Critical roles of Pten in B cell homeostasis and immunoglobulin class switch recombination

Pten is a tumor suppressor gene mutated in human cancers. We used the Cre-loxP system to generate a B cell-specific mutation of Pten in mice (bPten(flox/flox)mice). bPten(flox/flox) mice showed elevated numbers of B1a cells and increased serum autoantibodies. Among B2 cells in bPten(flox/flox) spleens, numbers of marginal zone B (MZB) cells were significantly increased while those of follicular B (FOB) cells were correspondingly decreased. Pten-deficient B cells hyperproliferated, were resistant to apoptotic stimuli, and showed enhanced migration. The survival kinase PKB/Akt was highly activated in Pten-deficient splenic B cells. In addition, immunoglobulin class switch recombination was defective and induction of activation-induced cytidine deaminase (AID) was impaired. Thus, Pten plays a role in developmental fate determination of B cells and is an indispensable regulator of B cell homeostasis.     

Hepatocyte-specific Pten deficiency results in steatohepatitis and hepatocellular carcinomas

PTEN is a tumor suppressor gene mutated in many human cancers, and its expression is reduced or absent in almost half of hepatoma patients. We used the Cre-loxP system to generate a hepatocyte-specific null mutation of Pten in mice (AlbCrePten(flox/flox) mice). AlbCrePten(flox/flox) mice showed massive hepatomegaly and steatohepatitis with triglyceride accumulation, a phenotype similar to human nonalcoholic steatohepatitis. Adipocyte-specific genes were induced in mutant hepatocytes, implying adipogenic-like transformation of these cells. Genes involved in lipogenesis and beta-oxidation were also induced, possibly as a result of elevated levels of the transactivating factors PPARgamma and SREBP1c. Importantly, the loss of Pten function in the liver led to tumorigenesis, with 47% of AlbCrePten(flox/flox) livers developing liver cell adenomas by 44 weeks of age. By 74-78 weeks of age, 100% of AlbCrePten(flox/flox) livers showed adenomas and 66% had hepatocellular carcinomas. AlbCrePten(flox/flox) mice also showed insulin hypersensitivity. In vitro, AlbCrePten(flox/flox) hepatocytes were hyperproliferative and showed increased hyperoxidation with abnormal activation of protein kinase B and MAPK. Pten is thus an important regulator of lipogenesis, glucose metabolism, hepatocyte homeostasis, and tumorigenesis in the liver.     

The loss of PTEN allows TCR alphabeta lineage thymocytes to bypass IL-7 and Pre-TCR-mediated signaling

The phosphatase and tensin homologue deleted on chromosome 10 (PTEN) negatively regulates cell survival and proliferation mediated by phosphoinositol 3 kinases. We have explored the role of the phosphoinositol(3,4,5)P3-phosphatase PTEN in T cell development by analyzing mice with a T cell-specific deletion of PTEN. Pten(flox/flox)Lck-Cre mice developed thymic lymphomas, but before the onset of tumors, they showed normal thymic cellularity. To reveal a regulatory role of PTEN in proliferation of developing T cells we have crossed PTEN-deficient mice with mice deficient for interleukin (IL)-7 receptor and pre-T cell receptor (TCR) signaling. Analysis of mice deficient for Pten and CD3gamma; Pten and gammac; or Pten, gammac, and Rag2 revealed that deletion of PTEN can substitute for both IL-7 and pre-TCR signals. These double- and triple-deficient mice all develop normal levels of CD4CD8 double negative and double positive thymocytes. These data indicate that PTEN is an important regulator of proliferation of developing T cells in the thymus.     

Acute lung injury with endotoxin or NO2 does not enhance development of airway epithelium from bone marrow.

