Promotion of conjunctival fibroblast-mediated collagen gel contraction by mast cells through up-regulation of matrix metalloproteinase release and activation.

Mast cells and conjunctival fibroblasts contribute to conjunctival wound healing and allergic ocular inflammation. The number of mast cells in the conjunctiva is increased in individuals with cicatricial fibrosis-causing ocular surface diseases and after glaucoma filtering surgery, suggesting that these cells may contribute to the scarring observed after such surgery. We studied the potential mechanism of fibroblast-mast cell interaction in the healing of conjunctival wounds using a three-dimensional collagen gel culture system. We found that mast cells derived from the bone marrow of mice embedded in a collagen gel did not induce gel contraction. However, an increase in mast cells was associated with increased collagen gel contraction mediated by mouse conjunctival fibroblasts. The extent of collagen degradation was not affected by the co-culture of mast cells and conjunctival fibroblasts. Gelatin zymography disclosed that mast cells increased the amounts of both the pro form of matrix metalloproteinase (MMP)-9 and the active form of MMP-2 in supernatants of conjunctival fibroblast cultures. Furthermore, the potentiating effect of mast cells on contraction of the collagen gel through conjunctival fibroblasts was attenuated by the addition of a synthetic MMP inhibitor. Thus, current results suggest that mast cells accelerate the conjunctival fibroblast-dependent contraction of collagen gel by increasing the release as well as activation of MMPs. Therefore, the interaction between mast cells and conjunctival fibroblasts may contribute to conjunctival scar formation after glaucoma filtering surgery.

Factors leading to failure to diagnose pulmonary malignant tumors using endobronchial ultrasound with guide sheath within the target lesion.

BACKGROUND: The diagnostic yield of peripheral pulmonary lesions has significantly increased with the use of radial endobronchial ultrasound with guide sheath within the lesion. Here, we retrospectively evaluated factors leading to misdiagnosis of pulmonary malignant tumors using endobronchial ultrasound with the guide sheath within the lesion. METHODS: We assessed the final histopathological diagnosis of biopsy samples taken from 130 patients with lung malignant tumors that underwent endobronchial ultrasound with guide sheath within the lesion. RESULTS: Among 130 patients, 8 (6%) showed no definite malignant findings in biopsy samples but the presence of malignant cells (primary lung cancer 7, diffuse large B cell lymphoma 1) was subsequently confirmed by histopathological study of specimens taken by computed tomography-guided needle biopsy or surgery. Of the eight cases with diagnostic failure, the size of the biopsy sample was insufficient in five due to technical difficulties during the diagnostic procedure, and the diagnosis of malignant tumor was difficult in five cases because of extensive scarring tissue or central necrosis. CONCLUSIONS: The results of this study showed that technical difficulties and/or pathological heterogeneity of the tumor might lead to failure to diagnose lung malignant tumor in cases using endobronchial ultrasound with guide sheath within the lesion.

Circulating extracellular vesicles are associated with lipid and insulin metabolism.

We have reported that hypertrophic adipocytes release extracellular vesicles (EVs) and the number of circulating adipocyte-derived EVs correlated with insulin and homeostasis model assessment-insulin resistance (HOMA-IR) in a pilot study using obese patients. Here, we explored the association between circulating EV level and various metabolic parameters, including obesity and lipid and glucose metabolisms, among 203 subjects (76 men and 127 women; median age, 54 yr) with or without risk factor for metabolic diseases, who received a 75-g oral glucose tolerance test (OGTT). Circulating EV number was significantly higher in men than in women ( P < 0.001). Circulating EV number in individuals with impaired OGTT pattern was significantly higher compared with those with normal OGTT patterns ( P < 0.05). Multiple regression analysis revealed that circulating EV number correlated most strongly and significantly with elevated triglyceride (TG; t = 8.55, P < 0.001). Additionally, circulating EV number correlated significantly with homeostasis model assessment-ß-cell function (HOMA-ß; t = 2.38, P < 0.05). Receiver operating characteristic curve revealed that the cutoff value of EV numbers in individuals with elevated serum TG levels (≧150 mg/dl) was identified (136,738 EVs/µl of plasma, P < 0.001, sensitivity 0.842, false-positive rate of 0.257). Perilipin and asialoglycoprotein receptor 1 were detected on a part of isolated circulating EVs, indicating EV release from adipocytes and hepatocytes, which were related to lipid and glucose metabolism. Circulating EVs represent a promising metabolic biomarker for lipid and glucose metabolism and have potential for monitoring metabolic status in humans, including individuals without metabolic risk factors.

Mice overexpressing latent matrix metalloproteinase-2 develop lung emphysema after short-term exposure to cigarette smoke extract.

