Search

There are two independent mol-ecules in the asymmetric unit of the title compound, C(16)H(10)F(6)N(4)O. The triazole ring is not coplanar with the quinoline ring system; the dihedral angle between the two planes being 74.47 (12) and 63.97 (13)° in the two mol-ecules. The crystal structure is characterized by inter-molecular C-H⋯F, C-H⋯N and C-H⋯O hydrogen bonding. Weak intra-molecular C-H⋯F inter-actions are observed. Disorder is observed in two F atoms of one of the trifluoro-methyl groups of one independent mol-ecule [occupancy ratios 0.77 (3):0.23 (3) and 0.77 (4):0.23 (4)] and in all three F atoms of one of the trifluoro-methyl groups of the second independent mol-ecule [occupancy ratios 0.520 (14):0.480 (14), 0.615 (17):0.385 (17) and 0.783 (11):0.217 (11)]. The O atom is also disordered over two positions with occupancies of 0.60 (13) and 0.40 (13) in the first mol-ecule.

The chemokine sink hypothesis pertaining to erythrocyte Duffy Antigen Receptor for Chemokines (DARC) during inflammation has received considerable attention, but lacks direct in vivo evidence. Here we demonstrate, using mice with a targeted deletion in CXCL5, that CXCL5 bound erythrocyte DARC and impaired its chemokine scavenging in blood. CXCL5 increased the plasma concentrations of CXCL1 and CXCL2 in part through inhibiting chemokine scavenging, impairing chemokine gradients and desensitizing CXCR2, which led to decreased neutrophil influx to the lung, increased lung bacterial burden and mortality in an Escherichia coli pneumonia model. In contrast, CXCL5 exerted a predominant role in mediating neutrophil influx to the lung during inflammation after LPS inhalation. Platelets and lung resident cells were the sources of homeostatic CXCL5 in blood and inflammatory CXCL5 in the lung respectively. This study presents a paradigm whereby platelets and red cells alter chemokine scavenging and neutrophil-chemokine interaction during inflammation., (Copyright 2010 Elsevier Inc. All rights reserved.)

Bacterial lung diseases are a major cause of morbidity and mortality both in immunocompromised and in immunocompetent individuals. Neutrophil accumulation, a pathological hallmark of bacterial diseases, is critical to host defense, but may also cause acute lung injury/acute respiratory distress syndrome. Toll-like receptors, nucleotide-binding oligomerization domain (NOD)-like receptors, transcription factors, cytokines, and chemokines play essential roles in neutrophil sequestration in the lungs. This review highlights our current understanding of the role of these molecules in the lungs during bacterial infection and their therapeutic potential. We also discuss emerging data on cholesterol and ethanol as environmentally modifiable factors that may impact neutrophil-mediated pulmonary innate host defense. Understanding the precise molecular mechanisms leading to neutrophil influx in the lungs during bacterial infection is critical for the development of more effective therapeutic and prophylactic strategies to control the excessive host response to infection.

The asymmetric unit of the title salt, (C(16)H(20)N(3)S)(2)[Co(NCS)(4)], comprises one monovalent isothio-pendylium cation and one-half of a divalent thio-cyanatocobaltate(II) anion (2 symmetry). The central thia-zine ring of the cation is slightly twisted in a boat-like fashion, with r.m.s. deviations from the mean plane of 0.272 (1) and 0.2852 (8) Å for the N and S atoms. The mol-ecular structure of the cation is stabilized by an intra-molecular N-H⋯N hydrogen bond. Within the complex anion, the Co(II) atom is tetra-hedrally surrounded by four N atoms of the thio-cyanate ligands. π-π stacking, with a distance of 3.7615 (10) Å between the centroids of benzene and pyridine rings, helps to consolidate the packing.

Lung diseases caused by bacteria are a leading cause of death in both immunocompromised and immunocompetent individuals as well as in children. Although neutrophil recruitment is critical to augment the host defence, excessive neutrophil accumulation results in life-threatening diseases, such as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). Therefore, it is important to modulate excessive neutrophil influx in ALI/ARDS to mitigate lung damage and mortality. A better understanding of the basic mechanisms underlying neutrophil influx is crucial to designing novel and innovative treatment strategies for ALI/ARDS. Recognition of bacteria in the lung is the critical first step leading to neutrophil influx. Pattern recognition receptors, such as Toll-like receptors and NOD-like receptors, play an important role in the recognition of bacterial pathogens. Understanding the molecular and cellular mechanisms associated with the recognition of bacterial pathogens by the host is critical for the development of effective therapeutic strategies to control parenchymal damage via modulating neutrophil accumulation in the lung.

In the title compound, C(17)H(11)ClO(2), the benzofuran ring system is almost planar (r.m.s. deviation = 0.011 Å) and forms a dihedral angle of 10.53 (6)° with the chloro-phenyl ring. No significant inter-molecular inter-actions are observed.

Klebsiella pneumoniae causes extensive lung damage. TLR signaling involves adaptors TRIF and MyD88. However, the relative contribution of TRIF and MyD88 signaling in host defense against pulmonary K. pneumoniae infection has not been elucidated. Therefore, we investigated the role of TRIF and MyD88 in K. pneumoniae pneumonia. TRIF(-/-) mice infected with K. pneumoniae showed impaired survival and reduced bacterial clearance, neutrophil influx, histopathologic evidence of inflammation, and TNF-alpha, IL-6, KC, MIP-2, but not LIX, expression in the lungs. In addition, K. pneumoniae-induced late NF-kappaB activation and phosphorylation of MAPKs was attenuated in the lungs of TRIF(-/-) mice. However, MyD88(-/-) mice infected with K. pneumoniae showed a much more remarkable phenotype, including impaired survival and reduced bacterial clearance, histopathology, and TNF-alpha, IL-6, KC, MIP-2, and LIX expression with almost no neutrophil influx in the lungs. In MyD88(-/-) mice, K. pneumoniae-induced early NF-kappaB and MAPK activation in the lungs was also reduced. Furthermore, the role of MyD88 is dominant over TRIF because TRIF/MyD88 double knockout mice displayed a more pronounced phenotype than TRIF(-/-) mice. Moreover, human alveolar macrophages pretreated with MyD88 blocking peptide showed attenuated TNF-alpha, IL-6, and IL-8 expression. Also, C57BL/6 mice pretreated with MyD88 blocking peptide exhibited attenuation in K. pneumoniae-induced neutrophil influx and enhanced bacterial burden in the lungs and dissemination. Overall, this investigation provides new insights into the TRIF and MyD88 signaling triggered by pulmonary K. pneumoniae infection in the lungs and demonstrate the therapeutic potential of MyD88 in reducing excessive neutrophil influx in human disease during Gram-negative bacterial pneumonia.

