Craniofacial malformation in R-spondin2 knockout mice.

In vertebrates, craniofacial formation is accomplished by synergistic interaction of many small elements which are generated independently from distinct germ layers. Because of its complexity, the imbalance of one signaling cascade such as Wnt/beta-catenin pathway easily leads to craniofacial malformation, which is the most frequent birth defect in humans. To investigate the developmental role of a newly identified activator of Wnt/beta-catenin signaling, Rspo2, we generated and characterized Rspo2(-/-) mice. We found CLP with mild facial skeletal defects in Rspo2(-/-) mice. Additionally, Rspo2(-/-) mice also exhibited distal limb loss and lung hypoplasia, and died immediately after birth with respiratory failure. We showed the apparent reduction of Wnt/beta-catenin signaling activity at the branchial arch and the apical ectodermal ridge in Rspo2(-/-) mice. These findings indicate that Rspo2 regulates midfacial, limb, and lung morphogenesis during development through the Wnt/beta-catenin signaling.

Role of soluble receptor for advanced glycation end products on endotoxin-induced lung injury.

RATIONALE: The interaction of receptor for advanced glycation end products (RAGE) and its ligands often leads to inflammatory processes or tissue injury, although the effect of the blockade of RAGE signaling on lung injury remains to be investigated. OBJECTIVES: Using a murine model of lung injury induced by intratracheal lipopolysaccharide (LPS), we evaluated RAGE expression in the airspace and the effect of recombinant soluble RAGE (sRAGE) on LPS-induced lung injury. METHODS: First, the expression of sRAGE in bronchoalveolar lavage (BAL) fluid was determined at 24 hours after intratracheal instillation of LPS or phosphate-buffered saline. Next, to evaluate the effect of sRAGE, BAL fluid was collected for cell counting and measurements of lung permeability and cytokine concentrations 24 hours after intratracheal LPS in the mice with or without intraperitoneal administration of sRAGE 1 hour after the instillation. In another series, lungs were sampled for histopathology and detection of apoptotic cells. The activation of nuclear factor (NF)-kappaB was analyzed 4 hours after LPS instillation. MEASUREMENTS AND MAIN RESULTS: In response to LPS challenge, a RAGE isoform of 48 kD was detected in the BAL fluid. Treatment with sRAGE significantly attenuated the increases in neutrophil infiltration, lung permeability, production of inflammatory cytokines, NF-kappaB activation, and apoptotic cells in the lung as well as development of pathologic changes after LPS instillation. CONCLUSIONS: RAGE plays an important role in the pathogenesis of LPS-induced lung injury in mice. It was suggested that sRAGE should be tested as a treatment modality in other models of acute lung injury.

Role of interleukin-6 in bleomycin-induced lung inflammatory changes in mice.

Interleukin-6 (IL-6) is known to be involved in the pathogenesis of various inflammatory diseases, but its role in bleomycin (BLM)-induced lung injury and subsequent fibrotic changes remains to be determined. We evaluated the role of IL-6 in the lung inflammatory changes induced by BLM using wild-type (WT) and IL-6-deficient (IL-6(-/-)) mice. The mice were treated intratracheally with 1 mg/kg BLM and killed 2, 7, or 21 days later. Lung Inflammation in the acute phase (Days 2 and 7) was assessed by differential cell counts in bronchoalveolar lavage (BAL) fluid and cytokine levels in the lung. Lung fibrotic changes were evaluated on Day 21 by histopathology and collagen assay. On Day 2, BLM administration induced significant increases in the numbers of total cells, macrophages, and neutrophils in BAL fluid, which were attenuated in IL-6(-/-) mice (P < 0.05). Lung pathology also showed inflammatory cell accumulation, which was attenuated in the IL-6(-/-) mice compared with WT mice. In WT mice, elevated levels of TGF-beta(1) and CCL3 were observed 2 and 7 days after BLM challenge, respectively. On Day 7, BLM-induced inflammatory cell accumulation did not differ between the genotypes. Lung pathology 21 days after BLM challenge revealed significant fibrotic changes with increased collagen content, which was attenuated in IL-6(-/-) mice. Although the TGF-beta(1) level in the lung did not differ between the genotypes on Day 21, CCL3 was significantly lower in IL-6(-/-) mice. These results indicate that IL-6 may play an important role in the pathogenesis of BLM-induced lung injury and subsequent fibrotic changes.

