[show abstract][hide abstract] ABSTRACT: Background. Methicillin-resistant Staphylococcus aureus (MRSA) can stimulate massive cytokine release. Ketamine suppresses tumor necrosis factor (TNF) secretion by MRSA-stimulated RAW264.7 macrophages, and the mechanism likely involves N-methyl-D-aspartic acid (NMDA) receptor antagonism. The downstream effects of NMDA-mediated TNF suppression, specifically the PI3K/Akt and mTOR modulation, have not been described. Methods. RAW264.7 cells were stimulated for 18 hrs with 10(5) to 10(7) CFU/mL inocula of either of two prototypical community-acquired- (CA-) MRSA isolates, USA300 strain LAC and USA400 strain MW2. Then we added the NMDA inhibitors ketamine or 2R-amino-5-phosphonopentanoate (AP5), NMDA substrate, LY294002, and rapamycin in various combinations. Results. NMDA inhibition suppressed TNF secretion by almost a third compared to the no-ketamine control. When NMDA substrate was added, the TNF secretion increased by 10%. Addition of LY294002 suppressed TNF production by macrophages by 20%. Rapamycin exhibited a concentration-dependent TNF induction-suppression response: induction at doses of 0.1 and 1 ng/mL and suppression at 10 and 100 ng/mL. Induction of TNF was abolished when LY294002 was added and the suppression became uniform. Ketamine-induced suppression of TNF secretion was intensified 10-15% when rapamycin was added, but not when LY294002 was added. Conclusion. These findings suggest that NMDA-induced TNF suppression can be augmented by concurrent mTOR inhibition.
International journal of inflammation. 01/2012; 2012:542727.
[show abstract][hide abstract] ABSTRACT: We tested the contribution of four staphylococcal components - PSM-α, PSM-β, δ-toxin, and PVL - in triggering macrophage secretion of tumor necrosis factor (TNF) and interleukins 6 (IL-6) and 12 (IL-12) by two prominent, circulating strains of community-associated, methicillin-resistant Staphylococcus aureus (CA-MRSA): LAC, USA300; MW2, USA400. RAW 264.7 murine macrophages were stimulated with live, antibiotic-exposed bacteria, and cytokine secretion was quantitated in supernatants. Deletion of PSM-α expression in LAC led to >50% reduction in macrophage TNF and IL-6 secretion and a 20% reduction in IL-12 secretion, while PSM-α deletion in MW2 did not significantly reduce macrophage TNF secretion but resulted in a 15-20% reduction in IL-6 and IL-12 secretion. Deletion of δ-toxin in either strain led to more than 50% reduction in macrophage IL-6 secretion and smaller reductions in macrophage TNF and IL-12 secretion (8-25%). Our data implicate both PSM-α and δ-toxin in stimulating macrophage cytokine responses to CA-MRSA bacteria.
[show abstract][hide abstract] ABSTRACT: Infections caused by community-associated strains of methicillin-resistant Staphylococcus aureus (CA-MRSA) are associated with a marked and prolonged host inflammatory response. In a sepsis simulation model, we tested whether the anesthetic ketamine inhibits the macrophage TNF response to antibiotic-exposed CA-MRSA bacteria via its antagonism of N-methyl-D-aspartate (NMDA) receptors. RAW264.7 cells were stimulated for 18 hrs with 105 to 107 CFU/mL inocula of either of two prototypical CA-MRSA isolates, USA300 strain LAC and USA400 strain MW2, in the presence of either vancomycin or daptomycin. One hour before bacterial stimulation, ketamine was added with or without MK-801 (dizocilpine, a chemically unrelated non-competitive NMDA receptor antagonist), APV (D-2-amino-5-phosphono-valerate, a competitive NMDA receptor antagonist), NMDA, or combinations of these agents. Supernatants were collected and assayed for TNF concentration by ELISA.
RAW264.7 cells exposed to either LAC or MW2 in the presence of daptomycin secreted less TNF than in the presence of vancomycin. The addition of ketamine inhibited macrophage TNF secretion after stimulation with either of the CA-MRSA isolates (LAC, MW2) in the presence of either antibiotic. The NMDA inhibitors, MK-801 and APV, also suppressed macrophage TNF secretion after stimulation with either of the antibiotic-exposed CA-MRSA isolates, and the effect was not additive or synergistic with ketamine. The addition of NMDA substrate augmented TNF secretion in response to the CA-MRSA bacteria, and the addition of APV suppressed the effect of NMDA in a dose-dependent fashion.