The title compound, C(9)H(10)N(2)O(3), crystallizes with one and a half mol-ecules in the asymmetric unit, one lying on a general position and the other on a twofold rotation axis. The dihedral angle between the two independent benzimidazole ring systems is 18.96 (5)°. In the crystal, mol-ecules are linked into a three-dimensional network by O-H⋯O hydrogen bonding involving N-hydroxy-methyl and carbonyl groups, and C-H⋯O hydrogen bonds.

Aim: To evaluate the efficacy of antioxidant lycopene in preventing pre-eclampsia in healthy primigravidas., Methods: A prospective randomized double blind placebo-controlled study was undertaken with informed written consent. A total of 159 primigravidas at gestational ages between 12 and 20 weeks, with singleton pregnancy and without any medical disorders were enrolled. Seventy-seven (subjects) of these women received 2 mg oral lycopene daily and 82 (controls) received a placebo daily until delivery. The women were followed up until delivery for the development of pre-eclampsia and maternal, fetal and neonatal outcomes., Results: The two study groups of healthy primigravidas (one receiving lycopene and the other placebo) were of similar physical and social parameters. The investigation showed no significant difference between the two groups in developing pre-eclampsia (18.18% with lycopene vs 18.29% with placebo, P value = 0.99). On the contrary, there was a significantly higher incidence of adverse effects like preterm labor (10.39% with lycopene vs 1.22% with placebo, P value = 0.02) and low birthweight (<2.5 kg) babies (22.08% with lycopene vs 9.76% with placebo, P value = 0.05)., Conclusion: The results of the present study indicate that the antioxidant lycopene has no benefit in prevention of pre-eclampsia in healthy primigravidas. Rather, there is an increased incidence of the adverse effects of preterm labor and low birthweight babies.

Bacterial pneumonia remains a serious disease. Pattern recognition receptors play an integral role in neutrophil accumulation during pneumonia. Although myeloid differentiation protein (MD)-2 has been recognized as a key molecule for LPS signaling, the role of MD-2 in neutrophil accumulation in the lung during bacterial infection has not been explored. Here, we investigate the role of MD-2 in Escherichia coli LPS-induced lung inflammation and E. coli-induced pneumonia. LPS-induced CD14-independent neutrophil accumulation was abolished in CD14/MD-2(-/-) mice. MD-2(-/-) mice challenged with LPS displayed attenuated neutrophil influx, NF-kappaB activation, cytokine/chemokine expression, and lung histopathology. MD-2(-/-) mice transplanted with MD-2(+/+) bone marrow demonstrated decreased neutrophil influx and cytokine/chemokine expression in the lungs when challenged by LPS. MD-2(-/-) mice infected with E. coli demonstrated reduced neutrophil influx and cytokine/chemokine expression in the lungs, whereas heat-killed E. coli did not induce either neutrophil accumulation or cytokine/chemokine expression in MD-2(-/-) mice infected with E. coli. Furthermore, MD-2(-/-) mice displayed increased bacterial burden in the lungs and enhanced bacterial dissemination. Toll-like receptor (TLR)-5(-/-) mice infected with E. coli exhibited attenuated neutrophil accumulation, whereas MD-2/TLR5(-/-) mice inoculated with E. coli showed further attenuated neutrophil influx and impaired bacterial clearance. Taken together, these new findings demonstrate: (1) the important role of MD-2 in the CD14-independent LPS-mediated cascade of neutrophil influx; (2) the relative importance of bone marrow- and non-bone marrow cell-derived MD-2 in LPS-induced inflammation; and (3) the essential role of MD-2-dependent and MD-2-independent (TLR5) signaling in E. coli-induced neutrophil accumulation and pulmonary host defense.

Background: Alcohol intoxication impairs innate immune responses to bacterial pneumonia, including neutrophil influx. Lipopolysaccharide (LPS)-induced chemokine (LIX or CXCL5) is a recently described chemokine produced by type-II alveolar epithelial (AE2) cells which facilitates neutrophil recruitment. The effect of acute alcohol intoxication on AE2 cell expression of LIX is unknown., Methods: C57BL/6 mice were given an intraperitoneal (i.p.) injection of ethanol (4 g/kg) or saline 30 minutes prior to intratracheal (i.t.) injection with 10 mug Escherichia coli LPS. In vitro stimulation of primary AE2 cells or murine AE2 cell line MLE-12 was performed with LPS and tumor necrosis factor-alpha (TNF-alpha)., Results: LIX protein is readily detectable in the lung but not in plasma following LPS administration, demonstrating "compartmentalization" of this chemokine during pulmonary challenge. In contrast to the CXC chemokines keratinocyte-derived chemokine and macrophage inflammatory protein-2, which are abundantly expressed in both lung tissue and alveolar macrophages, LIX expression is largely confined to the lung parenchyma. Compared to controls, intoxicated animals show a decrease in LIX and neutrophil number in bronchoalveolar lavage fluid following LPS challenge. Ethanol inhibits LIX at the transcriptional level. In vitro studies show that LPS and TNF-alpha are synergistic in inducing LIX by either primary AE2 or MLE-12 cells. Acute ethanol exposure potently and dose-dependently inhibits LIX expression by AE2 cells. Activation of nuclear factor-kappaB is critical to LIX expression in MLE-12 cells, and acute ethanol treatment interferes with early activation of this pathway as evidenced by impairing phosphorylation of p65 (RelA). Inhibition of p38 mitogen-activated protein kinase signaling, but not ERK1/2 activity, in MLE-12 cells by acute alcohol is likely an important cause of decreased LIX expression during challenge., Conclusions: These data demonstrate direct suppression of AE2 cell innate immune function by ethanol and add to our understanding of the mechanisms by which acute intoxication impairs the lung's response to microbial challenge.