Adult marrow-derived stem cells can localize to lung and acquire immunophenotypic characteristics of lung epithelial cells. Lung injury increases recruitment of the marrow-derived cells. We speculated that comparing patterns of lung engraftment following different lung injuries would provide insight into potential mechanisms by which marrow-derived cells were recruited to lung. To evaluate this, adult female C57Bl/6 mice irradiated and engrafted with marrow from adult male transgenic GFP mice were exposed to either intranasal inhalation of endotoxin (25 microg/mouse) or 3 days of 25 ppm NO(2) and then compared 1 or 3 months later to transplanted but otherwise uninjured mice. In all cases, the majority of marrow-derived cells recruited to lung were CD45(+) leukocytes. In lungs of transplanted but otherwise uninjured mice, small numbers of CD45(-) donor-derived cells in alveolar septae stained positively for pro-surfactant protein C. Rare donor-derived cells located in the airway epithelium stained positively with cytokeratin. Subsequent exposure of engrafted mice to NO(2) or endotoxin did not significantly increase the number or pattern of donor-derived CD45(-) cells found in recipient lungs. These results suggest that NO(2) or endotoxin lung injury does not result in significant engraftment of marrow-derived cells in lung.     

Pulmonary bioactivation of 1,1-dichloroethylene is associated with CYP2E1 levels in A/J, CD-1, and C57BL/6 mice

1,1-Dichloroethylene (DCE) elicits lung cytotoxicity and selectively targets Clara cells of bronchioles. The toxic effects are ascribed to CYP2E1-mediated formation of reactive intermediates including the DCE epoxide. Here we tested the hypothesis that differential CYP2E1 levels in the lungs of A/J, CD-1, and C57BL/6 mice lead to differences in the extents of DCE bioactivation and lung damage. Our results showed that lung CYP2E1 levels differed significantly in the three murine strains, and followed the rank order A/J > CD-1 > C57BL/6. Covalent binding of [(14)C]DCE to lung proteins in A/J mice was significantly higher than in either CD-1 or C57BL/6 mice. HPLC analysis of lung cytosol from DCE-treated mice showed that 2-S-glutathionyl acetate, a glutathione (GSH) conjugate derived from the epoxide (conjugate [C]), was the major metabolite formed. Levels of [C] detected in cytosol from A/J and CD-1 mice were significantly higher than in C57BL/6 mice. Immunohistochemical staining for [C] was pronounced in the lungs of A/J mice, was lower in CD-1 mice, and was lowest in C57BL/6 mice. Levels of GSH were similar in the lungs of all untreated mice. However, significant reduction in GSH was found in DCE-treated mice, with decreases comparable in all three strains. Bronchiolar Clara cell damage was more severe in A/J and CD-1 mice than in C57BL/6 mice. These results showed differences in CYP2E1 levels in the lungs of A/J, CD-1, and C57BL/6 mice that correlated with the extent to which the DCE epoxide is formed as well as with the severity of lung cytotoxicity.     

Bioactivation of 1,1-dichloroethylene by CYP2E1 and CYP2F2 in murine lung

1,1-Dichloroethylene (DCE) exposure evokes lung toxicity with selective damage to bronchiolar Clara cells. Recent in vitro studies have implicated CYP2E1 and CYP2F2 in the bioactivation of DCE to 2-S-glutathionyl acetate [C], a putative conjugate of DCE epoxide with glutathione. An objective of this study was to test the hypothesis that bioactivation of DCE is catalyzed by both CYP2E1 and CYP2F2 in murine lung. Western blot analysis of lung microsomal proteins from DCE-treated CD-1 mice showed time-dependent loss of immunodetectable CYP2F2 and CYP2E1 protein. Dose-dependent formation of conjugate [C] was observed in the lungs of CD-1 mice treated with DCE (75-225 mg/kg), but it was not detected after pretreatment with 5-phenyl-1-pentyne (5-PIP). Treatment of mice with 5-PIP and also with diallyl sulfone (DASO2) significantly inhibited hydroxylation of p-nitrophenol (PNP) and chlorzoxazone (CHZX). Incubation of recombinant CYP2F3 (a surrogate for CYP2F2) and recombinant CYP2E1 with PNP and CHZX confirmed that they are substrates for both of the recombinant enzymes. Incubation of the recombinant enzymes with DASO2 or 5-PIP significantly inhibited hydroxylation of both PNP and CHZX. Bronchiolar injury was elicited in CD-1 mice treated with DCE (75 mg/kg), but it was abrogated with 5-PIP pretreatment. Bronchiolar toxicity also was manifested in the lungs of CYP2E1-null and wild-type mice treated with DCE (75 mg/kg), but protection ensued after pretreatment with 5-PIP or DASO2. These results showed that bioactivation of DCE in murine lung occurred via the catalytic activities of both CYP2E1 and CYP2F2 and that bioactivation by these enzymes mediated the lung toxicity.     