Ketamine inhibits TNF secretion by MRSA-stimulated RAW264.7 macrophages and the mechanism likely involves NMDA receptor antagonism. These findings may have therapeutic significance in MRSA sepsis.
[show abstract][hide abstract] ABSTRACT: Inducible nitric oxide (NO) synthase (iNOS) is a stress response protein upregulated in inflammatory conditions, and NO may suppress cellular proliferation. We hypothesized that preventing L-arginine (L-arg) uptake in endothelial cells would prevent lipopolysaccharide/tumor necrosis factor-α (LPS/TNF)-induced, NO-mediated suppression of cellular proliferation. Bovine pulmonary arterial endothelial cells (bPAEC) were treated with LPS/TNF or vehicle (control), and either 10 mM L-leucine [L-leu; a competitive inhibitor of L-arg uptake by the cationic amino acid transporter (CAT)] or its vehicle. In parallel experiments, iNOS or arginase II were overexpressed in bPAEC using an adenoviral vector (AdiNOS or AdArgII, respectively). LPS/TNF treatment increased the expression of iNOS, arginase II, CAT-1, and CAT-2 mRNA in bPAEC, resulting in greater NO and urea production than in control bPAEC, which was prevented by L-leu. LPS/TNF treatment resulted in fewer viable cells than in controls, and LPS/TNF-stimulated bPAEC treated with L-leu had more viable cells than LPS/TNF treatment alone. LPS/TNF treatment resulted in cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase expression, which was attenuated by L-leu. AdiNOS reduced viable cell number, and treatment of AdiNOS transfected bPAEC with L-leu preserved cell number. AdArgII increased viable cell number, and treatment of AdArgII transfected bPAEC with L-leu prevented the increase in cell number. These data demonstrate that iNOS expression in pulmonary endothelial cells leads to decreased cellular proliferation, which can be attenuated by preventing cellular L-arg uptake. We speculate that CAT activity may represent a novel therapeutic target in inflammatory lung diseases characterized by NO overproduction.
[show abstract][hide abstract] ABSTRACT: The growing problem of antibiotic resistance has made the formerly routine therapy of many infectious diseases challenging,
and, in rare cases, impossible. The widespread nature of the problem has led some experts to speculate about a “post-antibiotic
era.” Furthermore, though antibiotic resistance occurs in nature and is an inevitable consequence of even the most prudent
antibiotic use, it is clear that our overuse and misuse of antibiotics is responsible for most of the recent increases in
antibiotic resistance (McGowan, 1983; Austin et al., 1999; Arnold and Straus, 2005). Judicious and rational antibiotic use
has the potential to limit the emergence of clinically important antibiotic resistance and may be able to reduce the impact
of resistance that has already developed, effectively prolonging the shelf life of today’s (and tomorrow’s) antibiotics (Dowell
et al., 1988). Nonetheless, the threat posed by antibiotic-resistant pathogens has been a wakeup call for modern medicine
– developing new antibiotics is important but strategies to prevent infectious diseases (by immunization or other public health
measures) will always be preferable when feasible.
Advances in experimental medicine and biology 01/2010; 659:73-82. · 1.83 Impact Factor
[show abstract][hide abstract] ABSTRACT: Pneumonia occurring as a secondary infection after influenza is a major cause of excess morbidity and mortality, despite the availability and use of antibiotics active against Streptococcus pneumoniae. We hypothesized that the use of a bacteriostatic protein synthesis inhibitor would improve outcomes by reducing the inflammatory response. BALB/cJ mice infected with influenza virus and superinfected with S. pneumoniae were treated with either the cell-wall-active antibiotic ampicillin or the protein synthesis inhibitor clindamycin or azithromycin. In the model, ampicillin therapy performed significantly worse (survival rate, 56%) than (1) clindamycin therapy used either alone (82%) or in combination with ampicillin (80%) and (2) azithromycin (92%). Improved survival appeared to be mediated by decreased inflammation manifested as lower levels of inflammatory cells and proinflammatory cytokines in the lungs and by observation of less-severe histopathologic findings. These data suggest that beta-lactam therapy may not be optimal as a first-line treatment for community-acquired pneumonia when it follows influenza.
