Nonhematopoietic cells are the primary source of bone marrow-derived lung epithelial cells

Previous studies have demonstrated that bone marrow (BM)-derived cells differentiate into nonhematopoietic cells of multiple tissues. To date, it remains unknown which population(s) of BM cells are primarily responsible for this engraftment. To test the hypothesis that nonhematopoietic stem cells in the BM are the primary source of marrow-derived lung epithelial cells, either wild-type hematopoietic or nonhematopoietic BM cells were transplanted into irradiated surfactant-protein-C (SPC)-null mice. Donor-derived, SPC-positive type 2 pneumocytes were predominantly detected in the lungs of mice receiving purified nonhematopoietic cells and were absent from mice receiving purified hematopoietic stem and progenitor cells. We conclude that cells contained in the nonhematopoietic fraction of the BM are the primary source of marrow-derived lung epithelial cells. These nonhematopoietic cells may represent a primitive stem cell population residing in adult BM.     

Bone marrow-derived mesenchymal stem cells in repair of the injured lung

We sought to determine whether an intact bone marrow is essential to lung repair following bleomycin-induced lung injury in mice, and the mechanisms of any protective effects conferred by bone marrow-derived mesenchymal stem cell (BMDMSC) transfer. We found that myelosupression increased susceptibility to bleomycin injury and that BMDMSC transfer was protective. Protection was associated with the differentiation of engrafted BMDMSC into specific and distinct lung cell phenotypes, with an increase in circulating levels of G-CSF and GM-CSF (known for their ability to promote the mobilization of endogenous stem cells) and with a decrease in inflammatory cytokines. In vitro, cells from injured, but not from normal, mouse lung produced soluble factors that caused BMDMSC to proliferate and migrate toward the injured lung. We conclude that bone marrow stem cells are important in the repair of bleomycin-injured lung and that transfer of mesenchymal stem cells protects against the injury. BMDMSC localize to the injured lung and assume lung cell phenotypes, but protection from injury and fibrosis also involves suppression of inflammation and triggering production of reparative growth factors.     

Bone marrow-derived mesenchymal stromal cells are attracted by multiple myeloma cell-produced chemokine CCL25 and favor myeloma cell growth in vitro and in vivo

Multiple myeloma (MM) is a malignancy of terminally differentiated plasma cells that are predominantly localized in the bone marrow (BM). Mesenchymal stromal cells (MSCs) give rise to most BM stromal cells that interact with MM cells. However, the direct involvement of MSCs in the pathophysiology of MM has not been well addressed. In this study, in vitro and in vivo migration assays revealed that MSCs have tropism toward MM cells, and CCL25 was identified as a major MM cell-produced chemoattractant for MSCs. By coculture experiments, we found that MSCs favor the proliferation of stroma-dependent MM cells through soluble factors and cell to cell contact, which was confirmed by intrafemoral coengraftment experiments. We also demonstrated that MSCs protected MM cells against spontaneous and Bortezomib-induced apoptosis. The tumor-promoting effect of MSCs correlated with their capacity to enhance AKT and ERK activities in MM cells, accompanied with increased expression of CyclinD2, CDK4, and Bcl-XL and decreased cleaved caspase-3 and poly(ADP-ribose) polymerase expression. In turn, MM cells upregulated interleukin-6 (IL-6), IL-10, insulin growth factor-1, vascular endothelial growth factor, and dickkopf homolog 1 expression in MSCs. Finally, infusion of in vitro-expanded murine MSCs in 5T33MM mice resulted in a significantly shorter survival. MSC infusion is a promising way to support hematopoietic recovery and to control graft versus host disease in patients after allogeneic hematopoietic stem cell transplantation. However, our data suggest that MSC-based cytotherapy has a potential risk for MM disease progression or relapse and should be considered with caution in MM patients.     

