Mechanical ventilation enhances Acinetobacter baumannii-induced lung injury through JNK pathways.

BACKGROUND: Patients in intensive care units (ICUs) often received broad-spectrum antibiotic treatment and Acinetobacter baumannii (A.b.) and Pseudomonas aeruginosa (P.a.) were the most common pathogens causing ventilator-associated pneumonia (VAP). This study aimed to examine the effects and mechanism of mechanical ventilation (MV) on A.b.-induced lung injury and the involvement of alveolar macrophages (AMs). METHODS: C57BL/6 wild-type (WT) and c-Jun N-terminal kinase knockout (JNK1-/-) mice received MV for 3 h at 2 days after nasal instillation of A.b., P.a. (1 × 106 colony-forming unit, CFU), or normal saline. RESULTS: Intranasal instillation of 106 CFU A.b. in C57BL/6 mice induced a significant increase in total cells and protein levels in the bronchoalveolar lavage fluid (BALF) and neutrophil infiltration in the lungs. MV after A.b. instillation increases neutrophil infiltration, interleukin (IL)-6 and vascular cell adhesion molecule (VCAM) mRNA expression in the lungs and total cells, IL-6 levels, and nitrite levels in the BALF. The killing activity of AMs against A.b. was lower than against P.a. The diminished killing activity was parallel with decreased tumor necrosis factor-α production by AMs compared with A.b. Inducible nitric oxide synthase inhibitor, S-methylisothiourea, decreased the total cell number in BALF on mice receiving A.b. instillation and ventilation. Moreover, MV decreased the A.b. and P.a. killing activity of AMs. MV after A.b. instillation induced less total cells in the BALF and nitrite production in the serum of JNK1-/- mice than those of WT mice. CONCLUSION: A.b. is potent in inducing neutrophil infiltration in the lungs and total protein in the BALF. MV enhances A.b.-induced lung injury through an increase in the expression of VCAM and IL-6 levels in the BALF and a decrease in the bacteria-killing activity of AMs. A lower inflammation level in JNK1-/- mice indicates that A.b.-induced VAP causes lung injury through JNK signaling pathway in the lungs.

GSKIP- and GSK3-mediated anchoring strengthens cAMP/PKA/Drp1 axis signaling in the regulation of mitochondrial elongation.

GSK3ß binding of GSKIP affects neurite outgrowth, but the physiological significance of PKA binding to GSKIP remains to be determined. We hypothesized that GSKIP and GSK3ß mediate cAMP/PKA/Drp1 axis signaling and modulate mitochondrial morphology by forming a working complex comprising PKA/GSKIP/GSK3ß/Drp1. We demonstrated that GSKIP wild-type overexpression increased phosphorylation of Drp1 S637 by 7-8-fold compared to PKA kinase-inactive mutants (V41/L45) and a GSK3ß binding-defective mutant (L130) under H2O2 and forskolin challenge in HEK293 cells, indicating that not only V41/L45, but also L130 may be involved in Drp1-associated protection of GSKIP. Interestingly, silencing either GSKIP or GSK3ß but not GSK3α resulted in a dramatic decrease in Drp1 S637 phosphorylation, revealing that both GSKIP and GSK3ß are required in this novel PKA/GSKIP/GSK3ß/Drp1 complex. Moreover, overexpressed kinase-dead GSK3ß-K85R, which retains the capacity to bind GSKIP, but not K85M which shows total loss of GSKIP-binding, has a higher Drp1 S637 phosphorylation similar to the GSKIP wt overexpression group, indicating that GSK3ß recruits Drp1 by anchoring rather than in a kinase role. With further overexpression of either V41/L45P or the L130P GSKIP mutant, the elongated mitochondrial phenotype was lost; however, ectopically expressed Drp1 S637D, a phosphomimetic mutant, but not S637A, a non-phosphorylated mutant, restored the elongated mitochondrial morphology, indicating that Drp1 is a downstream effector of direct PKA signaling and possibly has an indirect GSKIP function involved in the cAMP/PKA/Drp1 signaling axis. Collectively, our data revealed that both GSKIP and GSK3ß function as anchoring proteins in the cAMP/PKA/Drp1 signaling axis modulating Drp1 phosphorylation.

Pseudomonas aeruginosa colonization enhances ventilator-associated pneumonia-induced lung injury.