Effector/memory T cells (Tem) are required to maintain successful immunity, while regulatory T cells (Treg) are required to prevent excessive/uncontrolled inflammation and/or autoimmunity. Although both Tem and Treg cells are increased during aging, the relationship between the increased proportion of Foxp3(+) Treg cells and CD44(+) Tem cells with aging is not clearly understood. We found in this report that Foxp3(+) Treg cells are increased in parallel with CD44(+) Tem cells in SJL/J mice with aging, and that all Foxp3(+) Treg cells are of CD44(+) Tem phenotype, suggesting that the increased Foxp3(+) Treg cells originated from the expanded pool of CD44(+) Tem cells with aging. Our in vitro kinetic studies further suggested that Foxp3(+) Treg cells are converted through the CD44(+) stage. Furthermore, we observed that although the balance between Foxp3(+) Treg and CD44(+)Foxp3(-) Tem cells remained with aging, the aged mice have higher ratios of both Tem and Treg cells vs. naïve T cells resulting in the "shrunken" naïve T cell pools. Our results suggest that an age-associated imbalance of T cell repertoire is a mechanism that contributes to spontaneous occurrence of Hodgkin's-like lymphoma in aged SJL/J mice.

Liver X receptor (LXR) alpha and beta are members of the nuclear receptor superfamily of ligand-activated transcription factors. Best known for triggering "reverse cholesterol transport" gene programs upon their activation by endogenous oxysterols, LXRs have recently also been implicated in regulation of innate immunity. In this study, we define a role for LXRs in regulation of pulmonary inflammation and host defense and identify the lung and neutrophil as novel in vivo targets for pharmacologic LXR activation. LXR is expressed in murine alveolar macrophages, alveolar epithelial type II cells, and neutrophils. Treatment of mice with TO-901317, a synthetic LXR agonist, reduces influx of neutrophils to the lung triggered by inhaled LPS, intratracheal KC chemokine, and intratracheal Klebsiella pneumoniae and impairs pulmonary host defense against this bacterium. Pharmacologic LXR activation selectively modulates airspace cytokine expression induced by both LPS and K. pneumoniae. Moreover, we report for the first time that LXR activation impairs neutrophil motility and identify inhibition of chemokine-induced RhoA activation as a putative underlying mechanism. Taken together, these data define a novel role for LXR in lung pathophysiology and neutrophil biology and identify pharmacologic activation of LXR as a potential tool for modulation of innate immunity in the lung.

The original hygiene hypothesis suggests that early childhood respiratory infections preceding allergen exposure may decrease the prevalence of allergic diseases. We have recently demonstrated that Mycoplasma pneumoniae infection preceding allergen exposure reduced allergic responses in mice. However, the molecular mechanisms underlying the protective role of M. pneumoniae in allergic responses, particularly airway mucin production, remain unclear. Wild-type and Toll-like receptor 2 (TLR2)-deficient mice with a respiratory M. pneumoniae infection preceding allergen (ovalbumin) challenge were utilized to determine the regulatory role of TLR2-IFN-gamma signaling pathway in airway mucin expression. Furthermore, air-liquid interface cultures of mouse primary tracheal epithelial cells were performed to examine the effects of IFN-gamma on mucin expression. In wild-type mice, M. pneumoniae infection preceding allergen challenge significantly reduced airway mucins but increased IFN-gamma. In sharp contrast, in TLR2-deficient mice, M. pneumoniae preceding allergen challenge resulted in increased mucin protein without a noticeable change of IFN-gamma. In cultured mouse primary tracheal epithelial cells, IFN-gamma was shown to directly inhibit mucin expression in a dose-dependent manner. Our study demonstrates for the first time that a respiratory M. pneumoniae infection preceding allergen challenge reduces airway epithelial mucin expression in part through TLR2-IFN-gamma signaling pathway. A bacterial infection in asthmatic subjects with weakened TLR2-IFN-gamma signaling may result in an exaggerated airway mucin production.

Bacterial pneumonia remains a serious disease and is associated with neutrophil recruitment. Innate immunity is pivotal for the elimination of bacteria, and TLRs are essential in this process. Toll/IL-1R domain-containing adaptor inducing IFN-beta (TRIF) is an adaptor for TLR3 and TLR4, and is associated with the MyD88-independent cascade. However, the importance of TRIF in immune responses against pulmonary bacterial pathogens is not well understood. We investigated the involvement of TRIF in a murine model of Escherichia coli pneumonia. TRIF(-/-) mice infected with E. coli display attenuated neutrophil migration; NF-kappaB activation; and TNF-alpha, IL-6, and LPS-induced C-X-C chemokine production in the lungs. In addition, E. coli-induced phosphorylation of JNK, ERK, and p38 MAPK was detected in bone marrow-derived macrophages (BMMs) of TRIF(+/+) mice, but attenuated in BMMs of TRIF(-/-) mice. Furthermore, E. coli-induced TNF-alpha and IL-6 production was attenuated in BMMs of TRIF(-/-) mice. E. coli LPS-induced late MAPK activation, and TNF-alpha and IL-6 production were abolished in BMMs of TRIF(-/-) mice. Moreover, TRIF is not required for LPS-induced neutrophil influx, and keratinocyte cell-derived chemokine, MIP-2, and LPS-induced C-X-C chemokine production in the lungs. Using TLR3(-/-) mice, we ruled out the role of TLR3-mediated TRIF-dependent neutrophil influx during E. coli pneumonia. A TLR4-blocking Ab inhibited E. coli-induced TNF-alpha and IL-6 in BMMs of both TRIF(-/-) and TRIF(+/+) mice, suggesting that TRIF-mediated signaling involves TLR4. We also found that TRIF is critical to control E. coli burden in the lungs and E. coli dissemination. Thus, rapid activation of TRIF-dependent TLR4-mediated signaling cascade serves to augment pulmonary host defense against a Gram-negative pathogen.