Bone marrow-derived cells contribute to cell turnover in aging murine hearts

The paradigm that cardiac myocytes are non-proliferating, terminally differentiated cells was recently challenged by studies reporting the ability of bone marrow-derived cells (BMCs) to differentiate into cardiomyocytes after myocardial damage. However, little knowledge exists about the role of BMCs in the heart during physiological aging. Twelve-week-old mice (n=36) were sublethally irradiated and bone marrow from littermates transgenic for enhanced green fluorescent protein (eGFP) was transplanted. After 4 weeks, 18 mice were sacrificed at the age of 4 months and served as controls (group A); the remaining mice were sacrificed at the age of 18 months (group B). Group A did not exhibit a significant number of eGFP+ cells, whereas 9.4±2.8 eGFP+ cells/mm2 was documented in group B. In total, only five eGFP+ cardiomyocytes were detected in 20 examined hearts, excluding a functional role of BM differentiation in cardiomyocytes. Similarly, a relevant differentiation of BMCs in endothelial or smooth muscle cells was excluded. In contrast, numerous BM-derived fibroblasts and myofibroblasts were observed in group B, but none were detected in group A. The present study demonstrates that BMCs transdifferentiate into fibroblasts and myofibroblasts in the aging murine myocardium, suggesting their contribution to the preservation of the structural integrity of the myocardium, while they do not account for regenerative processes of the heart.     

Enhanced expression of stem cell markers and drug resistance in sphere-forming non-small cell lung cancer cells

There is growing evidence suggesting that cancer stem cells (CSCs) are playing critical roles in tumor progression, metastasis and drug resistance. However, the role of CSCs in non-small cell lung cancer (NSCLC) remains elusive. In this study, we enriched for stem-like cells from tumor spheres derived from NSCLC cell line A549 cultured in serum-free medium. Our results showed that sphere-derived cells expressed various stem cell markers such as CD44, CD133, Sox2 and Oct4. Compared with the corresponding cells in monolayer cultures, sphere-derived cells showed marked morphologic changes and increased expression of the stem cell markers CD133. Furthermore, we found that sphere-derived cells exhibited increased proliferation, cell-cycle progression as well as drug-resistant properties as compared to A549 adherent cells. Consistently, expression of several drug resistance proteins, including lung resistance-related protein (LRP), glutathion-S-transferase-π (GST-π) and multidrug resistance proteins-1 (MRP1) were all significantly enhanced in sphere-derived cells. These results indicate the enrichment of CSCs in sphere cultures and support their role in regulating drug resistance in NSCLC.     

Metaplastic breast carcinomas are enriched in markers of tumor-initiating cells and epithelial to mesenchymal transition

Metaplastic breast carcinomas constitute a distinct aggressive form of invasive breast cancer with histological evidence of epithelial to mesenchymal transition toward spindle, chondroid, or osseous cell types. During tumorigenesis, epithelial to mesenchymal transition promotes invasion and metastasis and has been linked to the presence of stem cells. We hypothesized that metaplastic carcinomas may express epithelial to mesenchymal transition markers and may be enriched in tumor-initiating cells specifically in the non-glandular metaplastic elements. In 27 primary metaplastic carcinomas of the breast we tested the expression of epithelial to mesenchymal transition inducers ZEB1 and E-cadherin and the presence of tumor-initiating cells by using aldehyde dehydrogenase-1 (ALDH-1) and CD44(+)/CD24(-/low) immunohistochemistry. Of the 27 metaplastic carcinomas, 20 (74%) had squamous and/or spindle areas and 7 (26%) had heterologous elements (6 chondroid and 1 osseous). ALDH-1-positive and CD44(+)/CD24(-/low)-expressing cells were detected in the non-glandular metaplastic components (Fisher's exact, P=0.0017). E-cadherin expression was reduced or absent (aberrant) in all metaplastic components whereas it was normal in the glandular areas. On the contrary, overexpression of ZEB1 was detected in 41% (11 of 27) of the non-glandular, metaplastic components, and in none of the glandular areas. The presence of tumor-initiating cells, aberrant E-cadherin, and ZEB1 upregulation was associated in over 90% of the spindle areas and heterologous elements (χ(2) test, P<0.05). We provide first in situ evidence that epithelial to mesenchymal transition inducers and tumor-initiating cells are present specifically in the non-glandular components of metaplastic carcinomas.     