BACKGROUND: Pseudomonas aeruginosa (PA) is the single-most common pathogen of ventilator-associated pneumonia (VAP). Large quantities of PA in the trachea of ventilated patients are associated with an increased risk of death. However, the role of PA colonization in PA VAP-induced lung injury remains elusive. This study examined the effect and mechanism of PA colonization in VAP-induced lung injury. METHODS: C57BL/6 wild-type (WT) and c-Jun N-terminal kinase knockout (JNK1(-/-)) mice received mechanical ventilation for 3 h at 2 days after receiving nasal instillation of PA (1 × 10(6) colony forming unit) or normal saline. RESULTS: Intranasal instillation of PA or mechanical ventilation induced the expression of interleukin-6 (IL-6) in the lungs. Phospho-JNK protein expression in the lungs was significantly increased in mice receiving mechanical ventilation after PA instillation as compared with those receiving ventilation alone. Mechanical ventilation after PA instillation significantly increased the expression of tumor necrosis factor-α (TNF-α), IL-1ß, and macrophage inflammatory protein-2 (MIP-2) proteins; neutrophil sequestration; and TNF-α, IL-1ß, and IL-6 levels in the lungs of WT mice, but not in JNK1(-/-) mice. CONCLUSION: PA colonization plays an important role in PA VAP-induced lung injury through the induction of JNK1-mediated inflammation. PA-induced VAP causes lung injury through JNK signaling pathway in the lungs. JNK inhibition in ICU patients with higher percentages of PA colonization may reduce VAP-induced lung injury and mortality.

Toll-like receptor stimulation induces nondefensin protein expression and reverses antibiotic-induced gut defense impairment.

Prior antibiotic exposure is associated with increased mortality in Gram-negative bacteria-induced sepsis. However, how antibiotic-mediated changes of commensal bacteria promote the spread of enteric pathogenic bacteria in patients remains unclear. In this study, the effects of systemic antibiotic treatment with or without Toll-like receptor (TLR) stimulation on bacterium-killing activity, antibacterial protein expression in the intestinal mucosa, and bacterial translocation were examined in mice receiving antibiotics with or without oral supplementation of dead Escherichia coli or Staphylococcus aureus. We developed a systemic ampicillin, vancomycin, and metronidazole treatment protocol to simulate the clinical use of antibiotics. Antibiotic treatment decreased the total number of bacteria, including aerobic bacteria belonging to the family Enterobacteriaceae and the genus Enterococcus as well as organisms of the anaerobic genera Lactococcus and Bifidobacterium in the intestinal mucosa and lumen. Antibiotic treatment significantly decreased the bacterium-killing activity of the intestinal mucosa and the expression of non-defensin-family proteins, such as RegIIIß, RegIIIγ, C-reactive protein-ductin, and RELMß, but not the defensin-family proteins, and increased Klebsiella pneumoniae translocation. TLR stimulation after antibiotic treatment increased NF-κB DNA binding activity, nondefensin protein expression, and bacterium-killing activity in the intestinal mucosa and decreased K. pneumoniae translocation. Moreover, germfree mice showed a significant decrease in nondefensin proteins as well as intestinal defense against pathogen translocation. Since TLR stimulation induced NF-κB DNA binding activity, TLR4 expression, and mucosal bacterium-killing activity in germfree mice, we conclude that the commensal microflora is critical in maintaining intestinal nondefensin protein expression and the intestinal barrier. In turn, we suggest that TLR stimulation induces nondefensin protein expression and reverses antibiotic-induced gut defense impairment.

Blocking TNF-α enhances Pseudomonas aeruginosa-induced mortality in burn mice through induction of IL-1ß.

PURPOSE: Tumor necrosis factor (TNFα) is a proinflammatory cytokine and has been a target for intervention in human sepsis. However, inhibition of TNF-α with a high dose of a TNF-receptor fusion protein in patients with septic shock worsened patient survival. This study was designed to investigate whether blocking TNF-α enhances mortality in infected burn mice through the induction of IL-1ß. METHODS: WT or Tnfrsf1a(-/-) mice received Pseudomonas aeruginosa injection in the back at 8h after burn injury. The animals were sacrificed at 24h after burn and lung tissues were harvested and examined for determining myeloperoxidase (MPO) activity, pulmonary microvascular dysfunction, NF-κB DNA binding activity, and IL-1ß expression. Also, the lung and blood were harvested for bacterial count assay. RESULT: Thermal injury alone induced NF-κB DNA binding activity and neutrophil infiltration in the lung in WT but not in Tnfrsf1a(-/-) mice. A 50% total body surface area (TBSA) burn induced a significant increase of mortality in WT compared with Tnfrsf1a(-/-) mice. In contrast, P. aeruginosa injection with a 30% TBSA burn pretreatment enhanced IL-1ß expression, bacterial counts in lung and blood, pulmonary microvascular dysfunction, and mortality in Tnfrsf1a(-/-) mice compared with WT mice. Injection of the IL-1 receptor antagonist, Anakinra, reduced P. aeruginosa infection with burn pretreatment-induced blood bacterial counts, IL-1ß levels as well as permeability of lung, and mortality in Tnfrsf1a(-/-) mice. CONCLUSIONS: Our findings suggest that thermal injury induces lung NF-κB activation and neutrophil sequestration through TNFα signaling. However, blocking TNF-α enhances P. aeruginosa infection-induced lung damage in burn mice via induction of IL-1ß. Using an IL-1 receptor antagonist combined with the neutralization of TNF-α could be a useful strategy for decreasing P. aeruginosa infection-induced mortality in burn patients.