Background: ECG and BNP have been assessed as screening tests for LVSD and heart failure. However, echocardiography also provides information about valvular disease and LVH. We assessed how good these screening tests are in identifying whether the subsequent echocardiogram will have any significant abnormality., Aims: To re-appraise the ECG and BNP as screening tests for echocardiography since there are important practical deficiencies in our current knowledge in this area., Methods: General practitioners referred suspected heart failure patients for clinical assessment, echocardiography, electrocardiography, and BNP measurement. The accuracy of each screening test and combinations of screening tests were calculated for LVSD, heart failure, valvular disease, and LVH., Results: The sensitivities of the ECG for LVSD, heart failure, LVH and valvular disease were 97%, 95%, 76%, and 69%, respectively. The corresponding figures for BNP were 86%, 82%, 59%, and 48%, respectively. When patients with atrial fibrillation and murmurs were excluded, the values for ECG were 94%, 87%, 53%, and 55%, while for BNP they were 83%, 73%, 50%, and 32%., Conclusions: ECG interpretation and BNP are adequate screening tests to detect LVSD or heart failure but fail to screen for other echocardiographic abnormalities, like valvular disease and LVH. This remains the case even if patients with atrial fibrillation or heart murmurs are excluded on the basis that they require echocardiography anyway.

Bacterial pneumonia is a leading cause of mortality and is associated with extensive neutrophil accumulation. Major pathogens associated with this disease include nonflagellated Klebsiella pneumoniae (Kp) and flagellated Pseudomonas aeruginosa (Pa). TLRs are essential for innate immune defense. TIRAP (Toll/IL-1R domain-containing adaptor protein) is an adaptor in TLR1, TLR2, TLR4, and TLR6 signaling, whereas MyD88 is an adaptor for all TLRs. However, the importance of TIRAP in pulmonary defense against Kp or Pa has not been examined. To demonstrate the role of TIRAP, TIRAP-deficient and wild-type littermates were intratracheally inoculated with Kp or Pa. We found that TIRAP(-/-) mice had substantial mortality, higher bacterial burden in the lungs, and enhanced dissemination following Kp challenge. Furthermore, Kp-induced neutrophil sequestration, histopathology, and MIP-2, TNF-alpha, IL-6, and LIX (lipopolysaccharide-induced CXC chemokine) production were attenuated in the lungs of TIRAP(-/-) mice. In contrast, TIRAP is not required for Pa-induced mortality, pulmonary bacterial burden, bacterial dissemination, neutrophil accumulation, or histopathology, yet it is necessary for MIP-2, TNF-alpha, and IL-6 production, but not LIX production. However, both Kp- and Pa-induced neutrophil influxes are MyD88 dependent. To determine the mechanisms associated with Pa-induced neutrophil accumulation, we inoculated mice with a flagellin C mutant of Pa (PaDeltafliC) or purified flagellin, a TLR5 agonist. PaDeltafliC-induced neutrophil sequestration and LIX expression are dependent on TIRAP, whereas flagellin-induced neutrophil influx and LIX expression are independent of TIRAP. These novel findings illustrate a pathogen-specific role for TIRAP in pulmonary defense and suggest that TLR5 plays an essential role for Pa-induced neutrophil influx via LIX production.

Pulmonary bacterial diseases are a leading cause of mortality in the U.S. Innate immune response is vital for bacterial clearance from the lung, and TLRs play a critical role in this process. Toll-IL-1R domain-containing adaptor protein (TIRAP) is a key molecule in the TLR4 and 2 signaling. Despite its potential importance, the role of TIRAP-mediated signaling in lung responses has not been examined. Our goals were to determine the role of TIRAP-dependent signaling in the induction of lung innate immune responses against Escherichia coli LPS and viable E. coli, and in lung defense against E. coli in mice. LPS-induced neutrophil sequestration; NF-kappaB translocation; keratinocyte cell-derived chemokine, MIP-2, TNF-alpha, and IL-6 expression; histopathology; and VCAM-1 and ICAM-1 expression were abolished in the lungs of TIRAP-/- mice. A cell-permeable TIRAP blocking peptide attenuated LPS-induced lung responses. Furthermore, immune responses in the lungs of TIRAP-/- mice were attenuated against E. coli compared with TIRAP+/+ mice. TIRAP-/- mice also had early mortality, higher bacterial burden in the lungs, and more bacterial dissemination following E. coli inoculation. Moreover, we used human alveolar macrophages to examine the role of TIRAP signaling in the human system. The TIRAP blocking peptide abolished LPS-induced TNF-alpha, IL-6, and IL-8 expression in alveolar macrophages, whereas it attenuated E. coli-induced expression of these cytokines and chemokines. Taken together, this is the first study illustrating the crucial role of TIRAP in the generation of an effective early immune response against E. coli LPS and viable E. coli, and in lung defense against a bacterial pathogen.

Cardiovascular risk scores are available but are often not calculated in busy clinics for numerous practical reasons. B-type natriuretic peptide (BNP) may be an alternative way of identifying subjects who have a high total cardiovascular risk score. We compared BNP level with Framingham 10-year Risk Scores for coronary heart disease and stroke and New Zealand Cardiovascular Risk Scores in 231 patients who had type 2 diabetes and no preexisting coronary heart disease or stroke. There was a significant correlation between log BNP and 10-year risk for coronary heart disease and stroke, and there were significantly higher BNP levels in those who had high cardiovascular risk as assessed by the New Zealand Risk Score. BNP may be a useful way of measuring total cardiovascular risk, thus having the potential to better target the most aggressive primary preventive therapies toward the most needy.

