B. Keith English

Specific Inhibitors of p38 and Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase Pathways Block Inducible Nitric Oxide Synthase and Tumor Necrosis Factor Accumulation in Murine Macrophages Stimulated with Lipopolysaccharide and Interferon-γ

Whether p38 and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase cascades are required for inducible nitric oxide synthase (iNOS) and tumor necrosis factor (TNF) accumulation in RAW 264.7 murine macrophages exposed to lipopolysaccharide (LPS) plus recombinant interferon-gamma (rIFN-gamma) was investigated. By use of Western blotting for iNOS detection and ELISA for quantitation of TNF secretion, three selective inhibitors of these pathways were tested (the p38 inhibitors SB202190 and SB203580 and the MEK 1,2/ERK inhibitor PD98059). Dose-related inhibition of iNOS production was demonstrated when inhibitors were added 1 h before, simultaneously with, or 1 h after LPS plus rIFN-gamma stimulation. In contrast, inhibition of TNF secretion was observed only when cells were preincubated with these agents. Thus, both the p38 and ERK pathways are involved in the up-regulation of iNOS and TNF production by murine macrophages, and specific inhibitors of these pathways block macrophage iNOS production even when added 1 h after activation of these cells.

Role of Mitogen-Activated Protein Kinases in CpG DNA-Mediated IL-10 and IL-12 Production: Central Role of Extracellular Signal-Regulated Kinase in the Negative Feedback Loop of the CpG DNA-Mediated Th1 Response

The mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK), and p38, are activated in response to infectious agents and innate immune stimulators such as CpG DNA, and regulate the subsequent initiation and termination of immune responses. CpG DNA activates p38 and ERK with slightly different kinetics in monocytic cells. The present studies investigated the roles of these two key mitogen-activated protein kinases in regulating the CpG DNA-induced production of pro- and anti-inflammatory cytokines in the macrophage-like cell line RAW264.7. p38 activity was essential for the induction of both IL-10 and IL-12 expression by CpG DNA. In contrast, CpG DNA-mediated ERK activation was shown to suppress IL-12 production, but to be essential for the CpG DNA-induced IL-10 production. Studies using rIL-10 and IL-10 gene-deficient mice demonstrated that the inhibitory effect of ERK on CpG DNA-mediated IL-12 production is indirect, due to the role of ERK in mediating IL-10 production. These results demonstrate that ERK and p38 differentially regulate the production of pro- and anti-inflammatory cytokines in APCs that have been activated by CpG DNA. CpG DNA-induced p38 activity is required for the resulting innate immune activation. In contrast, ERK plays a central negative regulatory role in the CpG DNA-mediated Th1 type response by promoting production of the Th2 type cytokine, IL-10.

Isolation and Characterization of Vancomycin-Tolerant Streptococcus pneumoniae from the Cerebrospinal Fluid of a Patient Who Developed Recrudescent Meningitis

The emergence of tolerance to vancomycin has recently been reported in Streptococcus pneumoniae, the most common cause of bacterial meningitis. A vancomycin- and cephalosporin-tolerant strain of S. pneumoniae, the Tupelo strain, was isolated from the cerebrospinal fluid of a patient who then developed recrudescence of meningitis despite treatment with vancomycin and a third-generation cephalosporin. The Tupelo strain evidenced no lysis in the exponential or stationary phase of growth when exposed to vancomycin and only minimal loss of viability. Further characterization revealed normal autolysin expression, localization, and triggering by detergents, indicating that the defect leading to tolerance in the Tupelo strain is in the control pathway for triggering of autolysis. Because tolerance is a precursor phenotype to resistance and may lead to clinical failure of antibiotic therapy, these observations may have important implications for vancomycin use in infections caused by S. pneumoniae.

Successful Medical Therapy for Deeply Invasive Facial Infection Due to Pythium insidiosum in a Child

Pythiosis occurs in animals and humans who encounter aquatic habitats that harbor Pythium insidiosum. Drug therapy for deeply invasive infections with this organism has been ineffective in humans and animals; patients have been cured only by radical surgical debridement. A 2-year-old boy developed periorbital cellulitis unresponsive to antibiotic and antifungal therapy. The cellulitis extended to the nasopharynx, compromising the airway and necessitating a gastrostomy for feeding. P. insidiosum was isolated from surgical biopsy specimens of the affected tissue. On the basis of in vitro susceptibility studies of the isolate, the patient was treated with a combination of terbinafine and itraconazole. The infection resolved over a period of a few months. The patient remained well 1.5 years after completing a 1-year course of therapy. Cure of deep P. insidiosum infection is feasible with drug therapy.

Treatment with Protein Synthesis Inhibitors Improves Outcomes of Secondary Bacterial Pneumonia after Influenza

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.