Bone marrow-derived cells expand memory CD8+ T cells in response to viral infections of the lung and skin

While naive CD8(+) T cells have been shown to require bone marrow-derived dendritic cells (DC) to initiate immunity, such a requirement for memory CD8(+) T cells has had limited assessment. By generating bone marrow chimeras that express the appropriate antigen-presenting molecules on either radiation-sensitive bone marrow-derived or radiation-resistant non-bone marrow-derived compartments, we showed that both primary and secondary immune responses to influenza virus infection of the lung were initiated in the draining LN. This required cells of bone marrow origin, most likely DC, for optimal expansion within the secondary lymphoid compartment. This was similarly the case with HSV-1 infection of the skin. As Langerhans cells are radioresistant, unlike other DC populations, these studies also demonstrate that the radiosensitive DC responsible for secondary expansion of HSV-specific memory are not Langerhans cells.     

In vivo demonstration and quantification of intracellular Bacillus anthracis in lung epithelial cells

Inhalational anthrax is initiated by the entry of Bacillus anthracis spores into the lung. A critical early event in the establishment of an infection is the dissemination of spores from the lung. Using in vitro cell culture assays, we previously demonstrated that B. anthracis spores are capable of entering into epithelial cells of the lung and crossing a barrier of lung epithelial cells without apparent disruption of the barrier integrity, suggesting a novel portal for spores to disseminate from the lung. However, in vivo evidence for spore uptake by epithelial cells has been lacking. Here, using a mouse model, we present evidence that B. anthracis spores are taken up by lung epithelial cells in vivo soon after spores are delivered into the lung. Immunofluorescence staining of thin sections of lungs from spore-challenged BALB/c mice revealed that spores were associated with the epithelial surfaces in the airway and the alveoli at 2 and 4 h postinoculation. Confocal analysis further indicated that some of the associated spores were surrounded by F-actin, demonstrating intracellular localization. These observations were further confirmed and substantiated by a quantitative method that first isolated lung cells from spore-challenged mice and then stained these cells with antibodies specific for epithelial cells and spores. The results showed that substantial amounts of spores were taken up by lung epithelial cells in vivo. These data, combined with those in our previous reports, provided powerful evidence that the lung epithelia were directly targeted by B. anthracis spores at early stages of infection.     

ALDH1-bright epithelial ovarian cancer cells are associated with CD44 expression, drug resistance, and poor clinical outcome

The role of aldehyde dehydrogenase 1 (ALDH1) as an ovarian cancer stem cell marker and its clinical significance have rarely been explored. We used an Aldefluor assay to isolate ALDH1-bright (ALDH1(br)) cells from epithelial ovarian cancer cell lines and characterized the properties of the stem cells. ALDH1(br) cells were enriched in ES-2 (1.3%), TOV-21G (1.0%), and CP70 (1.2%) cells. Both ALDH1(br) and ALDH1(low) cells repopulated stem cell heterogeneity, formed spheroids, and grew into tumors in immunocompromised mice, although these processes were more efficient in ALDH1(br) cells. In the ES-2 and CP70 cells, ALDH1(br) cells conferred more chemoresistance, and were more enriched in CD44 (by 1.74-fold and 5.18-fold, respectively) than in CD133 (by 1.39-fold and 1.17-fold, respectively), compared with ALDH1(low) cells. Immunohistochemical staining for ALDH1 on a tissue microarray containing 84 epithelial ovarian cancer samples revealed that patients with higher ALDH1 expression (>50%) had poor overall survival, compared with those with lower ALDH1 (P = 0.004) and yielded an odds ratio of death of 2.43 (95% CI = 1.12 to 5.28) by multivariate analysis. The results did not support ALDH1 alone as an ovarian cancer stem cell marker, but demonstrated that ALDH1 is associated with CD44 expression, chemoresistance, and poor clinical outcome. The use of a combination of ALDH1 with other stem cell markers may help define ovarian cancer stem cells more stringently.     