The Journal of Infectious Diseases 01/2009; 199(3):311-9. · 5.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: We hypothesized that the Src family tyrosine kinases (STKs) are involved in the upregulation of arginase and inducible nitric oxide synthase (iNOS) expression in response to inflammatory stimuli in pulmonary endothelial cells. Treatment of bovine pulmonary arterial endothelial cells (bPAEC) with lipopolysaccharide and tumor necrosis factor-alpha (L/T) resulted in increased urea and nitric oxide (NO) production, and this increase in urea and NO production was inhibited by the STK inhibitor PP1 (10 microM). The STK inhibitors PP2 (10 microM) and herbimycin A (10 microM) also prevented the L/T-induced expression of both arginase II and iNOS mRNA in bPAEC. Together, the data demonstrate a central role of STK in the upregulation of both arginase II and iNOS in bPAEC in response to L/T treatment. To identify the specific kinase(s) required for the induction of urea and NO production, we studied human pulmonary microvascular endothelial cells (hPMVEC) so that short interfering RNA (siRNA) techniques could be employed. We found that hPMVEC express Fyn, Yes, c-Src, Lyn, and Blk and that the protein expression of Fyn, Yes, c-Src, and Lyn could be inhibited with specific siRNA. The siRNA targeting Fyn prevented the cytokine-induced increase in urea and NO production, whereas siRNAs specifically targeting Yes, c-Src, and Lyn had no appreciable effect on cytokine-induced urea and NO production. These findings support our hypothesis that inflammatory stimuli lead to increased urea and NO production through a STK-mediated pathway. Furthermore, these results indicate that the STK Fyn plays a critical role in this process.
[show abstract][hide abstract] ABSTRACT: Secondary bacterial pneumonia following influenza is an old problem, which is re-emerging. Despite rapid advances in our armamentarium of antimicrobials, the case-fatality rate for this frequent complication of influenza remains high. In some settings, common treatment options may actually contribute to poor outcomes, as rapid lysis of pathogenic bacteria on the backdrop of an activated immune system responding to influenza may lead to inflammatory damage in the lung. An understanding of the inter-related contributions of the antecedent viral infection, the invading bacteria and the host immune response is necessary to formulate an appropriate therapeutic approach. Prevention and resolution of these fulminant infections will require new approaches, including alternate treatment strategies, combination therapies targeting several aspects of the pathogenic process and, potentially, immunomodulation. In the not-so-distant future, strategies aimed at disarming pathogens without eliminating them may be more effective than our current treatment paradigms.
[show abstract][hide abstract] ABSTRACT: Bacterial DNA (CpG DNA) induces macrophage activation and the production of inflammatory mediators, including tumor necrosis factor (TNF) and nitric oxide (NO) by these cells. However, the role of bacterial DNA in the macrophage response to whole bacteria is unknown. We used overlapping strategies to estimate the relative contribution of bacterial DNA to the upregulation of TNF and NO production in macrophages stimulated with antibiotic-treated group B streptococci (GBS). Selective inhibitors of the bacterial DNA/TLR9 pathway (chloroquine, an inhibitory oligonucleotide, and DNase I) consistently inhibited GBS-induced TNF secretion by 35-50% in RAW 264.7 macrophages and murine splenic macrophages, but had no effect on inducible nitric oxide synthase (iNOS) accumulation or NO secretion. Similarly, splenic and peritoneal macrophages from mice lacking TLR9 expression secreted 40% less TNF than macrophages from control mice after GBS challenge but accumulated comparable amounts of iNOS protein. Finally, studies in both RAW 264.7 cells and macrophages from TLR9-/- mice implicated GBS DNA in the upregulation of interleukins 6 (IL-6) and 12 (IL-12) but not interferon-beta (IFNbeta), a key intermediary in macrophage production of iNOS/NO. Our data suggest that the bacterial DNA/TLR9 pathway plays an important role in stimulating TNF rather than NO production in macrophages exposed to antibiotic-treated GBS, and that TLR9-independent upregulation of IFNbeta production by whole GBS may account for this difference.
Microbes and Infection 07/2008; 10(10-11):1106-13. · 2.92 Impact Factor
[show abstract][hide abstract] ABSTRACT: Edwardsiella tarda, a member of the family Enterobacteriaceae, is a Gram-negative bacillus that is most often pathogenic in aquatic environments. Human infections with Edwardsiella are rare, with most occurring in immunocompromised or immunosuppressed hosts. Reported infections include meningitis, cholecystitis, endocarditis, osteomyelitis, soft tissue infections, bacteremia and septicemia, dysentery, and gastroenteritis. This report describes a case of E. tarda gastroenteritis in a renal transplant patient receiving immunosuppressive therapy. The epidemiology, diagnosis, clinical presentation, and treatment options pertaining to E. tarda infections are examined.