NF-κB activation in myeloid cells mediates ventilator-induced lung injury.

BACKGROUND: Although use of the mechanical ventilator is a life-saving intervention, excessive tidal volumes will activate NF-κB in the lung with subsequent induction of lung edema formation, neutrophil infiltration and proinflammatory cytokine/chemokine release. The roles of NF-κB and IL-6 in ventilator-induced lung injury (VILI) remain widely debated. METHODS: To study the molecular mechanisms of the pathogenesis of VILI, mice with a deletion of IкB kinase in the myeloid cells (IKKß(Δmye)), IL-6(-/-) to WT chimeric mice, and C57BL/6 mice (WT) were placed on a ventilator for 6 hr.WT mice were also given an IL-6-blocking antibody to examine the role of IL-6 in VILI. RESULTS: Our results revealed that high tidal volume ventilation induced pulmonary capillary permeability, neutrophil sequestration, macrophage drifting as well as increased protein in bronchoalveolar lavage fluid (BALF). IL-6 production and IL-1ß, CXCR2, and MIP2 expression were also increased in WT lungs but not in those pretreated with IL-6-blocking antibodies. Further, ventilator-induced protein concentrations and total cells in BALF, as well as lung permeability, were all significantly decreased in IKKß(Δmye) mice as well as in IL6(-/-) to WT chimeric mice. CONCLUSION: Given that IKKß(Δmye) mice demonstrated a significant decrease in ventilator-induced IL-6 production, we conclude that NF-κB-IL-6 signaling pathways induce inflammation, contributing to VILI, and IкB kinase in the myeloid cells mediates ventilator-induced IL-6 production, inflammation, and lung injury.

TNF-alpha decreases infection-induced lung injury in burn through negative regulation of TLR4/iNOS.

BACKGROUND: Sepsis is an infectious process-induced generalized inflammatory response that mediates the excessive production of cytokines. However, anti-tumor necrosis factor (TNF)-α therapy has failed in decreasing mortality of sepsis patients due to undefined mechanisms. This study was designed to investigate whether absence of TNF receptor enhanced lung damage and mortality through toll-like receptors (TLRs) and inducible nitric oxide synthase (iNOS). MATERIALS AND METHODS: We injected Pseudomonas aeruginosa or lipopolysaccharide in the backs of wild-type, Tnfrsf1a(-/-) (deficient of TNF-α receptor 1), and TLR4(-/-) mice at 8 h after 30% total body surface area burn. The animals were sacrificed at 16 h after burn and lung tissues were harvested and examined for determining pulmonary microvascular dysfunction and interleukin (IL)-1ß, iNOS, and TLR4 expression. The blood of animals was harvested for bacterial count assay. The effect of S-methylisothiourea, an iNOS inhibitor, on P aeruginosa infection with thermal injury pretreatment-induced lung damage was also examined. RESULTS: P aeruginosa or lipopolysaccharide injection with thermal injury pretreatment enhanced TLR4, iNOS, and IL-1ß expression and pulmonary microvascular dysfunction in Tnfrsf1a(-/-) mice compared with wild-type mice. P aeruginosa infection with thermal injury pretreatment did not induce IL-1ß or iNOS expression and mortality in TLR4(-/-) mice. S-methylisothiourea treatment significantly decreased P aeruginosa infection with thermal injury pretreatment-induced lung injury, blood bacterial counts, pulmonary IL-1ß expression, and mortality in Tnfrsf1a(-/-) mice. CONCLUSIONS: Given that absence of the TNF-α receptor 1 is associated with increased lung permeability, we conclude that TNF-α decreases P aeruginosa infection-induced lung damage in burn mice through negative regulation of TLR4 as well as iNOS expression, and iNOS inhibitor might be useful in reversing anti-TNF-α therapy-induced lung injury in burn.

