Tomohiro Murakawa

Microvascular destruction identifies murine allografts that cannot be rescued from airway fibrosis

Small airway fibrosis (bronchiolitis obliterans syndrome) is the primary obstacle to long-term survival following lung transplantation. Here, we show the importance of functional microvasculature in the prevention of epithelial loss and fibrosis due to rejection and for the first time, relate allograft microvascular injury and loss of tissue perfusion to immunotherapy-resistant rejection. To explore the role of alloimmune rejection and airway ischemia in the development of fibroproliferation, we used a murine orthotopic tracheal transplant model. We determined that transplants were reperfused by connection of recipient vessels to donor vessels at the surgical anastomosis site. Microcirculation through the newly formed vascular anastomoses appeared partially dependent on VEGFR2 and CXCR2 pathways. In the absence of immunosuppression, the microvasculature in rejecting allografts exhibited vascular complement deposition, diminished endothelial CD31 expression, and absent perfusion prior to the onset of fibroproliferation. Rejecting grafts with extensive endothelial cell injury were refractory to immunotherapy. After early microvascular loss, neovascularization was eventually observed in the membranous trachea, indicating a reestablishment of graft perfusion in established fibrosis. One implication of this study is that bronchial artery revascularization at the time of lung transplantation may decrease the risk of subsequent airway fibrosis.

The ground glass opacity component can be eliminated from the T-factor assessment of lung adenocarcinoma †

OBJECTIVESnThe radiological ground glass opacity (GGO) component of an adenocarcinoma pathologically reflects a non-invasive adenocarcinoma in situ (AIS). Measuring the tumour diameter to include the GGO component may overestimate the T factor. In this retrospective study, we evaluated the effect of the GGO component on the recurrence of an adenocarcinoma.nnnMETHODSnWe reviewed patients who underwent a surgical resection of a lung adenocarcinoma and were pathologically proven to be T1-2N0M0, from 1999 to 2009. We conducted four different types of analyses (multivariate analysis, receiver operating characteristic [ROC] analysis, survival analysis according to subcategories and survival analysis of propensity score-matched pairs) to evaluate the impacts of GGO and the solid component on recurrence.nnnRESULTSnThe study included 241 patients, and there were 34 recurrences. Sixty-eight cases with AIS and minimally invasive adenocarcinoma exhibited 100% recurrence-free survival. A univariate and a multivariate analysis revealed that the maximum tumour diameter measured in the mediastinal window was a better prognostic factor than the maximum tumour diameter in the lung window. This finding was supported by an ROC curve analysis, a subgroup analysis and a propensity score-matched analysis. An ROC curve analysis revealed that GGO component exclusion resulted in improved prognostic performance for recurrence and pathological vessel invasion. A subgroup analysis and a propensity score-matched analysis demonstrated that tumours with similar solid component sizes and different GGO sizes exhibited equivalent recurrence-free survival.nnnCONCLUSIONSnThe GGO component showed little influence on recurrence. Recurrence-free survival was solely dependent on the solid component size. A T factor measured by the solid component may be a more accurate prognostic parameter.

CpG island methylation of microRNAs is associated with tumor size and recurrence of non-small-cell lung cancer

We investigated whether the CpG island methylation of certain microRNAs was associated with the clinicopathological features and the prognosis of non‐small‐cell lung cancer. The methylation of mir‐152, ‐9‐3, ‐124‐1, ‐124‐2, and ‐124‐3 was analyzed in 96 non‐small‐cell lung cancer specimens using a combined bisulfite restriction analysis. The median observation period was 49.5u2003months. The methylation of mir‐9‐3, ‐124‐2, and ‐124‐3 was individually associated with an advanced T factor independent of age, sex, and smoking habit. Moreover, the methylation of multiple microRNA loci was associated with a poorer progression‐free survival in a univariate analysis. Our result enlightens the accumulation of aberrant DNA methylation which occurs in concordance with the tumor progression. (Cancer Sci 2011; 102: 2126–2131)

Ex vivo characterization of γδ T-cell repertoire in patients after adoptive transfer of Vγ9Vδ2 T cells expressing the interleukin-2 receptor β-chain and the common γ-chain