Serum indicators for the diagnosis of pneumocystis pneumonia.

BACKGROUND: The diagnosis of pneumocystis pneumonia (PCP) is difficult because it requires microscopic examination to identify pneumocystis from induced sputum or BAL fluid. STUDY OBJECTIVE: To evaluate the usefulness of four serum markers-lactate dehydrogenase (LDH), (1-->3) beta-D-glucan (beta-D-glucan), KL-6, and C-reactive protein (CRP)-in the diagnosis of PCP. DESIGN: Case-control retrospective study. PATIENTS AND MEASUREMENTS: We reviewed the medical records of 295 consecutive patients who underwent BAL for the diagnosis of PCP. Differential cell counts in BAL fluid and serum levels of LDH, beta-D-glucan, KL-6, and CRP were examined. Oxygenation index was determined using arterial oxygen tension and inspiratory oxygen concentration. RESULTS: Based on the microscopic examination of BAL fluid, 57 patients were PCP positive and 238 patients were PCP negative. There were no significant differences in cell count or differentials in BAL fluid between the positive and negative cases. Serum levels of LDH, beta-D-glucan, and KL-6 were significantly higher in PCP-positive patients (p < 0.01). Receiver operating characteristic curves suggest that beta-D-glucan was the most reliable indicator. The cut-off level of beta-D-glucan was estimated to be 31.1 pg/mL, with which the positive and negative predictive values were 0.610 and 0.980, respectively. In PCP-positive patients, the oxygenation index was decreased and correlated with LDH. Both LDH and beta-D-glucan levels were correlated with the proportion of neutrophils in BAL fluid. CONCLUSIONS: Serum beta-D-glucan is a reliable marker for the diagnosis of PCP. Since BAL procedure is invasive, measuring beta-D-glucan should be considered as a primary modality for a diagnosis of PCP, especially for patients with severe respiratory failure.

Protective role of vascular endothelial growth factor in endotoxin-induced acute lung injury in mice.

BACKGROUND: Vascular endothelial growth factor (VEGF), a substance that stimulates new blood vessel formation, is an important survival factor for endothelial cells. Although overexpressed VEGF in the lung induces pulmonary edema with increased lung vascular permeability, the role of VEGF in the development of acute lung injury remains to be determined. METHODS: To evaluate the role of VEGF in the pathogenesis of acute lung injury, we first evaluated the effects of exogenous VEGF and VEGF blockade using monoclonal antibody on LPS-induced lung injury in mice. Using the lung specimens, we performed TUNEL staining to detect apoptotic cells and immunostaining to evaluate the expression of apoptosis-associated molecules, including caspase-3, Bax, apoptosis inducing factor (AIF), and cytochrome C. As a parameter of endothelial permeability, we measured the albumin transferred across human pulmonary artery endothelial cell (HPAEC) monolayers cultured on porous filters with various concentrations of VEGF. The effect of VEGF on apoptosis HPAECs was also examined by TUNEL staining and active caspase-3 immunoassay. RESULTS: Exogenous VEGF significantly decreased LPS-induced extravascular albumin leakage and edema formation. Treatment with anti-VEGF antibody significantly enhanced lung edema formation and neutrophil emigration after intratracheal LPS administration, whereas extravascular albumin leakage was not significantly changed by VEGF blockade. In lung pathology, pretreatment with VEGF significantly decreased the numbers of TUNEL positive cells and those with positive immunostaining of the pro-apoptotic molecules examined. VEGF attenuated the increases in the permeability of the HPAEC monolayer and the apoptosis of HPAECs induced by TNF-alpha and LPS. In addition, VEGF significantly reduced the levels of TNF-alpha- and LPS-induced active caspase-3 in HPAEC lysates. CONCLUSION: These results suggest that VEGF suppresses the apoptosis induced by inflammatory stimuli and functions as a protective factor against acute lung injury.

Effects of initial passage of endotoxin through the liver on the extent of acute lung injury in a rat model.