Gut flora enhance bacterial clearance in lung through toll-like receptors 4.

BACKGROUND: The influence of the gut flora on lung inflammatory reaction against bacterial challenge remains undefined. This study was designed to investigate whether gut flora enhances lung defense against E.coli pneumonia through TLR4 signaling. METHODS: C3H/HeN (WT) mice and C3H/HeJ (TLR4 deficient) mice were treated with antibiotics in drinking water for 4 weeks to deplete gut commensal microflora. At week 3, drinking water was supplemented with lipopolysaccharide (LPS); a ligand for TLR4, to trigger TLRs in intestinal tract. At the end of 4th week, E.coli was injected to trachea to induce E.coli pneumonia. RESULTS: We found that commensal depletion by antibiotic pretreatment before E.coli pneumonia challenge induced a 30% decrease of MPO activity in the lung, a significant decrease of bacterial killing activity of alveolar macrophage, and bacterial counts in C3H/HeN mice but not in C3H/HeJ (TLR4 deficient) mice. LPS, a TLR4 ligand, supplementation during antibiotic pretreatment reversed these effects and decreased E.coli pneumonia-induced mortality in C3H/HeN mice. Furthermore, commensal depletion induced a suppression of NF-κB DNA binding activity and an increase of KC, MIP-2, IL-1ß expression in the lung in C3H/HeN mice but not in C3H/HeJ mice. CONCLUSIONS: Taken together with that commensal depletion increased E.coli pneumonia-induced mortality and LPS supplementation decreased it, we conclude that gut flora enhances bacterial clearance against E.coli pneumonia through TLR4.

Nanodome solar cells with efficient light management and self-cleaning.

Here for the first time, we demonstrate novel nanodome solar cells, which have periodic nanoscale modulation for all layers from the bottom substrate, through the active absorber to the top transparent contact. These devices combine many nanophotonic effects to both efficiently reduce reflection and enhance absorption over a broad spectral range. Nanodome solar cells with only a 280 nm thick hydrogenated amorphous silicon (a-Si:H) layer can absorb 94% of the light with wavelengths of 400-800 nm, significantly higher than the 65% absorption of flat film devices. Because of the nearly complete absorption, a very large short-circuit current of 17.5 mA/cm(2) is achieved in our nanodome devices. Excitingly, the light management effects remain efficient over a wide range of incident angles, favorable for real environments with significant diffuse sunlight. We demonstrate nanodome devices with a power efficiency of 5.9%, which is 25% higher than the flat film control. The nanodome structure is not in principle limited to any specific material system and its fabrication is compatible with most solar manufacturing; hence it opens up exciting opportunities for a variety of photovoltaic devices to further improve performance, reduce materials usage, and relieve elemental abundance limitations. Lastly, our nanodome devices when modified with hydrophobic molecules present a nearly superhydrophobic surface and thus enable self-cleaning solar cells.

Peritonitis-induced peroxynitrite and lung damage depends on c-Jun NH2-terminal kinase signaling of hematopoietic cells.

OBJECTIVES: Abdominal sepsis is a common, life-threatening condition in critically ill patients, and pseudomonas peritonitis remains a serious clinical complication of peritoneal dialysis. This study was performed to determine whether peritonitis induces lung damage through the c-Jun NH2-terminal kinase. DESIGN: : Prospective, experimental study. SETTING: Research laboratory at a university hospital. SUBJECTS: Peritonitis models in the mice. INTERVENTIONS: Wild-type, c-Jun NH2-terminal kinase1, and c-Jun NH2-terminal kinase1 mice were subjected to peritonitis. A c-Jun NH2-terminal kinase inhibitor, SP600125 or leflunomide, was administered to mice immediately after peritonitis. MEASUREMENTS AND MAIN RESULTS: The changes of plasma dihydrorhodamine 123 oxidation level, the myeloperoxidase activity, and extravasations of Evans blue dye of lung in wild-type mice with or without c-Jun NH2-terminal kinase inhibitor; c-Jun NH2-terminal kinase1 mice and c-Jun NH2-terminal kinase1 mice; and chimeric mice (wild-type --> wild-type, c-Jun NH2-terminal kinase1 --> wild-type) with Pseudomonas aeruginosa-induced peritonitis were determined to evaluate the role of c-Jun NH2-terminal kinase signaling of the hematopoietic cells in peritonitis-induced lung damage. Our results showed that peritonitis induced dihydrorhodamine 123 oxidation, myeloperoxidase activity, activator protein-1 (AP-1) DNA binding activity, phosphorylated-c-Jun NH2-terminal kinase and inducible nitric oxide synthase expression, and Evans blue dye extravasations in lungs, and administration of specific c-Jun NH2-terminal kinase inhibitor decreased the peritonitis-induced dihydrorhodamine 123 oxidation and lung damage. Also, both c-Jun NH2-terminal kinase1 and c-Jun NH2-terminal kinase1 mice showed a decreased dihydrorhodamine 123 oxidation and lung damage after peritonitis. Finally, dihydrorhodamine 123 oxidation, reactive oxygen species, inducible nitric oxide synthase expression, and lung damage were decreased in c-Jun NH2-terminal kinase1 --> wild-type but not in wild-type --> c-Jun NH2-terminal kinase1 chimeric mice. CONCLUSIONS: Collectively, our data suggest that peritonitis-induced inducible nitric oxide synthase expression, peroxynitrite production, and lung damage depend on the c-Jun NH2-terminal kinase signaling of the hematopoietic cells.

