The relative effects of two beta-lactam antibiotics, penicillin-binding protein (PBP) 2-specific imipenem and PBP 3-specific ceftazidime, upon in vitro induction of lipopolysaccharide (LPS) release were investigated against smooth- and rough-LPS mutant isolates of Pseudomonas aeruginosa. Free LPS liberated from both isolates are 10- to 40-fold higher for ceftazidime-exposed cultures than control or imipenem-treated cultures after 4-8 h at 35 degrees C despite equivalent MICs. Lethalities of filtrates in mice correlated with in vitro endotoxin assay results. Sub-MIC levels of ceftazidime induced filamentation and LPS release without significant bacterial lysis. Amounts released not only matched the quantities achieved at inhibitory concentrations (e.g., 1-, 2-, and 50-times MIC) of ceftazidime but significantly exceeded levels of LPS liberated by exposure to imipenem, less than or equal to 100 times its MIC. Sub-MIC levels of imipenem released relatively small amounts of free LPS while reducing colony counts approximately 2 logs more than equivalent amounts of ceftazidime after 2 h. Data suggest that ceftazidime-induced filamentation releases larger quantities of bioreactive LPS than nonfilamentous fast-lysing imipenem.
The expression of the cannabinoid peripheral cannabinoid receptor (CB 2 ) receptor on peripheral immune cells suggests that compounds specific for CB 2 might be effective anti-inflammatory agents. In this report, we present the initial biological profiling of a novel triaryl bis-sulfone, Sch.336, which is selective for the human cannabinoid CB 2 receptor (hCB 2 ). Sch.336 is an inverse agonist at hCB 2 , as shown by its ability to decrease guanosine 5Ј-3-O-(thio)triphosphate (GTP␥S) binding to membranes containing hCB 2 , by the ability of GTP␥S to left-shift Sch.336 binding to hCB 2 in these membranes, and by the compound's ability to increase forskolin-stimulated cAMP levels in CHO cells expressing hCB 2 . In these systems, Sch.336 displays a greater potency than that reported for the CB 2 In vitro, Sch.336 impairs the migration of CB 2 -expressing recombinant cell lines to the cannabinoid agonist 2-arachidonylglycerol. In vivo, the compound impairs migration of cells to cannabinoid agonist HU210 [(6aR)-trans-3-(1,1-dimethylheptyl)-6a,7,10,10a-tetrahydro-1-hydroxy-6,6-dimethyl-6H-dibenzo [b,d] pyran-9-methanol]. Oral administration of the Sch.336 significantly inhibited leukocyte trafficking in several rodent in vivo models, induced either by specific chemokines or by antigen challenge. Finally, oral administration of Sch.336 blocked ovalbumin-induced lung eosinophilia in mice, a disease model for allergic asthma. We conclude that selective cannabinoid CB 2 inverse agonists may serve as novel immunomodulatory agents in the treatment of a broad range of acute and chronic inflammatory disorders in which leukocyte recruitment is a hallmark of disease pathology.The identification of a second cannabinoid receptor present primarily in peripheral immune tissues and cells (Munro et al., 1993) suggested an immunomodulatory role for endocannabinoids independent of the effects mediated by their interaction with the "brain" cannabinoid CB 1 receptor. Libraries of selective compounds now exist, based either on the structure of known ligands for the cannabinoid receptors or on results of random compound library screening (Huffman, 2000). With the help of CB 2 -specific compounds and by characterization of a CB 2 Ϫ/Ϫ mouse strain (Buckley et al., 2000), Article, publication date, and citation information can be found at
BackgroundObesity and inflammation are highly integrated processes in the pathogenesis of insulin resistance, diabetes, dyslipidemia, and non-alcoholic fatty liver disease. Molecular mechanisms underlying inflammatory events during high fat diet-induced obesity are poorly defined in mouse models of obesity. This work investigated gene activation signals integral to the temporal development of obesity.MethodsGene expression analysis in multiple organs from obese mice was done with Taqman Low Density Array (TLDA) using a panel of 92 genes representing cell markers, cytokines, chemokines, metabolic, and activation genes. Mice were monitored for systemic changes characteristic of the disease, including hyperinsulinemia, body weight, and liver enzymes. Liver steatosis and fibrosis as well as cellular infiltrates in liver and adipose tissues were analyzed by histology and immunohistochemistry.ResultsObese C57BL/6 mice were fed with high fat and cholesterol diet (HFC) for 6, 16 and 26 weeks. Here we report that the mRNA levels of macrophage and inflammation associated genes were strongly upregulated at different time points in adipose tissues (6-16 weeks) and liver (16-26 weeks), after the start of HFC feeding. CD11b+ and CD11c+ macrophages highly infiltrated HFC liver at 16 and 26 weeks. We found clear evidence that signals for IL-1β, IL1RN, TNF-α and TGFβ-1 are present in both adipose and liver tissues and that these are linked to the development of inflammation and insulin resistance in the HFC-fed mice.ConclusionsMacrophage infiltration accompanied by severe inflammation and metabolic changes occurred in both adipose and liver tissues with a temporal shift in these signals depending upon the duration of HFC feeding. The evidences of gene expression profile, elevated serum alanine aminotransferase, and histological data support a progression towards nonalcoholic fatty liver disease and steatohepatitis in these HFC-fed mice within the time frame of 26 weeks.