Amelioration of B16F10 melanoma lung metastasis in mice by a combination therapy with indomethacin and interleukin 2 [published erratum appears in J Exp Med 1987 Mar 1;165(3):935]

Our earlier work revealed that PGE-mediated inactivation of NK cells in tumor-bearing mice by host macrophages promoted spontaneous lung metastasis that could be prevented or ameliorated by chronic indomethacin therapy. Since PGE was found to suppress the in vitro development and/or activation of a family of tumoricidal lymphocytes such as CTL, NK, and LAK cells by one or both of two mechanisms, that is to say, a down regulation of IL-2-R and an inhibition of IL-2 production, the present study tested whether a combined therapy with indomethacin and IL-2 was more effective than one with indomethacin or IL-2 alone in ameliorating established experimental lung metastasis. B6 mice injected intravenously with 10(6) highly metastatic B16F10 melanoma cells showed profuse micrometastases in the lungs by day 5, and macrometastases by day 10 which were confluent on day 21. Chronic indomethacin therapy by the oral route (14 micrograms/ml in drinking water) starting on day 0 or day 5, or a single round of IL-2 therapy (25,000 U rIL-2, every 8 h for 5 d on days 10-14) reduced the number of metastatic nodules by two-thirds (from a median of 473 in control mice receiving vehicles alone) by day 21. A single round of IL-2 as above, combined with either protocol of indomethacin therapy, completely or nearly completely irradicated the lung metastases, corroborated by a histological examination. An evaluation of splenic killer cell activity measured with a 4-h 51Cr-release assay against NK-sensitive YAC-1 lymphoma and B16F10 melanoma or NK-resistant thymic lymphoma 9705 targets revealed negligible activity in control tumor-bearing mice, and a good restoration of activity against NK-sensitive targets with either protocols of indomethacin therapy. IL-2 alone or a combination of IL-2 and indomethacin given by either protocol generated strong killer activity against all these targets, most marked with the combination therapy. Splenic killer cell phenotype in normal as well as all treated animals was ASGM1+, Thy-1-, and Lyt-2-. The combination therapy resulted in the strongest mononuclear cell infiltration in the lungs, with areas of young granulation tissue suggestive of repair sites of original metastases.     

Evidence for lipid peroxidation by paraquat in the perfused rat lung

In order to evaluate the effect of paraquat on oxidative radical reactions in the lung, we studied MDA production and chemiluminescence (spontaneous and tBuOOH-induced) in the isolated rat lung. After 2 hr of perfusion with 3.0 mM paraquat, MDA content in lung homogenates was 16 +/- 7 nmol/gm dry weight higher than in control lungs (mean +/- S.E., n = 7, p less than 0.05 by paired test); during 30 min of perfusion, malondialdehyde efflux was 33 +/- 15 nmol/gm dry weight higher than in control perfusates (n = 6, p less than 0.05). Spontaneous chemiluminescence was not augmented by 2 hr of perfusion with concentrations of paraquat ranging from 0.75 to 6.0 mM. On the other hand, tBuOOH-induced chemiluminescence was 17% +/- 3 higher immediately after the addition of hydroperoxide and reached a 16% +/- 6 higher plateau for the paraquat-perfused lungs than for control lungs (n = 10, p less than 0.05). Spectral analysis of the light emitted during induced chemiluminescence demonstrated peak intensity between 630 and 730 nm for both control and paraquat-treated lungs. Increased MDA production and increased induced chemiluminescence indicate that perfusion with paraquat enhances lipid peroxidation in the isolated rat lung.     