The lung is continuously exposed to bacteria and their products, and has developed a complex defense mechanism, including neutrophil recruitment. In mice, keratinocyte cell-derived chemokine and macrophage inflammatory protein-2 are the major chemokines for neutrophil recruitment into the lung. We have previously described a role for C-X-C chemokine (CXCL5) in neutrophil trafficking during lipopolysaccharide (LPS)-induced lung inflammation in mice. The aims of the present study were to identify the cellular origin of CXCL5 and to determine the signaling cascades that regulate its expression in the lung during LPS-induced inflammation and in isolated LPS-stimulated CXCL5-expressing cells. Our immunohistochemical analysis indicates that alveolar epithelial type II (AEII) cells are the primary source of CXCL5 in the rodent lung. These in vivo observations were confirmed with primary AEII cells. In addition, our data indicate that the Toll-like receptor 4 (TLR4) signaling cascade involving TLR4, myeloid differentiation factor 88, and Toll-IL-1R domain-containing adapter protein is required to induce CXCL5 expression in the lung. Furthermore, p38 and c-Jun N-terminal kinases are involved in lung CXCL5 expression. Similarly, TLR4, and p38 and c-Jun N-terminal kinases, are associated with LPS-induced CXCL5 expression in AEII cells. These novel observations demonstrate that activation of AEII cells via TLR4-dependent signaling is important for the production of CXCL5 in the lung exposed to LPS.

Excessive airway mucin production contributes to airway obstruction in lung diseases such as asthma and chronic obstructive pulmonary disease. Respiratory infections, such as atypical bacterium Mycoplasma pneumoniae (Mp), have been proposed to worsen asthma and chronic obstructive pulmonary disease in part through increasing mucin. However, the molecular mechanisms involved in infection-induced airway mucin overexpression remain to be determined. TLRs have been recently shown to be a critical component in host innate immune response to infections. TLR2 signaling has been proposed to be involved in inflammatory cell activation by mycoplasma-derived lipoproteins. In this study, we show that TLR2 signaling is critical in Mp-induced airway mucin expression in mice and human lung epithelial cells. Respiratory Mp infection in BALB/c mice activated TLR2 signaling and increased airway mucin. A TLR2-neutralizing Ab significantly reduced mucin expression in Mp-infected BALB/c mice. Furthermore, Mp-induced airway mucin was abolished in TLR2 gene-deficient C57BL/6 mice. Additionally, Mp was shown to increase human lung A549 epithelial cell mucin expression, which was inhibited by the overexpression of a human TLR2 dominant-negative mutant. These results clearly demonstrate that respiratory Mp infection increases airway mucin expression, which is dependent on the activation of TLR2 signaling.

Rationale: A growing literature indicates that hydroxy-methylglutaryl coenzyme A reductase inhibitors (statins) modulate proinflammatory cellular signaling and functions. No studies to date, however, have addressed whether statins modulate pulmonary inflammation triggered by aerogenic stimuli or whether they affect host defense., Objectives: To test whether lovastatin modulates LPS-induced pulmonary inflammation and antibacterial host defense., Methods: To address these questions, and to confirm any effect of statins as dependent on inhibition of hydroxy-methylglutaryl coenzyme A reductase, we treated C57Bl/6 mice with three oral doses of 10 mg/kg lovastatin (or vehicle) and three intraperitoneal doses of 10 mg/kg mevalonic acid (or saline), and then exposed them to the following: (1) aerosolized LPS, (2) intratracheal keratinocyte-derived chemokine (KC), or (3) intratracheal Klebsiella pneumoniae., Measurements and Main Results: LPS- and KC-induced airspace neutrophils were reduced by lovastatin, an effect that was blocked by mevalonic acid cotreatment. Lovastatin was also associated with reduced parenchymal myeloperoxidase and microvascular permeability, and altered airspace and serum cytokines after LPS. Native pulmonary clearance of K. pneumoniae was inhibited by lovastatin and extrapulmonary dissemination was enhanced, both reversibly with mevalonic acid. Ex vivo studies of neutrophils isolated from lovastatin-treated mice confirmed inhibitory effects on Rac activation, actin polymerization, chemotaxis, and bacterial killing., Conclusion: Lovastatin attenuates pulmonary inflammation induced by aerosolized LPS and impairs host defense.

Acute lung injury (ALI) induced by lipopolysaccharide (LPS) is a major cause of mortality among humans. ALI is characterized by microvascular protein leakage, neutrophil influx, and expression of proinflammatory mediators, followed by severe lung damage. LPS binding to its receptors is the crucial step in the causation of these multistep events. LPS binding and signaling involves CD14 and Toll-like receptor 4 (TLR4). However, the relative contributions of CD14 and TLR4 in the induction of ALI and their therapeutic potentials are not clear in vivo. Therefore, the aim of the present study was to compare the roles of CD14 and TLR4 in LPS-induced ALI to determine which of these molecules is the more critical target for attenuating ALI in a mouse model. Our results show that CD14 and TLR4 are necessary for low-dose (300-microg/ml) LPS-induced microvascular leakage, NF-kappaB activation, neutrophil influx, cytokine and chemokine (KC, macrophage inflammatory protein 2, tumor necrosis factor alpha, interleukin-6) expression, and subsequent lung damage. On the other hand, when a 10-fold-higher dose of LPS (3 mg/ml) was used, these responses were only partially dependent on CD14 and they were totally dependent on TLR4. The CD14-independent LPS response was dependent on CD11b. A TLR4 blocking antibody abolished microvascular leakage, neutrophil accumulation, cytokine responses, and lung pathology with a low dose of LPS but only attenuated the responses with a high dose of LPS. These data are the first to demonstrate that LPS-induced CD14-dependent and -independent (CD11b-dependent) signaling pathways in the lung are entirely dependent on TLR4 and that blocking TLR4 might be beneficial in lung diseases caused by LPS from gram-negative pathogens.