C57BL/6J mice given low doses of lipopolysaccharide (LPS) (100 ng per mouse) plus D-galactosamine (8 mg per mouse) die within 24 h following LPS administration. We used this septic shock model to confirm the role of tumor necrosis factor in mortality using a monoclonal antibody to tumor necrosis factor to prevent lethality. Furthermore, we demonstrated that interleukin 6, rather than playing a lethal role, protected mice against death in this septic shock model. Antibody to interleukin 6 did not affect the fatal outcome in this low-LPS-dose model. However, pretreatment with antibody to tumor necrosis factor did protect the mice against death, in a dose-dependent manner. Moreover, mortality was enhanced by pretreatment with antibody to interleukin 6 when tumor necrosis factor was partly limited by anti-tumor necrosis factor treatment. Mortality was significantly reduced by pretreatment with both recombinant interleukin 6 and low doses of antibody to tumor necrosis factor; in the absence of the low dose of antibody to tumor necrosis factor, interleukin 6 alone did not confer any protection. These data demonstrate in vivo antagonistic activities of tumor necrosis factor and interleukin 6 and show that interleukin 6 can play a protective role against death from septic shock.
A highly encephalitogenic peptide whose structure resembles the sequence of amino acids surrounding the single tryptophan residue in the encephalitogenic A1 protein from bovine myelin was synthesized. This peptide is similar in the sequence to peptic peptide E and tryptic T27, derived directly from the A1 protein, and is as active on a molar basis as the A1 protein. The major disease-inducing site of the A1 protein resides in a linear sequence of nine amino acids: H-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys-OH. This region of the A1 protein is apparently the major encephalitogenic determinant since specific modification of the tryptophan residue in the A1 protein with 2-hydroxy-5-nitrobenzyl bromide destroyed its encephalitogenic activity.
Staphylococcal infections associated with catheter and prosthetic implants are difficult to eradicate and often lead to chronic infections. Development of novel antibacterial therapies requires simple, reliable, and relevant models for infection. Using bioluminescent Staphylococcus aureus, we have adapted the existing foreign-body and deep-wound mouse models of staphylococcal infection to allow real-time monitoring of the bacterial colonization of catheters or tissues. This approach also enables kinetic measurements of bacterial growth and clearance in each infected animal. Persistence of infection was observed throughout the course of the study until termination of the experiment at day 16 in a deep-wound model and day 21 in the foreign-body model, providing sufficient time to test the effects of antibacterial compounds. The usefulness of both animal models was assessed by using linezolid as a test compound and comparing bioluminescent measurements to bacterial counts. In the foreign-body model, a three-dose antibiotic regimen (2, 5, and 24 h after infection) resulted in a decrease in both luminescence and bacterial counts recovered from the implant compared to those of the mock-treated infected mice. In addition, linezolid treatment prevented the formation of subcutaneous abscesses, although it did not completely resolve the infection. In the thigh model, the same treatment regimen resulted in complete resolution of the luminescent signal, which correlated with clearance of the bacteria from the thighs.
Antibiotic mediated release of endotoxin (lipopolysaccharide, LPS) from Gram-negative bacteria is implicated in septic shock. The β-lactam (cell wall active) class, in particular, has been deemed responsible for release of greater quantities of LPS than other classes of antibiotics. However, it is becoming increasingly recognized that variations in the ability to liberate free LPS exist within the β-lactam subclasses. Until recently, LPS-release studies have primarily included the cephalosporin, monobactam, and penicillin β-lactams, but not the carbapenem subclass. We document here that carbapenems significantly liberate less LPS than other β-lactam subclasses, and that disparity in LPS release also occurs within the carbapenems as well (i.e. imipenem vs meropenem). The propensity to release LPS correlates with the cumulative PBP binding affinity of each β-lactam antibiotic regardless of subclass. A direct correlation has been established between antibiotic concentration, frequency of antibiotic exposure, differential LPS release, PBP specificity (morphology, rate of bacterial lysis or cell mass) and antibiotic efficacy in two mouse models of infection. In the first, differences in the pathogenic (endotoxic) potential of potently biologically active smooth LPS (S-LPS) and poorly biologically active rough chemotype LPS (R-LPS) expressing bacteria underscores the in vivo importance of endotoxin in mediating lethality following antibiotic chemotherapy. Endotoxin is, however, not innately toxic and thus its lethal effects are mediated through LPS stimulation of host cells (i.e. macrophages) to produce deleterious levels of cytokines. In the second experimental model, therefore, the toxic effects of antibiotic-liberated endotoxin have been abrogated by pretreatment of mice with carrageenan (CGN) prior to challenge with endotoxin or Gram-negative bacteria. CGN treatment eliminates or markedly reduces the numbers of LPS-responsive mediator cells thus affecting mortality. In both animal models, an in vivo role for LPS is demonstrated through differential changes in antibiotic efficacy (ED 50 ) in response to differential effects of LPS via modulation of the endotoxic sensitivity/responsiveness of the host. We conclude that antibiotic class, concentration, dosing intervals (timing) and perhaps, method of treatment (i.e. bolus vs infusion) may each be important in the survival of experimental animals severely infected with Gram-negative bacteria.
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