Immunotherapy for lung metastases of murine renal cell carcinoma: synergy between radiation and cytokine-producing tumor vaccines.

We investigated the combination therapy of local radiation of lung metastasis and vaccination with autologous tumor cells that produced interleukin (IL)-2, interferon-gamma (IFN-gamma), and granulocyte-macrophage colony-stimulating factor (GM-CSF) using the mouse Renca pulmonary metastasis model. Wild-type Renca (W/Renca) were transfected with pEF-BOS vector incorporating cDNAs for IL-2, IFN-gamma, or GM-CSF to express these cytokines. W/Renca, IL-2-producing Renca (Renca/IL-2), and IFN-gamma-producing Renca (Renca/IFN-gamma) produced subcutaneous tumor at the injection site in eight of eight, one of eight, and two of eight mice, respectively. No tumors were found in the GM-CSF-producing Renca (Renca/GM-CSF) group (zero of eight). Renca/IFN-gamma produced subcutaneous (s.c.) tumors in all Balb/c nude mice, but Renca/IL-2 and Renca/GM-CSF did not. To test the elicitation of antitumor activity, Balb/c mice were injected intravenously with 1 x 10(5) W/Renca on day 0, vaccinated, s.c., with 1 x 10(6) cells each of 5,000 rad preirradiated Renca/IL-2, Renca/IFN-gamma, and Renca/GM-CSF or 3 x 10(6) cells of preirradiated W/Renca on days 1, 7, and 14, and radiated with 300 rad to both lungs on day 5. The animals were killed on day 21 and tumor nodules in the lungs were enumerated. Neither local irradiation alone nor the combination of lung radiation and multiple vaccination with irradiated W/Renca significantly reduced the number of lung tumors. In contrast, the combination of lung radiation and the multiple vaccinations with cytokine-producing Renca cells significantly reduced the number of lung tumors. This regimen was more effective than the multiple vaccinations with cytokine-producing Renca cells alone. These studies demonstrate the efficacy of vaccination with autologous tumor cells expressing these cytokines and sensitization of the tumor target with radiation.     

Effects of two antibiotic regimens on course and persistence of experimental Chlamydia pneumoniae TWAR pneumonitis.

We studied the effects of two antibiotic regimens on the course of Chlamydia pneumoniae infection in the lungs of Swiss Webster mice. After intranasal challenge with isolates AR-388 (1.3 x 10(7) inclusion-forming units per mouse) and AR-39 (1.5 x 10(6) inclusion-forming units per mouse), groups of animals were treated with either doxycycline (10 mg/kg of body weight once a day for 3 days), azithromycin (10 mg/kg [single dose]), or saline. Responses were assessed by the isolation of organisms in cell culture, detection of TWAR DNA in lung tissues by PCR, and lung histology. Both regimens were effective in clearing infections induced by AR-388 (P = 0.02 and 0.007 for doxycycline and azithromycin, respectively) compared with controls. TWAR DNA was detected in 77 and 25% of culture-negative lungs 2 weeks after treatment of AR-388 and AR-39 infections, respectively. Histological changes showed interstitial pneumonitis and were similar over time for all groups. Single-dose azithromycin produced drug levels in lung tissues above the MICs for the test strains for a period three times longer than that of single-dose doxycycline. We concluded that short-term antibiotic regimens were successful for the treatment of experimental TWAR pneumonitis in mice. TWAR DNA was frequently recovered from lung tissues after apparently successful treatment.     