[show abstract][hide abstract] ABSTRACT: L-Arginine (L-arg) is metabolized to nitric oxide (NO) by inducible NO synthase (iNOS) or to urea and L-ornithine (L-orn) by arginase. NO is involved in the inflammatory response, whereas arginase is the first step in polyamine and proline synthesis necessary for tissue repair and wound healing. Mitogen-activated protein kinases (MAPK) mediate LPS-induced iNOS expression, and MAPK phosphatase-1 (MKP-1) plays a crucial role in limiting MAPK signaling in macrophages. We hypothesized that MKP-1, by attenuating iNOS expression, acts as a switch changing L-arg metabolism from NO production to L-orn production after endotoxin administration. To test this hypothesis, we performed studies in RAW264.7 macrophages stably transfected with an MKP-1 expression vector in thioglyollate-elicited peritoneal macrophages harvested from wild-type and Mkp-1(-/-) mice, as well as in vivo in wild-type and Mkp-1(-/-) mice. We found that overexpression of MKP-1 resulted in lower iNOS expression and NO production but greater urea production in response to LPS. Although deficiency of MKP-1 resulted in greater iNOS expression and NO production and lower urea production in response to LPS, neither the overexpression nor the deficiency of MKP-1 had any substantial effect on the expression of the arginases.
[show abstract][hide abstract] ABSTRACT: The aim of this study was to identify changes in the gene expression profile of Streptococcus pneumoniae in response to a subinhibitory concentration of penicillin in an effort to better understand mechanisms by which this organism copes with this stress.
S. pneumoniae serotype 2 strain D39 was grown for 1 h in the presence or absence of penicillin at a concentration equivalent to half the MIC (0.03 mg/L). RNA was isolated and gene expression profiles were compared using DNA microarrays. Differential expression of select genes was confirmed by real-time RT-PCR.
A total of 386 genes were found to be responsive to penicillin. Up-regulated genes included those of the ciaR-ciaH operon, luxS, genes encoding cell envelope proteins and genes of the pst locus. Down-regulated genes included genes involved in competence, genes encoding capsular polysaccharide biosynthesis proteins, genes involved in fatty acid chain elongation and genes of the polyamine transporter operon.
Altered expression of these genes reflects a protective response to perturbation of the bacterial cell wall by penicillin. Such genes may represent potential therapeutic targets for enhancing the activity of penicillin against this organism and provide insight into novel mechanisms of penicillin resistance.
Journal of Antimicrobial Chemotherapy 05/2007; 59(4):616-26. · 5.34 Impact Factor
[show abstract][hide abstract] ABSTRACT: MAPK phosphatase (MKP)-1 is an archetypal member of the dual specificity protein phosphatase family that dephosphorylates MAPK. We have previously demonstrated that MKP-1 acts as a negative regulator of p38 and JNK in immortalized macrophages after stimulation with peptidoglycan isolated from Gram-positive bacteria. To define the physiological function of MKP-1 during Gram-positive bacterial infection, we studied the innate immune responses to Gram-positive bacteria using Mkp-1 knockout (KO) mice. We found that Mkp-1(-/-) macrophages exhibited prolonged activation of p38 and JNK, but not of ERK, following exposure to either peptidoglycan or lipoteichoic acid. Compared with wild-type (WT) macrophages, Mkp-1(-/-) macrophages produced more proinflammatory cytokines such as TNF-alpha and IL-6. Moreover, after challenge with peptidoglycan, lipoteichoic acid, live or heat-killed Staphylococcus aureus bacteria, Mkp-1 KO mice also mounted a more robust production of cytokines and chemokines, including TNF-alpha, IL-6, IL-10, and MIP-1alpha, than did WT mice. Accordingly, Mkp-1 KO mice also exhibited greater NO production, more robust neutrophil infiltration, and more severe organ damage than did WT mice. Surprisingly, WT and Mkp-1 KO mice exhibited no significant difference in either bacterial load or survival rates when infected with live S. aureus. However, in response to challenge with heat-killed S. aureus, Mkp-1 KO mice exhibited a substantially higher mortality rate compared with WT mice. Our studies indicate that MKP-1 plays a critical role in the inflammatory response to Gram-positive bacterial infection. MKP-1 serves to limit the inflammatory reaction by inactivating JNK and p38, thus preventing multiorgan failure caused by exaggerated inflammatory responses.