B-1 cells are pivotal for in vivo inflammatory giant cell formation

The mechanisms that govern giant cell (GC) formation in inflammatory, neoplastic and physiologic conditions are far from being understood. Here, we demonstrate that B-1 cells are essential for foreign-body GC formation in the mouse. GCs were analysed on the surface of glass cover slips implanted into the subcutaneous tissue of the animals. It was demonstrated that GCs are almost absent on cover slips implanted into the subcutaneous tissue of BALB/c or CBA/N X-linked immunodeficient mice. As these animals do not have B-1 cells in the peritoneal cavity, they were reconstituted with B-1 cells obtained from cultures of adherent mouse peritoneal cells. Results showed that in B-1-reconstituted animals, the number of GCs on the implant surface surpassed the values obtained with preparations from wild animals. In animals selectively irradiated (pleural and peritoneal cavities) to deplete these cavities of B-1 cells, GCs were also not formed. Enriched suspensions of B-1 cells grown in culture were labelled with [(3)H]-tymidine and injected into the peritoneal cavity of naive mice before implantation of glass cover slips. After 4 days, about 17% of mononuclear cells had their nuclei labelled, and almost 70% of GCs had one or more of their nuclei labelled when analysed by histoautoradiographic technique. A few GCs expressed an immunoglobulin M when analysed by immunostaining and confocal microscopy. Overall, these data demonstrate that B-1 cells are pivotal in the mechanisms of foreign-body GC formation in the mouse.     

骨髓间充质干细胞参与A549肺腺癌的组织修复

背景：肿瘤被认为是一种特殊的不愈合创伤,骨髓间充质干细胞通过向肿瘤组织归巢和向间质成分分化,参与肿瘤间质重构,从而改变肿瘤微环境,影响肿瘤的生长和转移。 目的：在A549肺癌荷瘤小鼠模型上证实骨髓间充质干细胞参与其损伤修复,探讨骨髓间充质干细胞参与肿瘤组织修复的机制。 方法：体外分离、培养人骨髓间充质干细胞,使用流式细胞术鉴定,制造A549肺癌荷瘤小鼠模型。实验组采用瘤周注射人骨髓间充质干细胞,对照组注射等量PBS,对比观察动物生活情况,肿瘤生长大小。4周后取材,苏木精-伊红染色对比观察肿瘤组织,Masson染色对比胶原纤维含量,RT-PCR检测两组α平滑肌收缩蛋白的表达,免疫组织化学检测两组成纤维细胞特异蛋白、成纤维细胞活化蛋白的表达情况,反映两组肿瘤组织的间质纤维的程度。免疫组织化学方法对比两组中血管内皮生长因子、肝细胞生长因子、白细胞介素6、肌糖蛋白C的表达高低。 结果与结论：骨髓间充质干细胞促进荷瘤小鼠肿瘤的生长,实验组肿瘤组织生长速度明显快于对照组(P < 0.05)。RT-PCR检测α平滑肌收缩蛋白的表达：与对照组比较,实验组α平滑肌收缩蛋白 mRNA的表达水平显著升高。免疫组织化学方法检测实验组肿瘤组织中TAFs标志物：成纤维细胞特异蛋白、成纤维细胞活化蛋白的表达,IHC检测血管内皮生长因子、肝细胞生长因子、白细胞介素6、肌糖蛋白C的表达明显高于对照组,差异有显著性意义(P < 0.05)。说明骨髓间充质干细胞在肿瘤微环境中向成纤维细胞方向分化,参与肿瘤间质的形成和构建,分泌血管内皮生长因子、肝细胞生长因子、白细胞介素6、肌糖蛋白C等促进肿瘤的生长修复。     

Type I epithelial cells are the main target of whole-body hypoxic preconditioning in the lung