Reinforcing B16F10/GPI-IL-21 vaccine efficacy against melanoma by injecting mice with shZEB1 plasmid or miR200c agomir

In this study, we hypothesized that the inhibition of epithelial to mesenchymal transition (EMT) program by knockdown of Zinc-finger E-box binding homeobox 1 (ZEB1) or administration of miR200c agomir would strengthen the B16F10 cells transfected with GPI-anchored IL-21 (B16F10/GPI-IL-21) vaccine efficacy in inhibiting the melanoma metastasis. Our findings from the current study indicated that, when compared with the mice immunized with the B16F10/GPI-IL-21 vaccine alone, the mice immunized with B16F10/GPI-IL-21 vaccine combined with injection of shZEB1 plasmid or miR200c agomir not only meaningfully inhibited EMT of melanoma, reduced the EMT characteristic molecular expression in tumor tissues, but also significantly decreased the Treg cells and TGF-β1, enhanced the cytotoxicities of NK cells and cytotoxic T lymphocytes and the IFN-γ level. Furthermore, the immunotherapeutic combination resulted in inhibiting the melanoma growth and lung metastasis. Our study demonstrated that using the B16F10/GPI-IL-21 vaccine in combination with the down-regulated ZEB1 or miR200c administration effectively elicited anti-tumor immunity and reduced melanoma metastasis by inhibiting the EMT program in the B16F10 melanoma-bearing mice.     

体外分离培养小鼠肺间充质干细胞的生物学特性及其对肺损伤干预效果

背景:肺脏含有多种内源性干细胞,分别为来源于内胚层或中胚层的前体细胞,阐明肺组织间充质干细胞的特性有助于了解其在肺损伤中的生物学作用。目的:观察体外分离培养的小鼠肺组织间充质干细胞生长状态、表面标志、分化功能等生物学特性,探讨其对肺损伤鼠的干预效果。设计、时间及地点:细胞学体外观察与随机对照动物体内实验,于2005-10/2007-08在解放军军事医学科学院基础医学研究所细胞生物学实验室完成。材料:清洁级三四周龄C57BL/6雄鼠,用于分离培养肺组织间充质干细胞;清洁级6~8周龄C57BL/6雌鼠20只,分为细胞移植组、模型对照组,10只/组。方法:分离雄鼠肺组织,Ⅱ型胶原酶消化后,Ficoll法离心,收集界面层细胞,贴壁法分离培养肺组织间充质干细胞,当细胞接近70%~80%汇合时消化传代。细胞移植组、模型对照组小鼠按20mg/kg腔注射马利兰抑制骨髓干细胞的迁移后,均建立博莱霉素诱导的肺纤维化模型,造模后细胞移植组尾静脉注入5×105个肺间充质干细胞,模型对照组气管内注射100μLPBS。14d后取材,制备肺组织石蜡切片。主要观察指标:体外培养的肺组织间充质干细胞的生长状态、免疫表型、基因表达、分化潜能、集落形成能力以及静脉移植减轻肺损伤效果。结果:小鼠肺组织来源的间充质干细胞为成纤维样细胞,在体外能够长期培养和快速扩增;其细胞免疫表型为Sca-1 ,CD44 ,CD29 ,CD105 ,CD54 ,CD34-,CD45-,CD11b-,c-kit-,CD31-;不表达肺泡上皮细胞标志表面活性蛋白C、水通道蛋白5、clara细胞分泌蛋白,胚胎干细胞标志Oct-4和Nanog基因在培养扩增的细胞中持续表达;能够被诱导分化为脂肪细胞、成骨细胞和表达表面活性蛋白C的肺泡2型上皮样细胞;集落形成率约3%;经静脉输注后,细胞移植组肺损伤和纤维化程度较模型对照组减轻,Masson染色显示只有少量血管周围呈阳性,且经骨髓抑制处理后已接近正常肺组织。结论:体外培养的小鼠肺间充质干细胞能够快速扩增,可向脂肪细胞、成骨细胞和肺泡上皮细胞方向分化;静脉移植后有助于肺组织修复、减轻肺损伤和纤维化程度,且骨髓抑制不影响肺间充质干细胞的保护作用。     