Mortality associated with acute lung injury (ALI) induced by lipopolysaccharide (LPS) remains high in humans, warranting improved treatment and prevention strategies. ALI is characterized by the expression of proinflammatory mediators and extensive neutrophil influx into the lung, followed by severe lung damage. Understanding the pathogenesis of LPS-induced ALI is a prerequisite for designing better therapeutic strategies. In the present study, we used microarrays to gain a global view of the transcriptional responses of the lung to LPS in a mouse model of ALI that mimics ALI in humans. A total of 71 inflammation-associated genes were up-regulated in LPS-treated lungs, including a chemokine, LPS-induced CXC chemokine (LIX), whose role in the induction of ALI is unknown. Most of the inflammatory genes peaked at 2 h post-LPS treatment. Real-time reverse transcription-PCR confirmed the LPS-induced up-regulation of selected genes identified by microarray analysis, including LIX. The up-regulation of LIX, tumor necrosis factor alpha, and macrophage inflammatory protein 2 was confirmed at the protein level by enzyme-linked immunosorbent assays. To determine the role of LIX in the induction of ALI, we used both exogenous LIX and a LIX blocking antibody. Exogenous LIX alone elicited a neutrophil influx in the lungs, and the anti-LIX antibody attenuated the LPS-induced neutrophil accumulation in the lungs. Taken together, the results of our study demonstrate for the first time the temporal expression of inflammatory genes during LPS-induced ALI and suggest that early therapeutic intervention is crucial to attenuate lung damage. Moreover, we identified a role for LIX in the induction of ALI, and therefore LIX may serve as a novel therapeutic target for the minimization of ALI.

Bovine pneumonic pasteurellosis continues to be a major respiratory disease in feedlot cattle despite the recent advances in our understanding of the underlying complexities of causation. The etiological agent, Mannheimia haemolytica, possesses several virulence factors, including capsule, outer membrane proteins, adhesins, neuraminidase, endotoxin and exotoxic leukotoxin. Accumulating scientific evidence implicates leukotoxin as the primary factor contributing to clinical presentation and lung injury associated with this disease. Unlike other virulence factors, leukotoxin shows cell-type- and species-specific effects on bovine leukocytes. Recent investigations have delineated the mechanisms underlying the target-cell-specificity of leukotoxin and how this contributes to the pathogenesis of lung damage. This review summarizes current understanding of the secretion, regulation, mechanisms of action and evolutionary diversity of leukotoxin of M. haemolytica. Understanding the precise molecular mechanisms of leukotoxin is critical for the development of more effective prophylactic and therapeutic strategies to control this complex disease.

The leukotoxin (LktA) produced by Mannheimia haemolytica binds to bovine lymphocyte function-associated antigen 1 (LFA-1) and induces biological effects in bovine leukocytes in a cellular and species-specific fashion. We have previously shown that LktA also binds to porcine LFA-1 without eliciting any effects. These findings suggest that the specificity of LktA effects must entail both binding to LFA-1 and activation of signaling pathways which are present in bovine leukocytes. However, the signaling pathways leading to biological effects upon LktA binding to LFA-1 have not been characterized. In this context, several reports have indicated that ligand binding to LFA-1 results in activation of a nonreceptor tyrosine kinase (NRTK) signaling cascade. We designed experiments with the following objectives: (i) to determine whether LktA binding to LFA-1 leads to activation of NRTKs, (ii) to examine whether LktA-induced NRTK activation is target cell specific, and (iii) to determine whether LktA-induced NRTK activation is required for biological effects. We used a biologically inactive mutant leukotoxin (DeltaLktA) for comparison with LktA. Our results indicate that LktA induces tyrosine phosphorylation (TP) of the CD18 tail of LFA-1 in bovine leukocytes. The DeltaLktA mutant does not induce TP of the CD18 tail, albeit binding to bovine LFA-1. LktA-induced TP of the CD18 tail was attenuated by an NRTK inhibitor, herbimycin A; a phosphatidylinositol 3'-kinase (PI 3-kinase) inhibitor, wortmannin; and a Src kinase inhibitor, PP2, in a concentration-dependent manner. Furthermore, LktA induces TP of the CD18 tail in bovine, but not porcine, leukocytes. Moreover, LktA-induced intracellular calcium ([Ca2+]i) elevation was also inhibited by herbimycin A, wortmannin, and PP2. Thus, our data represent the first evidence that binding of LktA to bovine LFA-1 induces a species-specific NRTK signaling cascade involving PI 3-kinase and Src kinases and that this signaling cascade is required for LktA-induced biological effects.

Preparation of activated carbon from sewage sludge is a promising way to dispose of sewage sludge as well as to produce a low-cost adsorbent for pollutant removal. This research work aimed to optimise the condition for activated carbon preparation from anaerobically digested sewage sludge with the additive coconut husk. The sewage sludge sample was mixed with the additive coconut husk. The preparation condition variables investigated involved the concentration of the ZnCl2 solutions, heating temperature, dwell time and heating rate in pyrolysis and the mixing ratio of coconut husk to sewage sludge. Surface area, pore size distribution, aqueous phenol adsorption capacity and the production yield of the final products were determined and compared. Experimental results revealed that low concentrations of ZnCl2 solution tended to improve the microporosity of the final product. Heating temperature had a considerable impact on the surface area, pore size distribution and phenol adsorption capacity of the final products, whereas dwell time and heating rate performed comparatively insignificantly. The effect of increasing the mixing ratio of coconut husk to sewage sludge was principally to increase the microporosity of the final products. The activated carbon with the highest BET surface area was produced with the activation of 5 M ZnCl2 solution and, thereafter, pyrolysis at a heating temperature of 500 degrees C for 2 h with a heating rate of 10 degrees C/min. The mixing ratio of 1:4 in terms of coconut husk to sewage sludge based on their dried weights was found to be most cost effective.

Disposal of sewage sludge is an increasingly expensive and environmentally sensitive problem throughout the world. Preparation of activated carbon from sewage sludge offers an attractive re-use alternative to the traditional disposal routes. The objective of this research work was to compare anaerobically digested sewage sludge (DS) and undigested sewage sludge (US) as source materials in the preparation of activated carbons. Prior to the preparation the properties of the two types of sewage sludges were determined and compared. Subsequently the sludge samples were activated with 5 M ZnCl2 solution and thereafter pyrolysed at heating temperature of 650 degrees C for 2 h with the heating rate of 15 degrees C/min under a nitrogen atmosphere. The produced activated carbons were characterised by surface area and porosity analysis, CHN elemental composition and ash contents determination, and aqueous phase phenol adsorption tests. The results indicate that in comparison with the DS, the US had a higher carbon content and lower ash content, and accordingly yielded a better activated carbon with a higher BET surface area, pore volume, carbon content and phenol adsorption capacity.