Whole-body hypoxic preconditioning (WHPC) prolongs survival of mice exposed to severe hypoxia by attenuating pulmonary edema and preserving gas exchange. However, the cellular and molecular mechanism(s) of this protection remains unclear. The objective of this study was to identify the cellular target(s) of WHPC in the lung. Conscious mice were exposed to hypoxia (7% O(2)) for 6 hours with or without pretreatment of WHPC ([8% O(2)] x 10 min/[21% O(2)] x 10 min; 6 cycles). Hypoxia caused severe lung injury, as shown by the development of high-permeability-type pulmonary edema and the release of lactate dehydrogenase and creatine kinase into the airspace and the circulation. All these signs of hypoxic lung injury were significantly attenuated by WHPC. Hypoxia also caused a remarkable release of type I cell markers (caveolin-2 and receptor for advanced glycation end products) in lung lavage that was almost completely abolished by WHPC. Conversely, hypoxia-induced release of type II cell markers (surfactant-associated proteins A and D) was only marginal, and was unaffected by WHPC. Electron microscopic analysis demonstrated considerable hypoxic damage in alveolar type I cells and vascular endothelial cells. Notably, WHPC completely eliminated hypoxic damage in the former and alleviated it in the latter. Type II cells appeared normal. Furthermore, WHPC up-regulated protein expression of cytoprotective genes in the lung, such as heat shock proteins and manganese superoxide dismutase. Thus, WHPC attenuates hypoxic lung injury through protection of cells constituting the respiratory membrane, especially hypoxia-vulnerable type I epithelial cells. This beneficial effect may involve up-regulation of cytoprotective genes.     

Host DNA repair proteins in response to Pseudomonas aeruginosa in lung epithelial cells and in mice

Although DNA repair proteins in bacteria are critical for pathogens' genome stability and for subverting the host defense, the role of host DNA repair proteins in response to bacterial infection is poorly defined. Here, we demonstrate, for the first time, that infection with the Gram-negative bacterium Pseudomonas aeruginosa significantly altered the expression and enzymatic activity of 8-oxoguanine DNA glycosylase (OGG1) in lung epithelial cells. Downregulation of OGG1 by a small interfering RNA strategy resulted in severe DNA damage and cell death. In addition, acetylation of OGG1 is required for host responses to bacterial genotoxicity, as mutations of OGG1 acetylation sites increased Cockayne syndrome group B (CSB) protein expression. These results also indicate that CSB may be involved in DNA repair activity during infection. Furthermore, OGG1 knockout mice exhibited increased lung injury after infection with P. aeruginosa, as demonstrated by higher myeloperoxidase activity and lipid peroxidation. Together, our studies indicate that P. aeruginosa infection induces significant DNA damage in host cells and that DNA repair proteins play a critical role in the host response to P. aeruginosa infection, serving as promising targets for the treatment of this condition and perhaps more broadly Gram-negative bacterial infections.     

多重耐药基因在睫状体上皮细胞容积调节中的作用

目的：研究多重耐药（ＭＤＲ１）基因与细胞容积调节的关系。方法：用反义寡核苷酸阻抑牛眼睫状体非色素上皮细胞ＭＤＲ１基因表达,在激光共聚焦显微镜下检测ＭＤＲ１基因反义寡核苷酸（ＭＤＲ－ａｎｔｉ）的导入,用反射／散射技术测量由低渗液引起的细胞容积变化。结果：阳离子脂质体促进ＭＤＲ－ａｎｔｉ导入细胞,ＭＤＲ－ａｎｔｉ导入量与剂量呈依赖性增强关系（ｒ＝０９７,Ｐ＜００１）。人ＭＤＲ－ａｎｔｉ使低渗液引起的细胞调节性容积减小反应延迟、缓慢,容积恢复不完全（６０％）,而鼠ＤＭＲ－ａｎｔｉ没有此作用。结论：人ＭＤＲ－ａｎｔｉ抑制细胞调节性容积减小,提示ＭＤＲ１基因参与细胞容积调节。     

The role of Axl in drug resistance and epithelial-to-mesenchymal transition of non-small cell lung carcinoma