We hypothesized that the extent of acute lung injury (ALI) caused by lipopolysaccharide (LPS) is modified with its initial passage through the liver. We tested this hypothesis by administering LPS, 5 mg/kg, or saline to 120 male Wistar rats via the portal vein (PV) or the inferior vena cava (IVC) over 1 h. Four experimental groups of rats were administered saline into the PV, saline into the IVC, LPS into the PV (LPS-PV group), and LPS into the IVC (LPS-IVC group), respectively. At 15 and 30 min after onset of 51Chromium-LPS infusion, the gamma counts in the liver were higher in the LPS-PV group than that in the LPS-IVC group. The ratio of 125Iodine-albumin counts in lung tissue to that in plasma per unit of weight (as an assessment of pulmonary microvascular permeability) at 240 min after onset of LPS stimulation, the accumulation of polymorphonuclear cell (assessed by myeloperoxidase activity) and the concentration of tumor necrosis factor alpha in the lung at 60 and 240 min after onset of LPS infusion, were higher in the LPS-IVC group than in the LPS-PV group. Significant differences in several factors indicative of inflammation and in the extent of LPS-induced ALI were observed after the onset of LPS infusion, depending on whether it was delivered via the PV or the IVC. These observations suggest that the entrapping of LPS during its initial passage through the hepatic circulation may attenuate LPS-induced ALI within 4 h of initiation of LPS stimulation.

[Clinical features of Pneumocystis pneumonia in patients with systemic lupus erythematosus].

Systemic lupus erythematosus (SLE) is often associated with various opportunistic infections, particularly during treatment with corticosteroids or immunosuppressants. We studied the clinical characteristics of 15 patients with SLE who underwent diagnostic bronchoalveolar lavage (BAL) and compared 6 patients with confirmed Pneumocystis pneumonia (PcP+), with 9 patients without Pneumocystis pneumonia (PcP-). The serum concentrations of beta-D-glucan and KL-6 were significantly higher in PcP+ than in PcP- patients, whereas serum LDH was similar in both groups. The serum concentrations of complement, a marker of SLE activity, and of IgG did not predict the presence of PcP. In all patients, the overall cell and lymphocyte counts were increased in the BAL fluid, without any significant difference between the PcP+ and PcP- groups. Ground-glass opacities on chest computed tomography, and oxygenation impairment (PaO2/FiO2<200Torr) were more common in PcP+ than PcP- patients. We concluded that, in patients with SLE, serum beta-D-glucan and KL-6 might be useful in the diagnosis of PcP, particularly when severe hypoxemia precludes BAL.

[Small cell lung cancer with Sjögren's syndrome and Lambert-Eaton myasthenic syndrome].

A 72-year-old man was admitted to our hospital with complaints of dry mouth, muscle weakness of the lower limbs and gait disturbance. The patient had dry mouth, dry eyes, positive anti-SS-B antibody and salivary gland inflammation. Sjögren's syndrome was diagnosed. Since muscle weakness of the lower limbs and gait disturbance were not compatible with Sjögren's syndrome, we considered the possibility of paraneoplastic syndrome. Serum levels of CEA, NSE and ProGRP were elevated. Chest roentgenogram and CT showed a nodular lesion in the right upper lobe of the lung and swollen lymph nodes in the hilum and mediastinum. Small cell lung cancer was diagnosed by bronchoscopy. Anti-P/Q-type Ca2+ channel antibody was positive. Electromyogram showed a reduction in the amplitude of the evoked muscle action potential response after slow repetitive stimulation and did not show a reduction after rapid repetitive stimulation. Based on these findings, we made a diagnosis of Lambert-Eaton myasthenic syndrome (LEMS). Concurrent chemoradiotherapy induced an improvement of muscle weakness of the lower limbs. LEMS is frequently associated with a malignant tumor and an autoimmune disorder. We thought that in this patient, the presentation of LEMS was apparent because he had both Sjögren's syndrome and small cell lung cancer.

[A case of stage IV primary lung cancer with no metastasis except in the choroid plexus].

A 69-year-old man become aware of myiodesopsia. He visited the Department of Ophthalmology at Keio University Hospital and choroidal metastasis of the right eye was diagnosed. A tumor shadow was detected in the lower lobe of the left lung on chest radiographs, and the serum CEA concentration was found to be significantly increased. Through bronchoscopic examinations, he was confirmed to have primary lung cancer (histological classification: adenocarcinoma). Although detailed systemic examinations were performed, no metastasis to lymph nodes or distant organs except the choroid plexus was detected, which is very rare in a case of stage IV primary lung cancer. In conclusion, it is suggested to be very important to look for ophthalmological abnormalities, even in the case of apparently early lung cancer that seems to be resectable.