Production of Lymphotoxin and Tumor Necrosis Factor by Human Neonatal Mononuclear Cells

ABSTRACT: Lymphotoxin (LT) and tumor necrosis factor (TNF) are cytokines with many common biologic effects including antiviral activity and induction of fever and the acute phase response; despite common effects, they are molecularly distinct. Because neonates are unduly susceptible to viral infection and frequently fail to mount a febrile response to infection, we hypothesized that neonatal cells would produce less LT and TNF than adult cells. We analyzed LT and TNF production by blood mononuclear cells and purified T cells using Northern blot analysis to detect specific messenger ribonucleic acid and specific assays to detect LT and TNF protein in culture superna-tants. Compared to LT, TNF messenger ribonucleic acid and protein were produced more rapidly both by total mononuclear cells and by T cells in response to mitogen stimulation. Although there was intersubject variability, adult and neonatal mononuclear cells and T cells (n = 6) produced similar amounts of LT and TNF messenger ribonucleic acid and protein with similar kinetics. In experiments with phytohemagglutinin-stimulated mononuclear cells from ten additional subjects, supernatant LT was somewhat greater in neonatal cultures (neonatal = 62.8 ± 60.5, adult = 13.2 ± 10.7 units/ml, p < 0.05), and TNF was somewhat greater in adult cultures (neonatal = 708 ± 429, adult = 1987 ± 392 pg/ml, p < 0.01) at 24 h; results at 48 h and 72 h were similar. Thus, neonatal MC produced as much or more LT than did adult MC. Although the decreased production of TNF by neonatal MC was statistically significant, these cells did produce substantial amounts of this cytokine. Because the reduction in TNF production by neonatal MC in response to nonspecific stimuli was modest, it is unlikely to be the principal mechanism but may contribute to the diminished febrile response and increased susceptibility to infection of the human neonate.

Knockout of Mkp-1 Enhances the Host Inflammatory Responses to Gram-Positive Bacteria

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-α 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-α, IL-6, IL-10, and MIP-1α, 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.

Decreased Granulocyte-Macrophage Colony-Stimulating Factor Production by Human Neonatal Blood Mononuclear Cells and T Cells

ABSTRACT: Impaired production and delivery of neutrophils to the site of infection have been implicated in the increased susceptibility of the neonate to infection. Because granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) play critical roles in the production of neutrophils from marrow precursors, we assessed the ability of leukocytes from neonates and adults to produce GM-CSF, G-CSF, and, for comparison, macrophage colony-stimulating factor (M-CSF) after stimulation with concanavalin A ± phorbol myristate acetate [blood mononuclear cells (MC) and T lymphocytes] or lipopolysaccharide (monocytes). MC and monocytes from adult and neonatal subjects produced mRNA for GM-CSF, G-CSF, and M-CSF, whereas T cells produced only GM-CSF mRNA. Neonatal MC and T cells accumulated only −30% as much GM-CSF mRNA as did adult MC and T cells. In contrast, the accumulation of GM-CSF mRNA by neonatal and adult monocytes was similar. Neonatal MC also accumulated similar amounts of G-CSF mRNA and somewhat more M-CSF mRNA than did adult MC; results with monocytes were similar to those with MC. Results of colony-stimulating activity bioassays on supernatants from neonatal and adult MC stimulated with concanavalin A paralleled the mRNA results. Although the overall number of colonies generated using neonatal and adult supernatants was similar, neonatal MC supernatants generated significantly more (p < 0.05) monocyte-containing colonies (72 ±19 versus 46 ± 11), significantly fewer (p < 0.05) eosinophil-containing colonies (7 ± 6 versus 23 ± 13), and similar numbers of granulocyte-containing colonies (59 ± 23 versus 63 ± 11) compared with adult MC supernatants. Because GM-CSF is a major determinant of eosinophil production in these assays, these data suggested diminished amounts of GM- CSF in the neonatal culture supernatants. Similarly, GM- CSF concentrations in neonatal MC and T cell culture supernatants averaged 40 to 50% of the concentrations in adult culture supernatants as determined by ELISA (p < 0.01). Whether the modestly diminished GM-CSF production by neonatal T cells contributes t the observed deficiency of granulocyte production in neonates, which occurs when demand is increased in response to infection, remains to be determined.

The use and abuse of antibiotics and the development of antibiotic resistance

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.

Pneumococcal meningitis in children: relationship of antibiotic resistance to clinical characteristics and outcomes.

Background. The relationship of antibiotic susceptibility to clinical outcome in children with pneumococcal meningitis is uncertain. Previous studies have been limited by inclusion of relatively few patients infected with nonsusceptible pneumococci and inconsistent use of empiric vancomycin. Methods. Medical records of 86 children with culture-confirmed pneumococcal meningitis at a single institution from October, 1991, to October, 1999, were retrospectively reviewed, and differences in presentation and outcome based on antibiotic susceptibility of pneumococcal isolates were assessed. Results. Of 86 isolates 34 were nonsusceptible to penicillin (12 resistant). Of 60 isolates for which cefotaxime susceptibility data were available, 17 were nonsusceptible (12 resistant). Antibiotic susceptibility was not significantly associated with death, intensive care unit admission, mechanical ventilation, focal neurologic deficits, seizures, secondary fever, abnormal neuroimaging studies or hospital days. Children with penicillin-resistant isolates had significantly higher median blood leukocyte counts (24 100/&mgr;l vs. 15 700/&mgr;l, P = 0.03) and lower median CSF protein concentrations (85 mg/dl vs. 219 mg/dl, P = 0.04), were more likely to have a CSF glucose concentration of ≥50 mg/dl (7 of 11 vs. 15 of 68, P = 0.009) and had lower rates of sensorineural hearing loss (1 of 8 vs. 25 of 40, P = 0.02) than children with isolates that were not resistant to penicillin. Children with cefotaxime-nonsusceptible isolates had an increased median duration of primary fever compared with those with nonsusceptible strains (6 days vs. 3.5 days, P = 0.02). Conclusions. In children with pneumococcal meningitis, penicillin resistance was associated with a reduced risk of hearing loss, while cefotaxime resistance was associated with a longer duration of fever. Other outcome measures were not significantly influenced by the antibiotic susceptibility of pneumococcal isolates.