Pasteurella (Mannheimia) haemolytica leukotoxin (Lkt) and lipopolysaccharide (LPS) are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Previous studies have characterized in vitro responses of bovine alveolar macrophages (AMs) to Lkt and LPS. Activation of AMs with Lkt or LPS causes induction of proinflammatory cytokines, and Lkt causes cytolysis of AMs at higher concentrations. Since AMs are exposed to both of these bacterial virulence factors during disease, previous studies may have underestimated the possibility of functional interactions between Lkt and LPS. The purpose of this study was to characterize the effect of simultaneous exposure to both Lkt and LPS on AM cytolysis and proinflammatory cytokine expression. Using cellular leakage of lactate dehydrogenase as an indirect measure of cytolysis, we studied AM responses to Lkt alone, LPS alone and Lkt+LPS. We found that 80-200 pg/ml LPS, which does not itself cause cytolysis, synergistically enhanced the cytolysis induced by 2-5 Lkt units (LU)/ml Lkt. Northern blot analysis demonstrated that synergism between Lkt and LPS resulted in increased levels of IL-8 mRNA, and that the kinetic patterns of TNF-alpha and IL-8 mRNA expression induced by Lkt+LPS differed from those induced by each agent separately. Finally, the WEHI 164 (clone 13) bioassay was used to show that Lkt/LPS synergism resulted in enhanced secretion of biologically active TNF-alpha. These results provide direct evidence of synergism between Lkt and LPS in AM cytolysis and inflammatory cytokine expression. Additional studies to characterize the molecular basis of this phenomenon are indicated., (Copyright 2001 Academic Press.)

Pasteurella (Mannheimia) haemolytica leukotoxin (Lkt) is the major factor that contributes to lung injury in bovine pneumonic pasteurellosis. Lkt is a pore-forming exotoxin that has the unique property of inducing cytolysis only in ruminant leukocytes and platelets. Cytolysis of many cell types is mediated by arachidonic acid (AA) and its generation by phospholipases is regulated by G-protein-coupled receptors. However, the contribution of Lkt-induced AA generation to cytolysis and the signalling cascade underlying AA generation in bovine leukocytes have not been determined. We have determined whether AA mediates Lkt-induced cytolysis and delineated the signalling mechanisms underlying AA generation in bovine leukocytes. Bovine lymphoma cells were used as an experimental system to investigate the Lkt-induced [(3)H] AA release, an index of AA generation and lactate dehydrogenase release, an index of cytolysis. The results indicate that Lkt induces AA release and cytolysis in a concentration- and time-dependent fashion. The AA analog, 5,8,11,14-eicosatetraynoic acid inhibited Lkt-induced cytolysis, but not AA release. Lkt-induced AA release and cytolysis were inhibited by pertussis toxin, inhibitors of cytosolic phospholipase A(2)(cPLA(2)), phospholipase C and protein kinase C (PKC), and by chelation of intracellular calcium. Furthermore, Western blot analysis revealed the presence of G(i), G(s)and G(q)type G-proteins. These results demonstrate that AA metabolites from cPLA(2)activation contribute to Lkt-induced cytolysis and G(i)type G-proteins, Ca(2+)and PKC, regulate the cPLA(2)activity., (Copyright 2001 Crown Copyright.)

Objective: To evaluate a new pattern of electrical of electrical stimulation as a treatment for stress incontinence., Design: A randomized, double-blind, controlled trial., Setting: The study took place on three clinical sites., Subjects: Patients (n = 27) with urodynamically proven stress incontinence recruited via consultant referral., Interventions: Patients were randomly allocated to one of two groups: the new pattern of stimulation or sham stimulation., Main Outcome Measures: Patients were assessed pre, mid and post treatment using: perineometry, digital assessment and pad testing. The following were only used pre and post treatment: seven-day frequency/volume chart, SF-36, the Incontinence Impact Questionnaire and the Urogenital Distress Inventory., Results: No significant between-group differences were highlighted except when quality of life was assessed with the Urogenital Distress Inventory (p = 0.01). A significant reduction in scores was observed in the stimulation group (p = 0.03) However, improvements were seen in both the strength and endurance characteristics of the pelvic floor musculature, although these changes were not translated into a reduction in symptoms., Conclusion: Although promising, the improvement in pelvic floor function did not result in a reduction in symptoms in all patients. Further research is required to investigate the effects of the new stimulation in combination with pelvic floor exercises and to compare the new stimulation pattern with existing forms of electrical stimulation.

Much research has been conducted into the use of electrical stimulation to restore function in weak/atrophied muscle and it is used widely in the field of muscle rehabilitation. As stress incontinence is a condition which is the result of pelvic floor muscle weakness, it is thought that the symptoms of this condition may be alleviated once the strength and endurance characteristics of this muscle group have been improved. Many studies have been conducted to evaluate the efficacy of various types of electrical stimulation, although definitive conclusions have yet to be drawn. Current forms of electrical stimulation for stress incontinence involve the use of uniform frequencies. In animal studies this type of stimulation has been shown to have drawbacks that are unacceptable when trying to rehabilitate muscle. Consequently, there is a need to develop more physiological patterns of stimulation that will enhance both strength and endurance characteristics without causing premature fatigue.

