoassay. Univariate and multivariate analyses were conducted to identify variables associated with HAV seropositivity. RESULTS: HAV antibodies were found in 48.3% of the 499 employees studied, being lowest in pediatricians (38.7%), and increased with years of work at the hospital, job percentage and contact with pediatric patients. However, multivariate analysis showed that only the sociodemographic variables (age and crowding during childhood) were independently and significantly associated with seropositivity. CONCLUSIONS: HAV seropositivity was associated mainly with sociodemographic variables. Most children's hospital employees, especially pediatricians and other young (<40 years) employees, are seronegative and therefore susceptible to HAV. Vaccination of the high risk groups should be considered.

Enhancement of pentylenetetrazole-induced seizures by Shigella dysenteriae in LPS-resistant C3H/HeJ mice: role of the host response.

Convulsions and encephalopathy are common complications of Shiga toxin (Stx)-producing Shigella and enterohemorrhagic Escherichia coli infections. In previous studies, we demonstrated that Stx and lipopolysaccharide (LPS) act in concert to enhance mice sensitivity to pentylenetetrazole (PTZ)-induced seizures via mechanisms involving tumor necrosis factor alpha (TNFalpha), interleukinl beta and nitric oxide. To further elucidate the role of the host response in Shigella-related seizures, we studied the ability of Shigella dysenteriae and its products to modulate seizures in C3H/HeJ (lps(d/d)) and in C3H/HeN (lps(n/n) mice. Injection of S. dysenteriae 60R sonicate elevated plasma TNFalpha and enhanced the convulsive response to PTZ in both mouse strains. Induced TNFalpha levels were markedly lower in LPS-hyporesponsive C3H/HeJ mice than in LPS- responsive C3H/HeN mice: 7.4 ng/ml vs 44 ng/ml (induced by 4LD50). Accordingly, a higher dose of S. dysenteriae sonicate was needed to sensitize the C3H/HeJ mice to seizures. Stx or LPS alone did not enhance seizures in either strain. Stx together with LPS enhanced seizures in LPS-responsive mice, but not in LPS-hyporesponsive mice in which they induced only a minor elevation in TNFalpha levels (1.5 ng/ml). As compared to LPS-responsive mice, the LPS-hyporesponsive mice were less susceptible to the lethal effects of Shigella sonicate and were resistant to the lethal effect of purified Stx with LPS. These results demonstrate the crucial role of the host response with regard to the sensitivity to to LPS, and specifically TNFalpha production, in Shigella lethality and Shigella-related seizures.

Risk of exposure to hepatitis A virus among day-care workers in Israel: implications for preventive measures.

Hepatitis A is an important infectious disease in Israel. The high incidence of hepatitis A, the declining prevalence of antibodies against hepatitis A virus throughout the past decade, and the availability of an active immunization for hepatitis A have engendered the identification of occupations in which workers are at increased risk of acquiring the disease. A prevalence study for hepatitis A antibodies was conducted during the first half of 1998 in a random sample of 37 day-care centers in different parts of Israel. A short interview was conducted, and serum samples were then collected from each worker. The control group comprised a sample of factory workers, blood donors, and women in maternity wards. Ninety percent (402 of 446) of the day-care workers had antibodies to hepatitis A. In a logistic-regression analysis, seropositivity was associated with increased age, minimal education, and lack of military service. When the authors compared a subsample of workers aged 18-40 yr with the control group, seropositivity was associated with the following: birth in Israel, duration of residence in Israel, very minimal education, military service, and employment in a day-care center. These results, together with earlier reports, indicate that day-care workers are at high risk of occupational exposure to hepatitis A. These results may imply that seronegative workers have a 2-fold chance of acquiring hepatitis A. Appropriate preventive measures should be considered, and active immunization of susceptible workers should be undertaken.