Axl, a member of receptor tyrosine kinases (RTKs), has been established as a strong candidate for targeted therapy of cancer. Some reports showed that Axl is a promising therapeutic target to enhance EGFR TKI response in selected EGFR WT NSCLC patients. The present study was aimed to investigate the role of Axl in non-small cell lung carcinoma (NSCLC) drug resistance and the progress of epithelial-to-mesenchymal transition (EMT). MTT was used to detect the cytotoxicity of chemotherapeutic drugs in NSCLC cells, and Western blot to detect the expression of Axl in EGFR wild type NSCLC cell lines. The EMT markers were also determined by Western blot. We found that when downregulating Axl in EGFR WT NSCLC cells, the cells showed a more sensitive response to erlotinib than those overexpressed Axl. The further study showed that when downregulating Axl, the EMT markers E-cadherin was increased while N-cadherin and vimentin were decreased. Those data showed that the inhibition of Axl could reverse the EMT. Combined therapeutic strategies of the inhibitor of Axl and EGFR TKI could be more effective in the treatment of NSCLC drug resistance patients. The EMT signature and Axl might be predictive biomarkers of drug response and therapeutic targets in patients with NSCLC.     

Human T cell proliferative responses to particulate microbial antigens are supported by populations enriched in dendritic cells.

The efficacy of dendritic cells in antigen presentation was studied in eight healthy subjects using a lymphoproliferation assay. Both particulate (Mycobacterium leprae, H37Ra) and soluble (PPD, tetanus toxoid) bacterial antigens were used as stimulants over a concentration range of accessory cells (monocytes (MO) and dendritic cells (DC)) varying from 10 to 0.1% in co-cultures using T-enriched cells. In general, co-cultures with T + MO and T + DC at all concentrations of accessory cells showed significant improvement of antigen-induced lymphoproliferation over PBMC cultures. The improvement in delta ct/min of T + DC co-cultures as compared to T + MO with parallel concentrations of accessory cells (P less than 0.05 to less than 0.01) was significant. Of the bacterial antigens used to test the antigen-presenting ability of DC, the particulate antigen (H37Ra) showed the most impressive improvement (380%) of T cell proliferation in DC reconstituted cultures as compared to monocytes. PPD, soluble protein derived from a similar tuberculosis strain of mycobacteria, was not presented as effectively as the particulate equivalent even though the donors of the appropriate cell combinations showed skin test reactivity to this antigen.     

Pneumococcal interactions with epithelial cells are crucial for optimal biofilm formation and colonization in vitro and in vivo

The human nasopharynx is the main reservoir for Streptococcus pneumoniae (the pneumococcus) and the source for both horizontal spread and transition to infection. Some clinical evidence indicates that nasopharyngeal carriage is harder to eradicate with antibiotics than is pneumococcal invasive disease, which may suggest that colonizing pneumococci exist in biofilm communities that are more resistant to antibiotics. While pneumococcal biofilms have been observed during symptomatic infection, their role in colonization and the role of host factors in this process have been less studied. Here, we show for the first time that pneumococci form highly structured biofilm communities during colonization of the murine nasopharynx that display increased antibiotic resistance. Furthermore, pneumococcal biofilms grown on respiratory epithelial cells exhibited phenotypes similar to those observed during colonization in vivo, whereas abiotic surfaces produced less ordered and more antibiotic-sensitive biofilms. The importance of bacterial-epithelial cell interactions during biofilm formation was shown using both clinical strains with variable colonization efficacies and pneumococcal mutants with impaired colonization characteristics in vivo. In both cases, the ability of strains to form biofilms on epithelial cells directly correlated with their ability to colonize the nasopharynx in vivo, with colonization-deficient strains forming less structured and more antibiotic-sensitive biofilms on epithelial cells, an association that was lost when grown on abiotic surfaces. Thus, these studies emphasize the importance of host-bacterial interactions in pneumococcal biofilm formation and provide the first experimental data to explain the high resistance of pneumococcal colonization to eradication by antibiotics.