Growth inhibition of established B16-F10 lung metastases by sequential aerosol delivery of p53 gene and 9-nitrocamptothecin

Growth inhibition of established tumor metastases in the lungs poses a difficult challenge for most clinical settings in spite of extensive multi-modality approaches. Aerosol delivery of drugs and genes holds promise for the treatment of disseminated lung metastases, since aerosol delivery can target the lungs specifically and uniformly. We previously demonstrated that aerosol delivery of dilauroylphosphatidylcholine liposome formulation of 9-nitrocamptothecin (9NC-DLPC) inhibits B16-F10 melanoma lung metastases. Aerosol delivery of polyethleneimine-p53 DNA (PEI-p53) complexes results in a similar anti-tumor effect in the B16-F10 model. In both these previous studies, the protocols were designed to inhibit development of lung metastases. In this study we demonstrate, using the B16-F10 melanoma lung metastasis model, that sequential aerosol delivery of PEI-p53 and 9NC-DLPC acts additively to inhibit growth of established B16-F10 tumor metastases in the lungs. Mice injected with B16-F10 cells and treated with a combination of 9NC-DLPC (twice weekly) and PEI-p53 (once weekly) aerosol complexes starting on day 11 after tumor inoculation, exhibited a highly significant (P < 0.01) reduction in the number of visible tumor foci as compared with untreated mice or mice treated with either single agent alone, or with a combination of 9NC and a control plasmid. There was a highly significant reduction in the tumor burden, as well as the lung weights for the 9NC and p53 combination group (P < 0.001 as compared with other groups). Moreover, the doses of p53 gene and 9NC in the combination group were reduced at least two-fold as compared with our previous single agent studies, but still achieved significant tumor inhibition. Furthermore, the sequential aerosol delivery of p53 and 9NC lead to a 30-40% increase in the mean survival time of these mice, as compared with animals in different control groups. The data suggest that the combination of 9NC and p53 gene delivered by aerosol is an attractive strategy for growth inhibition of established tumor metastases in the lungs.     

Adenovirus-mediated fibroblast growth factor 1 expression in the lung induces epithelial cell proliferation: consequences to hyperoxic lung injury in rats

High concentrations of oxygen can induce pulmonary toxicity and cause injury to alveolar epithelial and endothelial cells. The present study was performed to determine whether the potent epithelial and endothelial fibroblast growth factor 1 (FGF-1) protected against hyperoxia-induced lung injury. Recombinant adenovirus carrying the gene encoding human secreted FGF-1 (Ad. FGF1) increased the proliferation of lung epithelial cells in vitro. Ad.FGF1 or control vector with an empty expression cassette (Ad.V152) was administered intratracheally to Wistar rats. With Ad.FGF1 (10(9), 5 x 10(9), 10(10), or 5 x 10(10) viral particles [VP]), FGF-1 protein was found in bronchoalveolar lavage fluid 4 days postinfection at levels proportional to the viral dose and was detected in plasma after doses of 10(10) VP or more were administered. Histological examination of the lungs showed intense proliferation and apoptosis of alveolar and bronchial epithelial cells, with few inflammatory cells. The alveolar architecture returned to normal within 17 days. Rats pretreated with Ad.FGF1 (10(9) or 5 x 10(9) VP) 2 days before exposure to hyperoxia (95% O2) survived, whereas rats pretreated with Ad.V152 died within 3 days. In conclusion, adenovirus-mediated FGF-1 overexpression in the lungs causes epithelial cell proliferation and has beneficial effects in hyperoxic lung injury.     

Respiratory epithelial cell expression of human transforming growth factor-alpha induces lung fibrosis in transgenic mice.