BACKGROUND AIMSnAdoptive immunotherapy is emerging as a potent anti-tumor treatment modality; Vγ9Vδ2 T cells may represent appropriate agents for such cancer immunotherapy. To improve the currently limited success of Vγ9Vδ2 T-cell-based immunotherapy, we examined the in vivo dynamics of these adoptively-transferred cells and hypothesized that interleukin (IL)-15 is the potential factor for Vγ9δ2 T cell in vivo survival.nnnMETHODSnWe conducted a clinical trial of adoptive Vγ9Vδ2 T-cell transfer therapy in six colorectal cancer patients who received pulmonary metastasectomy. Patients peripheral blood mononuclear cells were stimulated with zoledronate (5 μmol/L) and IL-2 (1000 IU/mL) for 14 d. Harvested cells, mostly Vγ9Vδ2 T cells, were given intravenously weekly without additional IL-2 eight times in total. The frequency, phenotype and common γ-chain cytokine receptor expression of Vγ9Vδ2 T cells in peripheral blood was monitored by flow cytometry at each time point during treatment and 4 and 12 weeks after the last administration.nnnRESULTSnAdoptively transferred Vγ9Vδ2 T cells expanded well without exogenous IL-2 administration or lymphodepleting preconditioning. They maintained effector functions in terms of interferon-γ secretion and prompt release of cytotoxic granules in response to PMA/ionomycin or isopentenyl pyrophosphate-positive cells. Because they are IL-2Rα(-)IL-7Rα(-)IL-15Rα(-)IL-2Rβ(+)γc(+), it is likely that IL-2 or IL-15 is required for their maintenance.nnnCONCLUSIONSnThe persistence of large numbers of functionally active adoptively transferred Vγ9Vδ2 T cells in the absence of exogenous IL-2 implies that an endogenous factor, such as IL-15 transpresentation, is adequate to support these cells in vivo.

Simultaneous LFA-1 and CD40 Ligand Antagonism Prevents Airway Remodeling in Orthotopic Airway Transplantation: Implications for the Role of Respiratory Epithelium as a Modulator of Fibrosis

Airway remodeling is a prominent feature of certain immune-mediated lung diseases such as asthma and chronic lung transplant rejection. Under conditions of airway inflammation, the respiratory epithelium may serve an important role in this remodeling process. Given the proposed role of respiratory epithelium in nonspecific injury models, we investigated the respiratory epithelium in an immune-specific orthotopic airway transplant model. MHC-mismatched tracheal transplants in mice were used to generate alloimmune-mediated airway lesions. Attenuation of this immune injury and alteration of antidonor reactivity were achieved by the administration of combined anti-LFA-1/anti-CD40L mAbs. By contrast, without immunotherapy, transplanted airways remodeled with a flattening of respiratory epithelium and significant subepithelial fibrosis. Unopposed alloimmune injury for 10 days was associated with subsequent epithelial transformation and subepithelial fibrosis that could not be reversed with immunotherapy. The relining of donor airways with recipient-derived epithelium was delayed with immunotherapy resulting in partially chimeric airways by 28 days. Partial epithelial cell chimerism was sufficient to prevent luminal fibrosis. However, epithelial chimerism was also associated with airway remodeling. Therefore, there appears to be an intimate relationship between the morphology and level of chimerism of the respiratory epithelium and the degree of airway remodeling following alloimmune injury.

Epithelial to Mesenchymal Transition in Murine Tracheal Allotransplantation: An Immunohistochemical Observation