Regulation of the HIF-1alpha level is essential for hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are sustained in a specific microenvironment known as the stem cell niche. Mammalian HSCs are kept quiescent in the endosteal niche, a hypoxic zone of the bone marrow (BM). In this study, we show that normal HSCs maintain intracellular hypoxia and stabilize hypoxia-inducible factor-1alpha (HIF-1alpha) protein. In HIF-1alpha-deficient mice, the HSCs lost their cell cycle quiescence and HSC numbers decreased during various stress settings including bone marrow transplantation, myelosuppression, or aging, in a p16(Ink4a)/p19(Arf)-dependent manner. Overstabilization of HIF-1alpha by biallelic loss of an E3 ubiquitin ligase for HIF-1alpha (VHL) induced cell cycle quiescence in HSCs and their progenitors but resulted in an impairment in transplantation capacity. In contrast, monoallelic loss of VHL induced cell cycle quiescence and improved BM engraftment during bone marrow transplantation. These data indicate that HSCs maintain cell cycle quiescence through the precise regulation of HIF-1alpha levels.

Role of toll-like receptor 4 in acute neutrophilic lung inflammation induced by intratracheal bacterial products in mice.

BACKGROUND: Toll-like receptors (TLRs) represent a conserved family of innate immune recognition receptors. Among TLRs, TLR4 is important for the recognition of Gram-negative bacteria, whereas TLR2 recognizes cell wall constituents of Gram-positive microorganisms, such as peptidoglycan (PGN). METHODS: To evaluate the role of TLR4 in the pathogenesis of acute lung injury induced by Escherichia coli endotoxin (lipopolysaccharide; LPS) or PGN, we compared inflammatory cell accumulation in bronchoalveolar lavage (BAL) fluid and lung pathology between C3H/HeJ (TLR4 mutant) and wild-type C3H/HeN mice. The levels of proinflammatory cytokines and chemokines in plasma and BAL fluid and nuclear factor-κB (NF-κB) translocation in the lung were also evaluated. RESULTS: In C3H/HeJ mice, LPS-induced neutrophil emigration was significantly decreased compared with C3H/HeN mice, whereas PGN-induced neutrophil emigration did not differ. Differential cell count in BAL fluid revealed comparable neutrophil recruitment in the alveolar space. In TLR4 mutant mice, LPS-induced upregulation of tumor necrosis factor-alpha (TNF-α), KC, and CXCL10 in plasma and BAL fluid was attenuate, which was not different after PGN. NF-κB translocation in the lung was significantly decreased in C3H/HeJ compared with C3H/HeN mice, whereas PGN-induced NF-κB translocation was not different. CONCLUSION: These results suggest that TLR4 mediates inflammatory cascade induced by Gram-negative bacteria that is locally administered.

Stem cell defects in ATM-deficient undifferentiated spermatogonia through DNA damage-induced cell-cycle arrest.

Mammalian spermatogenesis is maintained by stem cell capacity within undifferentiated spermatogonial subpopulation. Here, using a combination of surface markers, we describe a purification method for undifferentiated spermatogonia. Flow cytometric analysis revealed that this population is composed of Plzf-positive cells and exhibits quiescence and the side population phenotype, fulfilling general stem cell criteria. We then applied this method to analyze undifferentiated spermatogonia and stem cell activity of Atm(-/-) mice. Atm(-/-) testis shows progressive depletion of undifferentiated spermatogonia accompanied by cell-cycle arrest. In Atm(-/-) undifferentiated spermatogonia, a self-renewal defect was observed in vitro and in vivo. Accumulation of DNA damage and activation of the p19(Arf)-p53-p21(Cip1/Waf1) pathway were observed in Atm(-/-) undifferentiated spermatogonia. Moreover, suppression of p21(Cip1/Waf1) in an Atm(-/-) background restored transplantation ability of undifferentiated spermatogonia, indicating that ATM plays an essential role in maintenance of undifferentiated spermatogonia and their stem cell capacity by suppressing DNA damage-induced cell-cycle arrest.

Endothelin-1 level in epithelial lining fluid of patients with acute respiratory distress syndrome.