Commensal microflora induce host defense and decrease bacterial translocation in burn mice through toll-like receptor 4.

BACKGROUND: Major burn is associated with decreased gut barrier function and increased bacterial translocation (BT). This study is to investigate whether commensal microflora induce host defense and decrease BT in burn mice. METHODS: First, we treated Wild type (WT) mice with antibiotics in drinking water for 4 weeks to deplete gut commensal microflora. At week 3, drinking water was supplemented with lipopolysaccharide (LPS); a ligand for TLR4, to trigger TLRs in gut. The intestinal permeability, glutathione level, NF-kappaB DNA-binding activity, TLR4 expression of intestinal mucosa, BT to mesenteric lymph nodes (MLNs), and bacterial killing activity of peritoneal cells were measured after thermal injury. Second, lung of animals were harvested for MPO activity and TNFalpha mRNA expression assay. Third, WT animals were treated with oral antibiotics with or without LPS supplement after burn. At 48 hr after burn, TLR4 expression of intestinal mucosa and bacterial killing activity of cells were examined. Finally, bacterial killing activity and BT to MLNs after thermal injury in C3H/HeJ (TLR4 mutant) mice were measured. RESULTS: Burn induced BT to MLNs in WT mice. Commensal depletion decreased TLR4 expression as well as NF-kappaB activation of intestine, myeloperoxidase (MPO) activity as well as TNFalpha expression of lung, and bacterial killing activity of peritoneal cells. Oral LPS supplement markedly reduced 81% of burn-induced BT and increased TLR4 expression, MPO activity of lung, as well as bacterial killing activity of peritoneal cells. LPS supplement did not change BT or bacterial killing activity in C3H/HeJ mice. CONCLUSIONS: Collectively, commensal microflora induce TLR4 expression of intestine and bacterial killing activity of inflammatory cells in burn. TLR4 ligand increases bacterial killing activity and decreases burn-induced BT. Taken together with the abolition of LPS effect in TLR4 mutant mice, we conclude that commensal microflora induce host defense and decrease bacterial translocation in burn mice through toll-like receptor 4.

Carbon-silicon core-shell nanowires as high capacity electrode for lithium ion batteries.

We introduce a novel design of carbon-silicon core-shell nanowires for high power and long life lithium battery electrodes. Amorphous silicon was coated onto carbon nanofibers to form a core-shell structure and the resulted core-shell nanowires showed great performance as anode material. Since carbon has a much smaller capacity compared to silicon, the carbon core experiences less structural stress or damage during lithium cycling and can function as a mechanical support and an efficient electron conducting pathway. These nanowires have a high charge storage capacity of approximately 2000 mAh/g and good cycling life. They also have a high Coulmbic efficiency of 90% for the first cycle and 98-99.6% for the following cycles. A full cell composed of LiCoO(2) cathode and carbon-silicon core-shell nanowire anode is also demonstrated. Significantly, using these core-shell nanowires we have obtained high mass loading and an area capacity of approximately 4 mAh/cm(2), which is comparable to commercial battery values.

GSKIP, an inhibitor of GSK3beta, mediates the N-cadherin/beta-catenin pool in the differentiation of SH-SY5Y cells.