BACKGROUNDnAberrant epithelial repair is a crucial event in the airway remodeling that characterizes obliterative bronchiolitis (OB) in transplanted lungs. Recent data from experiments using epithelial cell lines and human airway tissues from lung transplant recipients suggest that epithelial to mesenchymal transition (EMT) plays an important role in OB. The aim of this study was to clarify whether EMT is involved in airway remodeling in an animal model.nnnMETHODSnWe performed orthotopic tracheal transplantation from BALB/c to C57BL/6 mice with from BALC/c to BALB/c mouse grafts as controls. Five allogeneic and 3 syngeneic recipients were humanely killed at predetermined postoperative days 2-12 as well as 14 and 21. Histology was evaluated using hematoxylin-eosin (H&E) staining. We studied the expression of specific markers, including E-cadherin, an epithelial marker; α-smooth muscle actin (SMA), and S100A4, mesenchymal markers, and zinc finger E-box-binding homeobox 1 (ZEB1), an EMT-related transcription factor.nnnRESULTSnHistologic assessment of serial H&E stains of allogeneic grafts showed remarkable pseudostratified respiratory epithelium with subepithelial inflammatory cell infiltration, as well as denuded and flattened epithelium and subepithelial fibrosis. The dynamic epithelial changes occurred earlier than the subepithelial fibrosis. Immunohistochemical evaluation indicated the emergence of α-SMA- positive epithelial cells that were most prominent on day 7. The expression of E-cadherin was attenuated in α-SMA-positive epithelial cells. S100A4 was also expressed in epithelial cells. A few days before the intraepithelial expression of α-SMA, ZEB1 emerged in the nuclei of epithelial cells.nnnCONCLUSIONSnWe observed expression of an EMT-related transcription factor and mesenchymal markers along with the attenuation of epithelial marker expression in epithelial cells, several days before prominent subepithelial fibrosis formation, results that suggest epithelial cells to play an important fibrosis role in airway remodeling during epithelial to mesenchymal transition.

Chest wall deformity found in patients with primary spontaneous pneumothorax.

Background the current consensus is that primary spontaneous pneumothorax frequently occurs in young male patients who are tall and thin. However, upon careful observation, many chest wall deformities have been recognized in primary spontaneous pneumothorax patients. We investigated the distinctive features of the chest wall configuration of primary spontaneous pneumothorax patients by comparing them with controls (normal and Marfan syndrome patients). Methods the anteroposterior and transverse diameters of the bilateral hemithoraxes of each patient at predetermined levels were measured by computed tomography. The asymmetry, which was based on the right/left anteroposterior and right/left transverse diameter ratio, and thickness based on the anteroposterior/transverse ratio, were examined. Results severe anteroposterior asymmetry and transverse asymmetry were observed in Marfan syndrome patients, and moderate anteroposterior asymmetry and transverse asymmetry were observed in primary spontaneous pneumothorax patients in comparison with normal control patients. Primary spontaneous pneumothorax and Marfan patients showed flatter thoraxes than normal control patients. Conclusions primary spontaneous pneumothorax patients tend to have asymmetric and flat thoraxes. Subclinical thoracic wall deformity observed in the primary spontaneous pneumothorax group may partly contribute to alveolar pressure heterogeneity, and may be related to subpleural bleb formation.

Outcomes of combined modality therapy for patients with stage III or IV mediastinal malignant germ cell tumors

PurposeThe treatment of primary mediastinal germ cell tumors with cisplatin-based chemotherapy followed by surgery is an established practice; however, the prognosis has remained poor. This study reviews the survival outcomes of patients with primary mediastinal germ cell tumors to evaluate the efficacy of our treatment.MethodsWe retrospectively reviewed 11 consecutive patients with primary mediastinal germ cell tumors.ResultsWe had treated four patients with seminomas and seven patients with non-seminomas. Ten patients had undergone cisplatin-based chemotherapy. All patients underwent complete resection. Two patients showed a failure of first-line chemotherapy and thus received salvage chemotherapies, including paclitaxel plus ifosfamide followed by high-dose carboplatin plus etoposide (TI-CE) with stem cell transplantation. One of them died of relapse 29xa0months later; while the other patient remained disease-free for 56xa0months postoperatively. The postoperative overall 3-year survival rates of the patients with non-seminomas and seminomas were 83 and 100xa0%, respectively.ConclusionComplete resection after establishing normalized or decreased at a low-level serum tumor markers plateau plays a crucial role in the management of patients with primary mediastinal malignant germ cell tumors.

Fibroblasts of recipient origin contribute to airway fibrosis in murine tracheal transplantations