Increased production of EGF or TGF-alpha by the respiratory epithelial cells has been associated with the pathogenesis of various forms of lung injury. Growth factors and cytokines are thought to act locally, via paracrine and autocrine mechanisms, to stimulate cell proliferation and matrix deposition by interstitial lung cells resulting in pulmonary fibrosis. To test whether TGF-alpha mediates pulmonary fibrotic responses, we have generated transgenic mice expressing human TGF-alpha under control of regulatory regions of the human surfactant protein C (SP-C) gene. Human TGF-alpha mRNA was expressed in pulmonary epithelial cells in the lungs of the transgenic mice. Adult mice bearing the SP-C-TGF-alpha transgene developed severe pulmonary fibrosis. Fibrotic lesions were observed in peribronchial, peribronchiolar, and perivascular regions, as well as subjacent to pleural surfaces. Lesions consisted of fibrous tissue that included groups of epithelial cells expressing endogenous SP-C mRNA, consistent with their identification as distal respiratory epithelial cells. Peripheral fibrotic regions consisted of thickened pleura associated with extensive collagen deposition. Alveolar architecture was disrupted in the transgenic mice with loss of alveoli in the lung parenchyma. Pulmonary epithelial cell expression of TGF-alpha in transgenic mice disrupts alveolar morphogenesis and produces fibrotic lesions mediated by paracrine signaling between respiratory epithelial and interstitial cells of the lung.     

Calorie restriction enhances adult mouse lung stem cells function and reverses several ageing‐induced changes

Ageing is associated with decreased lung function and an increased incidence of lung infections. Several studies have suggested that long‐term calorie restriction (CR) promotes health and longevity and results in the reduced risk of several diseases. The effect of CR is thought to be through improving the function of tissue stem cells. Stem cell function is known to decline with ageing. In this study, we examined the effects of ageing on lung epithelial and stem cells and the effect of CR on young and old lungs. We found that ageing results in a decrease in tracheal basal stem cells. CR induced an increase in basal stem cells in both young and old mice. In addition, ageing induced lung inflammation, and CR tended to reduce baseline lung inflammatory cell infiltration in young mice and significantly reduced ageing‐induced lung inflammation. Furthermore, ageing reduced the number and function of mitochondria in lung and increased the level of mitochondrial reactive oxygen species. CR increased the number and function of mitochondria both in young and old mice. Moreover, ageing reduced lung stem cell colony‐forming efficiency (CFE), and CR increased the CFE in both young and old mice. Finally, CR improved epithelial cell survival in injured lungs of young mice. In conclusion, ageing causes several structural and functional changes/impairments in lung epithelial cells. CR induces several potentially beneficial changes in lung epithelial cells, even when it is initiated at an older age, including reversal of some ageing‐induced changes.     

Characterization of a murine model of endotoxin-induced acute lung injury

Endotoxin-induced microvascular lung injury in mice is a commonly used experimental model of the acute respiratory distress syndrome (ARDS). The present paper aimed to characterize this popular model in a comprehensive and systematic fashion. Male C57bl/6 mice (n = 5) were administered an LD55 dose of E. coli endotoxin (15 mg/kg, i.p.), and lungs were harvested at several time points and evaluated for injury as well as for expression of a variety of inflammatory mediators. Endotoxin induced many features characteristic of acute microvascular lung injury. These included early (1-2 h) expression of inflammatory mediators (IL-1alpha, IL-1beta, IL-4, IL-6, IL-10, TNF-alpha, interferon-alpha, interferon gamma, and MCP-1) and leukocyte accumulation in lung tissue (lung myeloperoxidase activity 18.5 +/- 7.8 U/g tissue, P < 0.05), followed by pulmonary edema (lung water content index 17.4% +/- 2.5%, P < 0.05) and mortality. Histopathological evaluation of lung tissue was compatible with these findings. The characterization of this murine model of endotoxin-induced microvascular injury will facilitate its utilization in ARDS research.     