Emerging evidence has shown that GSK3beta plays a pivotal role in regulating the specification of axons and dendrites. Our previous study has shown a novel GSK3beta interaction protein (GSKIP) able to negatively regulate GSK3beta in Wnt signaling pathway. To further characterize how GSKIP functions in neurons, human neuroblastoma SH-SY5Y cells treated with retinoic acid (RA) to differentiate to neuron-like cells was used as a model. Overexpression of GSKIP prevents neurite outgrowth in SH-SY5Y cells. GSKIP may affect GSK3beta activity on neurite outgrowth by inhibiting the specific phosphorylation of tau (ser396). GSKIP also increases beta-catenin in the nucleus and raises the level of cyclin D1 to promote cell-cycle progression in SH-SY5Y cells. Additionally, overexpression of GSKIP downregulates N-cadherin expression, resulting in decreased recruitment of beta-catenin. Moreover, depletion of beta-catenin by small interfering RNA, neurite outgrowth is blocked in SH-SY5Y cells. Altogether, we propose a model to show that GSKIP regulates the functional interplay of the GSK3beta/beta-catenin, beta-catenin/cyclin D1, and beta-catenin/N-cadherin pool during RA signaling in SH-SY5Y cells.

Phase transformation of biphasic Cu2S-CuInS2 to monophasic CuInS2 nanorods.

We synthesized wurtzite CuInS(2) nanorods (NRs) by colloidal solution-phase growth. We discovered that the growth process starts with nucleation of Cu(2)S nanodisks, followed by epitaxial overgrowth of CuInS(2) NRs onto only one face of Cu(2)S nanodisks, resulting in biphasic Cu(2)S-CISu heterostructured NRs. The phase transformation of biphasic Cu(2)S-CuInS(2) into monophasic CuInS(2) NRs occurred with growth progression. The observed epitaxial overgrowth and phase transformation is facile for three reasons. First, the sharing of the sulfur sublattice by the hexagonal chalcocite Cu(2)S and wurtzite CuInS(2) minimizes the lattice distortion. Second, Cu(2)S is in a superionic conducting state at the growth temperature of 250 degrees C wherein the copper ions move fluidly. Third, the size of the Cu(2)S nanodisks is small, resulting in fast phase transformation. Our results provide valuable insight into the controlled solution growth of ternary chalcogenide nanoparticles and will aid in the development of solar cells using ternary I-III-VI(2) semiconductors.

Lactobacillus plantarum reverse diabetes-induced Fmo3 and ICAM expression in mice through enteric dysbiosis-related c-Jun NH2-terminal kinase pathways.

Diabetes mellitus (DM) is characterized by increased fatality associated with the atherogenetic process. Circulating trimethylamine-N-oxide (TMAO) levels are closely associated with atherosclerosis. The flavin mono-oxygenase family (Fmo) members oxidize trimethylamine (TMA) to TMAO. The effect and the regulatory mechanism of intestinal microflora on diabetes-induced Fmo3 and intercellular adhesion molecule (ICAM) expression were examined in streptozotocin-induced diabetic mice (STZDM) and Akita mice (C57BL/6J-Ins2Akita). STZDM-JNK1-/- and Ins2Akita-JNK1-/- mice were produced and used to study the role of pJNK in the regulatory mechanisms. Diabetic mice exhibited decreased Lactobacilli growth and reactive oxygen species (ROS) production in the intestinal mucosa; increased levels of pJNK and iNOS proteins in the intestinal mucosa; increased levels of serum nitrate, IL-1ß, and TNF-α expression in Kupffer cells; increased Fmo3 expression in the liver; and increased ICAM expression in the aorta. Reversal of diabetes-induced enteric dysbiosis by prebiotic (FOS) or probiotic (dead L. plantarum) treatment decreased diabetes-induced pJNK and iNOS expression in the intestine, Fmo3 expression in the liver, IL-1ß expression in Kupffer cells, and ICAM expression in the aorta and liver. Ins2Akita-JNK1-/- and STZDM-JNK1-/- mice demonstrated decreased levels of serum NO, IL-1ß expression in Kupffer cells, Fmo3 expression in the liver, and ICAM expression in the aorta. GF mice cohoused with DM mice demonstrated an increase in ICAM expression in the liver. In conclusion, diabetes induced the expression of both Fmo3 and ICAM expression and possible vascular impairment through enteric dysbiosis. Diabetes-induced Fmo3 and ICAM expression could be reversed by pJNK inhibition or by correcting enteric dysbiosis.

Central roles of the roof plate in telencephalic development and holoprosencephaly.