BACKGROUND AND OBJECTIVES: Endothelin-1 (ET-1), a potent vasoconstrictor peptide produced by endothelial cells, has been implicated in the dysfunction of various organs. To determine the role of ET-1 in acute lung injury (ALI) and ARDS, ET-1 levels were measured in epithelial lining fluid (ELF) and plasma obtained from patients with ALI/ARDS. METHODS: A cross-sectional study of patients with ALI/ARDS in the intensive care unit of two university hospitals was performed. Patients with ALI/ARDS underwent bronchoscopic microsampling to collect ELF on the day of onset of the disease. Patients who underwent bronchoscopy to examine a small peripheral pulmonary nodule served as controls. RESULTS: In the 23 patients with ALI/ARDS, the ET-1 level in ELF was significantly greater than that in plasma (P < 0.001). In contrast, ET-1 was not detectable in the ELF from six of the seven control subjects. The albumin concentration of ELF, used as a marker of endothelial and epithelial permeability, correlated with the ET-1 level in ELF (P < 0.001). The oxygenation index (PaO(2)/FiO(2)) was also correlated with ET-1 concentration in ELF (P < 0.001). CONCLUSION: In patients with ALI/ARDS, ET-1 is produced mainly in the lung and is associated not only with pulmonary vasoconstriction but also the development of permeability oedema, leading to the impairment of oxygenation.

Attenuation of endotoxin-induced acute lung injury by the Rho-associated kinase inhibitor, Y-27632.

A small GTPase, Rho, plays key roles in cell adhesion, motility, and contraction after stimulation. Among Rho effectors isolated, the family of Rho-associated coiled-coil-forming protein kinases (ROCK) is implicated in Rho-mediated cell adhesion and smooth muscle contraction. The effect of a specific inhibitor of ROCK, Y-27632, was evaluated in a murine model of acute lung injury induced by intravenous injection of Escherichia coli endotoxin (lipopolysaccharide [LPS]). Lung edema was evaluated by measuring extravascular leakage of radio-labeled serum albumin, and neutrophil emigration into the lung parenchyma by morphometric observation and measuring myeloperoxidase activity. Pretreatment with Y-27632 attenuated both lung edema and neutrophil emigration after LPS. We also measured albumin transfer through cultured endothelial cell monolayers on a porous filter. Tumor necrosis factor-alpha significantly increased albumin transfer, which was attenuated by pretreatment with Y-27632. Fluorescence microscopy revealed that morphologic changes in endothelial cells induced by tumor necrosis factor-alpha were inhibited by Y-27632. In contrast, the increased fraction of neutrophils with polymerized actin after formyl-methionyl-leucyl-phenylalanine was not altered by Y-27632. These data suggest that ROCK may play an important role in the pathogenesis of LPS-induced lung injury and that ROCK inhibition could attenuate cytoskeletal rearrangement of endothelial cells, leading to decreased neutrophil emigration into the lung parenchyma.

Effect of CD14 blockade on endotoxin-induced acute lung injury in mice.

CD14 functions as a cell surface receptor for endotoxin (lipopolysaccharide [LPS]) and is thought to have an essential role in innate immune responses to infection. Previous studies have revealed attenuation of the systemic response after sepsis by blocking CD14. In this study, we tested the hypothesis that CD14 blockade protects against inflammatory responses associated with LPS pneumonia. We examined the effect of an anti-murine CD14 monoclonal antibody (4C1) on the development of acute lung injury induced by intratracheal LPS in mice. We also measured the production of cytokines (tumor necrosis factor-alpha, interleukin-6, and macrophage inflammatory protein-2) and nitric oxide by murine peritoneal macrophages exposed to LPS in vitro. Nuclear factor (NF)-kappa B translocation was evaluated in nuclear extracts from lung homogenates. 4C1 significantly attenuated pulmonary edema and neutrophil emigration after LPS administration. The production of cytokines and nitric oxide by LPS-stimulated macrophages was significantly decreased by 4C1 treatment. NF-kappa B translocation induced by LPS instillation was also suppressed by 4C1. These results suggest that blockade of CD14 might attenuate acute lung injury after intratracheal instillation of LPS through the suppression of NF-kappa B translocation. The inhibitory effect of CD14 blockade on cytokine production and nitric oxide release of macrophages might contribute to the attenuation of lung injury.