PINK1 deficiency impairs mitochondrial homeostasis and promotes lung fibrosis

Although aging is a known risk factor for idiopathic pulmonary fibrosis (IPF), the pathogenic mechanisms that underlie the effects of advancing age remain largely unexplained. Some age-related neurodegenerative diseases have an etiology that is related to mitochondrial dysfunction. Here, we found that alveolar type II cells (AECIIs) in the lungs of IPF patients exhibit marked accumulation of dysmorphic and dysfunctional mitochondria. These mitochondrial abnormalities in AECIIs of IPF lungs were associated with upregulation of ER stress markers and were recapitulated in normal mice with advancing age in response to stimulation of ER stress. We found that impaired mitochondria in IPF and aging lungs were associated with low expression of PTEN-induced putative kinase 1 (PINK1). Knockdown of PINK1 expression in lung epithelial cells resulted in mitochondria depolarization and expression of profibrotic factors. Moreover, young PINK1-deficient mice developed similarly dysmorphic, dysfunctional mitochondria in the AECIIs and were vulnerable to apoptosis and development of lung fibrosis. Our data indicate that PINK1 deficiency results in swollen, dysfunctional mitochondria and defective mitophagy, and promotes fibrosis in the aging lung.     

Keratinocyte growth factor gene transduction ameliorates acute lung injury and mortality in mice

At present there is no known effective pharmacological therapy for acute lung injury (ALI). Because keratinocyte growth factor (KGF) promotes epithelial cell growth, intratracheal administration of KGF has the possibility of restoring lung tissue integrity in injured lungs and improving patient outcomes. However, treatment using recombinant KGF protein is limited by its short effective duration. Thus, we investigated the effectiveness of intratracheal KGF gene transduction using adenoviral vector in ALI. We constructed an adenoviral vector expressing mouse KGF (mKGF), and 1.0 x 10(9 ) plaque-forming units of mKGF cDNA-expressing (Ad-KGF) and control (Ad-1w1) adenoviral vector was intratracheally instilled, using a MicroSprayer, into anesthetized BALB/c mice. Three days later, the mice were exposed to >90% oxygen for 72 hr, and the effect of KGF on hyperoxia-induced lung injury was examined. In the Ad-KGF group, KGF was strongly expressed in the airway epithelial cells, while peribronchiolar and alveolar inflammation caused by adenoviral vector instillation was minimal. The KGF overexpression not only induced proliferation of surfactant protein C-positive cuboidal cells, especially in the terminal bronchiolar and alveolar walls, but also prevented lung injury including intraalveolar exudation/hemorrhage, albumin permeability increase, and pulmonary edema. The arterial oxygen tension and the survival rate were significantly higher in the KGF-transfected group. These findings suggest that KGF gene transduction into the airway epithelium is a promising potential treatment for ALI.     

Imipenem, doxycycline and amikacin in monotherapy and in combination in Acinetobacter baumannii experimental pneumonia

Acinetobacter baumannii is a common cause of nosocomial pneumonia and other nosocomial infections. Multiresistant A. baumannii has also a high prevalence, which can make effective treatment difficult. We designed a new model of A. baumannii experimental pneumonia using C57BL/6 immunocompetent mice. This model was used to compare the efficacy of imipenem, doxycycline and amikacin in monotherapy, and the combination of imipenem plus amikacin and doxycycline plus amikacin. Doxycycline plus amikacin were synergic in vitro after 24 h incubation, whereas imipenem plus amikacin showed no in vitro synergy. The number of sterile lungs and the lung clearance of A. baumannii were greater in the group treated with imipenem than in those treated with amikacin or doxycycline in monotherapy (P < 0.05). The combination of imipenem plus amikacin and doxycycline plus amikacin was no more effective than imipenem alone in the clearance of organisms from lungs (2.42 +/- 1.46 cfu/g versus 2.7 +/- 1.5 cfu/g versus 1.23 +/- 1.02 cfu/g). These results suggest that the addition of amikacin does not improve the results obtained by imipenem monotherapy. Doxycycline plus amikacin is an alternative to imipenem in the therapy of A. baumannii pneumonia.