The roof plate is a well known signaling center in CNS development, but its roles in the developing telencephalon and the common holoprosencephaly (HPE) malformation have been uncertain. Using cellular ablations in mice, we show that roof plate cell loss causes failed midline induction and HPE in the dorsal telencephalon. This morphologic phenotype is accompanied by selective deficits in midline gene expression and a reduced activity gradient for bone morphogenetic proteins (Bmps), the major signals produced by the roof plate. In dissociated cells and mutant explants, exogenous Bmp4 is sufficient to mimic roof plate selectivity in midline gene regulation and to rescue roof plate-dependent midline patterning. Previously unrecognized neuroanatomical defects predicted by the mouse model are then confirmed in human HPE patients. These findings establish selective roles for roof plate-dependent Bmp signaling in dorsal telencephalic patterning and HPE and define novel candidate genes for the human disorder.

Pseudomonas aeruginosa Ventilator-Associated Pneumonia Induces Lung Injury through TNF-α/c-Jun NH2-Terminal Kinase Pathways.

Ventilator-associated pneumonia (VAP) is a common nosocomial infection among intensive care unit (ICU) patients. Pseudomonas aeruginosa (PA) is the most common multidrug-resistant Gram-negative pathogen and VAP caused by PA carries a high rate of morbidity and mortality. This study examined the molecular mechanism of PA VAP-induced lung injury. C57BL/6 wild-type (WT) mice and JNK1 knockout (JNK1-/-) mice received mechanical ventilation (MV) for 3 h at 2 days after receiving nasal instillation of PA. The WT and JNK1-/- mice also received MV after the induction of lung injury by instillation of supernatants from PA-stimulated alveolar macrophages (AMs). AMs isolated from WT, IκB-kinase (IKK)ßΔMye (IKKß was selectively deleted in macrophages), and JNK1-/- mice were ex vivo stimulated with live PA and supernatants were collected for cytokine assay. Intranasal instillation of 106 PA enhanced MV-induced NF-κB DNA binding activity in the lungs and nitrite levels in BALF. MV after PA instillation significantly increased the expression of ICAM and VCAM in the lungs and TNF-α, IL-1ß, and IL-6 levels in bronchoalveolar lavage fluid (BALF) of WT mice, but not in JNK1-/- mice. MV after supernatant instillation induced more total protein concentration in BALF and neutrophil sequestration in the lungs in WT mice than JNK1-/- mice and cytokine assay of supernatants indicated that TNF-α is a critical regulator of PA VAP-induced lung injury. Ex vivo PA stimulation induced TNF-α production by AMs from WT as well as JNK1-/- mice but not IKKßΔMye mice. In summary, PA colonization plays an important role in PA VAP-induced lung injury through the induction of JNK1-mediated inflammation. These results suggest that the pathogenesis mechanism of PA VAP involves production of TNF-α through activation of IKK/NF-κB pathways in AMs and JNK signaling pathway in the lungs.

Inhibition of nitric oxide production reverses diabetes-induced Kupffer cell activation and Klebsiella pneumonia liver translocation.

Klebsiella pneumoniae (KP) is the most common pathogen of pyogenic liver abscess in East and Southeast Asia and diabetes mellitus (DM) is a major risk factor. The effect and mechanism of diabetes on KP liver abscess was examined in streptozotocin-induced diabetic mice and Akita mice (C57BL/6J-Ins2Akita). KP translocation to liver and plasma alaine transaminase levels were increased and liver clearance of KP was decreased in DM mice. Diabetic mice exhibited overgrowth of Enterococcus as well as E.coli and decreased lactobacilli/bifidas growth in intestine, increased intestinal iNOS protein and nitrite levels in portal vein, and increased IL-1ß and TNF-α expression of Kupffer cells. Fructooligosaccharides (FOS) or dead L. salivarius (dLac) supplementation reversed diabetes-induced enteric dysbiosis, NO levels in portal vein, and KP translocation to liver. L-NAME treatment decreased intestinal iNOS protein expression as well as Kupffer cell activation and increased liver clearance of KP in DM mice. Dead E.coli (2×108 CFU/ml) feeding for one week induced iNOS and TLR4 expression of intestine in germ-free (GF) mice. Dead bacteria feeding induced IL-1ß and TNF-α expression of Kupffer cells in GF mice but not in GF TLR4-/- mice. In conclusion, balance of intestinal microflora is important for preventing intestinal iNOS expression, Kupffer cell activation, and KP liver translocation in diabetes. Reversal of diabetes-induced enteric dysbiosis with FOS or dead L. salivarius decreases diabetes-induced intestinal iNOS expression and KP liver translocation. Diabetes induces Kupffer cell activation and KP liver translocation through enteric dysbiosis and nitric oxide production.