Pasteurella (Mannheimia) haemolytica leukotoxin (Lkt) causes cell type- and species-specific effects in ruminant leukocytes. Recent studies indicate that P. haemolytica Lkt binds to bovine CD18, the common subunit of all beta2 integrins. We designed experiments with the following objectives: to identify which member of the beta2 integrins is a receptor for Lkt; to determine whether Lkt binding to the receptor is target cell (bovine leukocytes) specific; to define the relationships between Lkt binding to the receptor, calcium elevation, and cytolysis; and to determine whether a correlation exists between Lkt receptor expression and the magnitude of target cell cytolysis. We compared Lkt-induced cytolysis in neutrophils from control calves and from calves with bovine leukocyte adhesion deficiency (BLAD), because neutrophils from BLAD-homozygous calves exhibit reduced beta2 integrin expression. The results demonstrate for the first time that Lkt binds to bovine CD11a and CD18 (lymphocyte function-associated antigen 1 [LFA-1]). The binding was abolished by anti-CD11a or anti-CD18 monoclonal antibody (MAb). Lkt-induced calcium elevation in bovine alveolar macrophages (BAMs) was inhibited by anti-CD11a or anti-CD18 MAb (65 to 94% and 37 to 98%, respectively, at 5 and 50 Lkt units per ml; P < 0.05). Lkt-induced cytolysis in neutrophils and BAMs was also inhibited by anti-CD11a or anti-CD18 MAb in a concentration-dependent manner. Lkt bound to porcine LFA-1 but did not induce calcium elevation or cytolysis. In neutrophils from BLAD calves, Lkt-induced cytolysis was decreased by 44% compared to that of neutrophils from control calves (P < 0.05). These results indicate that LFA-1 is a Lkt receptor, Lkt binding to LFA-1 is not target cell specific, Lkt binding to bovine LFA-1 correlates with calcium elevation and cytolysis, and bovine LFA-1 expression correlates with the magnitude of Lkt-induced target cell cytolysis.

In bovine alveolar macrophages (BAMs), exposure to leukotoxin (Lkt) and endotoxin (LPS) from Pasteurella haemolytica results in expression of inflammatory cytokine genes and intracellular calcium ([Ca2+]i) elevation. Leukotoxin from P. haemolytica interacts only with leukocytes and platelets from ruminant species. Upregulation of cytokine genes in different cells by LPS involves activation of the transcription factor NF-kappaB (NF-kappaB), resulting in its translocation from the cytoplasm to the nucleus. Using immunocytochemical staining and confocal imaging, we studied whether NF-kappaB activation represents a common mechanism for the expression of multiple cytokine genes in BAMs (Lkt-susceptible cells) stimulated with Lkt and LPS. Bovine pulmonary artery endothelial cells and porcine alveolar macrophages were used as nonsusceptible cells. The role of Ca2+ and tyrosine kinases in NF-kappaB activation and inflammatory cytokine gene expression was studied, since an inhibitor of tyrosine kinases attenuates LPS-induced [Ca2+]i elevation in BAMs. The results are summarized as follows: (a) Lkt induced NF-kappaB activation and [Ca2+]i elevation only in BAMs, while LPS effects were demonstrable in all cell types; (b) chelation of [Ca2+]i blocked NF-kappaB activation and IL-1beta, TNFalpha, and IL-8 mRNA expression; and (c) tyrosine kinase inhibitor herbimycin A blocked expression of all three cytokine genes in BAMs stimulated with Lkt, while only the expression of IL-1beta was blocked in BAMs stimulated with LPS. We conclude that cytokine gene expression in BAMs requires NF-kappaB activation and [Ca2+]i elevation, and Lkt effects exhibit cell type- and species specificity., (Copyright 1999 Academic Press.)

Leukotoxin and endotoxin derived from Pasteurella haemolytica serotype 1 are the primary virulence factors contributing to the pathogenesis of lung injury in bovine pneumonic pasteurellosis. Activation of bovine alveolar macrophages with endotoxin or leukotoxin results in the induction of cytokine gene expression, with different kinetics (H. S. Yoo, S. K. Maheswaran, G. Lin, E. L. Townsend, and T. R. Ames, Infect. Immun. 63:381-388, 1995; H. S. Yoo, B. S. Rajagopal, S. K. Maheswaran, and T. R. Ames, Microb. Pathog. 18:237-252, 1995). Furthermore, extracellular Ca2+ is required for leukotoxin-induced cytokine gene expression. However, the involvement of Ca2+ in endotoxin effects and the precise signaling mechanisms in the regulation of intracellular Ca2+ by leukotoxin and endotoxin are not known. In fura-2-acetoxymethyl ester-loaded alveolar macrophages, intracellular Ca2+ regulation by leukotoxin and endotoxin was studied by video fluorescence microscopy. Leukotoxin induced a sustained elevation of intracellular Ca2+ in a concentration-dependent fashion by influx of extracellular Ca2+ through voltage-gated channels. In the presence of fetal bovine serum, endotoxin elevated intracellular Ca2+ even in the absence of extracellular Ca2+. Leukotoxin-induced intracellular Ca2+ elevation was inhibited by pertussis toxin, inhibitors of phospholipases A2 and C, and the arachidonic acid analog 5,8,11,14-eicosatetraynoic acid. Intracellular Ca2+ elevation by endotoxin was inhibited by inhibitors of phospholipase C and protein tyrosine kinase, but not by pertussis toxin, or the arachidonic acid analog. To the best of our knowledge, this is the first report of Ca2+ signaling by leukotoxin through a G-protein-coupled mechanism involving activation of phospholipases A2 and C and release of arachidonic acid in bovine alveolar macrophages. Ca2+ signaling by endotoxin, on the other hand, involves activation of phospholipase C and requires tyrosine phosphorylation. The differences in the Ca2+ signaling mechanisms may underlie the reported temporal differences in gene expression during leukotoxin and endotoxin activation.

A patient with Mönckeberg's calcinosis is presented in whom a pneumatic limb tourniquet failed to be effective because of calcification of the femoral artery wall. Bleeding from the operation site was noticed to be appreciably greater while the tourniquet was inflated since the cuff, though not occluding the femoral artery, acted as a very effective venous tourniquet. Theoretical risks which might be associated with the use of tourniquets in the presence of arterial calcification are fracture of the calcified vessel wall and systemic overdose of local anaesthetic agent following attempted regional intravenous block. The problem of tourniquet failure in general, and the dangers which might be associated with the use of tourniquets in patients with incompressible arteries, are briefly discussed.