The Journal of Immunology 04/2007; 178(8):5312-20. · 5.52 Impact Factor
[show abstract][hide abstract] ABSTRACT: Exposure of any of six clinical isolates of Staphylococcus aureus to daptomycin alone or in combination with vancomycin or oxacillin (compared with vancomycin or oxacillin alone) led to a dampened macrophage inflammatory response with diminished tumor necrosis factor secretion and reduced accumulation of inducible nitric oxide synthase protein.
Antimicrobial Agents and Chemotherapy 07/2006; 50(6):2225-7. · 4.57 Impact Factor
[show abstract][hide abstract] ABSTRACT: Experts recommend that children with suspected pneumococcal meningitis should empirically receive combination therapy with vancomycin plus either ceftriaxone or cefotaxime. The relationship between timing of the first dose of vancomycin relative to other antibiotics and outcome in these children, however, has not been addressed.
Medical records of children with pneumococcal meningitis at a single institution from 1991-2001 were retrospectively reviewed. Vancomycin start time was defined as the number of hours from initiation of cefotaxime or ceftriaxone therapy until the administration of vancomycin therapy. Outcome variables were death, sensorineural hearing loss, and other neurologic deficits at discharge. Associations between independent variables and outcome variables were assessed in univariate and multiple logistic regression analyses.
Of 114 subjects, 109 received empiric vancomycin therapy in combination with cefotaxime or ceftriaxone. Ten subjects (9%) died, whereas 37 (55%) of 67 survivors who underwent audiometry had documented hearing loss, and 14 (13%) of 104 survivors were discharged with other neurologic deficits. Subjects with hearing loss had a significantly shorter median vancomycin start time than did those with normal hearing (<1 vs 4 hours). Vancomycin start time was not significantly associated with death or other neurologic deficits in univariate or multivariate analyses. Multiple logistic regression revealed that hearing loss was independently associated with vancomycin start time <2 hours, blood leukocyte count <15000/microL, and cerebrospinal fluid glucose concentration <30 mg/dL.
Early empiric vancomycin therapy was not clinically beneficial in children with pneumococcal meningitis but was associated with a substantially increased risk of hearing loss. It may be prudent to consider delaying the first dose of vancomycin therapy until > or =2 hours after the first dose of parenteral cephalosporin in children beginning therapy for suspected or confirmed pneumococcal meningitis.
[show abstract][hide abstract] ABSTRACT: Several lines of evidence have suggested that protein tyrosine phosphatases, including CD45 and SHP-1, regulate macrophage activation. Macrophages from mice lacking SHP-1 (motheaten mice) are hyper-responsive to many stimuli, suggesting that SHP-1 may negatively regulate macrophage activation. Herein we report that the repressible/inducible over-expression of wild-type SHP-1 in a subclone of RAW 264.7 macrophages (RAW-TT10 cells) inhibited both TNF secretion and iNOS protein accumulation in response to stimulation with lipopolysaccharide (LPS) and recombinant murine interferon-gamma and led to diminished LPS-mediated tyrosine phosphorylation of vav1. In contrast, expression of a truncated SHP-1 construct previously shown to interfere with endogenous SHP-1 function modestly augmented LPS-mediated TNF and iNOS production and did not inhibit vav1 tyrosine phosphorylation. Taken together, these data provide the first direct evidence that SHP-1 inhibits macrophage activation by LPS and suggest that this effect may be mediated in part by dephosphorylation of vav1.
Biochemical and Biophysical Research Communications 05/2006; 342(2):547-55. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: During the past century the excitement of discovering antibiotics as a treatment of infectious diseases has given way to a sense of complacency and acceptance that when faced with antimicrobial resistance there will always be new and better antimicrobial agents to use. Now, with clear indications of a decline in pharmaceutical company interest in anti-infective research, at the same time when multi-drug resistant micro-organisms continue to be reported, it is very important to review the prudent use of the available agents to fight these micro-organisms. Injudicious use of antibiotics is a global problem with some countries more affected than others. There is no dearth of interest in this subject with scores of scholarly articles written about it. While over the counter access to antibiotics is mentioned as an important contributor towards injudicious antibiotic use in developing nations, as shown in a number of studies, there are many provider, practice and patient characteristics which drive antibiotic overuse in developed nations such as the United States. Recognizing that a thorough review of this subject goes far and beyond the page limitations of a review article we provide a summary of some of the salient aspects of this global problem with a focus towards readers practicing in developing nations.
The Indian Journal of Pediatrics 05/2006; 73(4):343-50. · 0.72 Impact Factor