Modulation of angiogenic processes in cultured endothelial cells by low density lipoproteins subfractions from patients with familial hypercholesterolemia.

OBJECTIVE: Electronegative low density lipoprotein (LDL) subfractions are cytotoxic to endothelial cells. To continue our study of homozygotic familial hypercholesterolemic (FH)-LDL, we report the effects of FH-LDL subfractions (FH-L1 to FH-L5) on the angiogenic processes in cultured endothelial cells. METHODS AND RESULTS: Subconfluent bovine aortic endothelial cells (BAEC) were treated with LDL subfractions (20 microg/ml), and the effects on angiogenic functions, including cell proliferation, migration, apoptosis, tube formation, secretion of matrix metalloproteinases (MMPs), and vascular endothelial growth factor (VEGF) were determined. The electronegative FH-L4 and FH-L5 inhibited cell proliferation while the other FH-LDL subfractions and LDL from normocholesterolemic subjects (N-LDL) had negligible effects. Like Cu2+ ox-LDL, FH-L5 strongly inhibited endothelial cell viability and FH-L4 had a milder effects. Similarly, FH-L4 and FH-L5 but not the other subfractions retarded cell migration, induced cell apoptosis, and perturbed tube formation by BAEC in matrigel. FH-L5 inhibited secretion of MMP-2 and MMP-9 by BAEC without affecting their endogenous levels. In contrast, FH-L5 increased the VEGF expression in endothelial cells. CONCLUSIONS: Our results show for the first time that FH-L5, a circulating LDL subfraction from hypercholesterolemic patients, modulates various angiogenic processes, thereby dysregulating endothelial function in a way that may be atherogenic.

Thermal injury-induced priming effect of neutrophil is TNF-alpha and P38 dependent.

Priming response of neutrophil in clinical-related conditions and its mechanism has not been clarified. This study is to determine if thermal injury-induced priming effect of neutrophil is TNF-alpha and p38 dependent. In Experiment 1, bone marrow neutrophil of wild-type (WT) mice and TNF receptor superfamily, member 1A (Tnfrsf1a-/-) mice were harvested and treated with TNF-alpha, platelet activating factor (PAF) first, then with or without N-formyl-Met-Leu-Phe (fMLP). Reactive oxygen species (ROS) production and p38 phosphorylation were evaluated. In Experiment 2, ROS of neutrophil from WT and Tnfrsf1a-/- mice at 3 or 15 h after thermal injury with or without fMLP treatment were assayed. In Experiment 3, p38 and p44/42 phosphorylation, CXCR2 and macrophage inflammatory protein-2 expression, apoptotic ratio, and activating protein-1 (AP-1) and nuclear factor-kappa B (NF-kappaB) activation of neutrophil from WT and Tnfrsf1a-/- mice at 3 h after thermal injury were tested. FMLP treatment after TNF-alpha or PAF incubation of neutrophil increased ROS of PAF-treated but not TNF-alpha-treated neutrophil. PAF treatment increased ROS of neutrophil in WT and Tnfrsf1a-/- mice. FMLP increased ROS of neutrophil of WT mice at 3 h after thermal but not that of Tnfrsf1a-/- mice. TNF-alpha and PAF increased p38 phosphorylation of neutrophil in WT but not that in Tnfrsf1a-/- mice. Thermal injury increased p38 phosphorylation, NF-kappaB activation, and decreased apoptosis of neutrophil at 3 h after thermal injury in WT but not in Tnfrsf1a-/- mice. Thermal injury also induced AP-1 activation and ROS production on neutrophil at 3 and 15 h after thermal injury, respectively, in WT and Tnfrsf1a-/- mice. Collectively, fMLP stimulates ROS of neutrophil through TNF-alpha signaling; PAF stimulates that of neutrophil through both TNF-alpha-dependent and TNF-alpha-independent pathway. Thermal injury induces a TNF-alpha-dependent priming effect and a TNF-alpha-independent activation effect on neutrophil at 3 and 15 h after thermal injury, respectively. NF-kappaB signaling pathway plays an important role in neutrophil activation. Thermal injury also induces TNF-alpha-dependent delay apoptosis and TNF-alpha-independent AP-1 activation of neutrophil at 3 h after thermal injury. Taken together with the TNF-alpha-dependent p38 and NF-kappaB activation in primed neutrophil, we conclude that thermal injury-induced priming effect of polymorphonuclear neutrophil is TNF-alpha and p38 dependent.