Dear Sirs, Bronchiolitis obliterans (BO) is a major limitation in the long-term success of lung transplantation. BO is characterized as fibrotic obliterations in small airways [1]. Fibroblasts are the key players in fibrosis. They produce extracellular matrix components, and those deposition results in fibrosis. The source of fibroblasts in transplanted organs is an unresolved question. Determining the origin of airway fibroblasts is considered to be a key step in establishing ways to prevent fibrosis. According to previous reports [2–4], three possible sources of fibroblasts in transplanted organs are recipient bone marrow cells [2], regional fibroblasts in the grafts [3], or transitioned donor cells that had undergone epithelial to mesenchymal transition (EMT) [4]. In this study, we investigated whether fibroblasts in rejected airways originated from donor or recipient cells using orthotopic tracheal transplantation (OTT) and heterotopic tracheal transplantation (HTT) mouse models with transgenic C57Bl/6 mice that ubiquitously expressed green fluorescent protein (GFP) (B6-Tg(GFP)). Subepithelial fibrosis in OTT allografts [5] and intraluminal fibrosis in HTT allografts [6] are observed on or after the 28th day. All animals received humane care in compliance with the ‘Guide for Animal Experimentation, University of Tokyo, revised 2007’ and the ‘Act on Welfare and Management of Animals’ published by Japanese ministry. All of the mice were purchased from Japan SLC, Inc. BALB/c and B6-Tg(GFP) female mice were used as donors or recipients. The heterotopic and orthotopic tracheal transplantations were performed under the operating microscope as previously reported [5,6]. For HTT, the donor tracheas were placed into the subcutaneous space of the anterior neck area. For OTTs and HTTs, the following strain combinations were used: transplantations from BALB/c to B6-Tg(GFP) mice, those from B6-Tg(GFP) to BALB/c mice, and from BALB/c to BALB/c as a syngeneic control. All grafts harvested on the 28th day were frozen within an optimal cutting temperature compound (Sakura Finetek Japan, Tokyo, Japan). The hematoxylin and eosin (H&E) stains were performed regularly after formalin fixation. Fibroblasts in allografts were characterized using anti-mouse a-SMA Cy3-conjugated antibody (C6198; Sigma-Aldrich, St. Louis, MO, USA). The emergence of fibroblasts suggested the occurrence of fibrosis. For GFP staining, an anti-mouse GFP fluorescein isothiocyanate-labeled antibody (A21311; Life technologies, Carlsbad, CA, USA) was used. After fixation and blocking, the sections were incubated with diluted conjugated antibodies (a-SMA 1:200, GFP 1:200). Additionally, DAPI (D1306; Life technologies, Carlsbad, CA, USA) was used as a nuclear counterstain. The images were acquired using a microscope (BIOREVO-9000; Keyence, Osaka, Japan). The percentages (GFP co-localized area)/ (subepithelial or intraluminal a-SMA expression area) were calculated using Image J Software (version 1.4.3.67; National Institute of Health, USA) for the OTTs and HTTs. The results are displayed as a box plot that was created with SPSS 11.0 (Dr. SPSS II for Windows, standard version; SPSS Inc., Chicago, IL, USA). In both OTT groups, subepithelial thickening and the emergence of fibroblasts occurred on the 28th day, and the reproducibility was similar in each group. For transplanted group from BALB/c to B6-Tg(GFP), the subepithelial a-SMA-positive cells were also GFP positive (Fig. 1a). In contrast, the subepithelial a-SMA-positive cells were GFP negative for the group transplanted from B6-Tg(GFP) to BALB/c (Fig. 1b). The proportion of GFP co-localization with the subepithelial aSMA staining is shown in Fig. 1e. Partial luminal obliterations were observed in both HTT groups on the 28th day. For transplanted group from BALB/c to B6-Tg(GFP), the majority of the a-SMA-positive cells in the graft lumens were GFP positive (Fig. 1c). In contrast, another allogeneic group, the luminal a-SMApositive fibroblasts were GFP negative (Fig. 1d). The calculated colocalized percentages for the luminal fibrosis groups Presented at the 33rd Annual meeting of the International Society for Heart and Lung Transplantation, Montr eal, 23–27 April 2013.

Toll-like Receptor 4 Signaling Affects Myofibroblasts Expression in Mice Tracheal Allograft

Toll-like Receptor 4 Signaling Affects Myofibroblasts Expression in Mice Tracheal Allograft M. Kawashima1, M. Sato1, T. Murakawa2, M. Anraku1, C. Konoeda1, A. Hosoi3, K. Kakimi3, J. Nakajima1. 1Department of Thoracic Surgery, The University of Tokyo, Tokyo, Japan, 2Department of Thoracic Surgery, Kansai Medical University, Osaka, Japan, 3Department of Immunotherapeutics, The University of Tokyo, Tokyo, Japan,