Chronic obstructive pulmonary disease is the major growing cause of mortality and morbidity worldwide, and it is going to become the third most common cause of death by 2020. Chronic obstructive pulmonary disease is pathologically characterized by lung emphysema and small airway inflammation. Animal models are very important to get insights into the disease pathogenesis but current models of chronic obstructive pulmonary disease take a long time to develop. The need of a new model is compelling. In the present study we focus on the role of matrix metalloproteinases in the pathogenesis of chronic obstructive pulmonary disease and hypothesized that lung overexpression of latent matrix metalloproteinases-2 would allow the development of emphysema after short-term exposure to cigarette smoke extract inhalation. Human latent matrix metalloproteinases-2 transgenic mouse expressing high level of the protein in the lungs and wild type mouse were exposed to aerosolized cigarette smoke extract for two weeks. Transgenic mice showed significant lung emphysematous changes, increased infiltration of inflammatory cells and enhanced lung concentrations of inflammatory cytokines in the lungs compared to their wild type counterparts after inhalation of cigarette smoke extract. This novel mouse model will be a very useful tool for evaluating the mechanistic pathways and for development of novel therapies in cigarette smoke-associated lung emphysema.

Inhibition of Cell Apoptosis and Amelioration of Pulmonary Fibrosis by Thrombomodulin.

Pulmonary fibrosis is the terminal stage of a group of idiopathic interstitial pneumonias, of which idiopathic pulmonary fibrosis is the most frequent and fatal form. Recent studies have shown that recombinant human thrombomodulin (rhTM) improves exacerbation and clinical outcome of idiopathic pulmonary fibrosis, but the mechanism remains unknown. This study evaluated the mechanistic pathways of the inhibitory activity of rhTM in pulmonary fibrosis. Transgenic mice overexpressing human transforming growth factor-ß1 that develop spontaneously pulmonary fibrosis, and wild-type mice treated with bleomycin were used as models of lung fibrosis. rhTM was administered to mice by i.p. injection or by the intranasal route. Therapy with rhTM significantly decreased the concentration of high mobility group box1, interferon-γ, and fibrinolytic markers, the expression of growth factors including transforming growth factor-ß1, and the degree of lung fibrosis. rhTM significantly suppressed apoptosis of lung epithelial cells in in vivo and in vitro experiments. The results of the present study demonstrated that rhTM can inhibit bleomycin-induced pulmonary fibrosis and transforming growth factor-ß1-driven exacerbation and progression of pulmonary fibrosis, and that apart from its well-recognized anticoagulant and anti-inflammatory properties, rhTM can also suppress apoptosis of lung epithelial cells.

Renal Injury during Long-Term Crizotinib Therapy.

Crizotinib is highly effective against anaplastic lymphoma kinase-positive and c-ros oncogen1-positive non-small cell lung cancer. Renal dysfunction is associated with crizotinib therapy but the mechanism is unknown. Here, we report a case of anaplastic lymphoma kinase positive non-small cell lung cancer showing multiple cysts and dysfunction of the kidneys during crizotinib administration. We also present results demonstrating that long-term crizotinib treatment induces fibrosis and dysfunction of the kidneys by activating the tumor necrosis factor-α/nuclear factor-κB signaling pathway. In conclusion, this study shows the renal detrimental effects of crizotinib, suggesting the need of careful monitoring of renal function during crizotinib therapy.

Homeostasis of thymus-derived Foxp3+ regulatory T cells is controlled by ultraviolet B exposure in the skin.

Accumulating evidence shows that immunological tolerance induced by Ag administration together with UVB irradiation is dependent on Foxp3(+) CD4(+) regulatory T (Treg) cells. However, the mechanisms by which UVB controls Treg cells in the skin are currently unclear. In this study, we have shown that exposure to UVB induced expansion of Treg cells up to 50-60% of the CD4(+) T cells in the irradiated skin. The Treg cell expansion in the skin lasted for 2 wk after exposure, which contributed to homeostasis of Treg cells in the periphery later. UVB-expanded Treg cells formed clusters with dendritic cells and proliferated in situ. Furthermore, the expanded Treg cells appeared to derive from neuropilin 1(+) thymus-derived Treg (tTreg) cells in the periphery because UVB-expanded Treg cells possessed Treg cell-specific CpG hypomethylation pattern, as seen in tTreg cells. These results collectively indicate that homeostasis of tTreg cells is controlled by UVB exposure in the skin. UVB therapy may be useful for not only inflammatory skin disorders, but also autoimmunity, transplantation, and allergy.

Thrombomodulin inhibits the activation of eosinophils and mast cells.

Eosinophils and mast cells play critical roles in the pathogenesis of bronchial asthma. Activation of both cells leads to the release of pro-inflammatory mediators in the airway of asthmatic patients. Recently, we have shown that inhaled thrombomodulin inhibits allergic bronchial asthma in a mouse model. In the present study, we hypothesize that thrombomodulin can inhibit the activation of eosinophils and mast cells. The effect of thrombomodulin on the activation and release of inflammatory mediators from eosinophils and mast cells was evaluated. Thrombomodulin inhibited the eotaxin-induced chemotaxis, upregulation of CD11b and degranulation of eosinophils. Treatment with thrombomodulin also significantly suppressed the degranulation and synthesis of inflammatory cytokines and chemokines in eosinophils and mast cells. Mice treated with a low-dose of inhaled thrombomodulin have decreased number of eosinophils and activated mast cells and Th2 cytokines in the lungs compared to untreated mice. The results of this study suggest that thrombomodulin may modulate allergic responses by inhibiting the activation of both eosinophils and mast cells.

Inhibition of very late antigen-4 and leukocyte function-associated antigen-1 in experimental autoimmune uveoretinitis.

B10.RIII mice were immunized with interphotoreceptor retinoid binding protein peptide to induce uveitis. Mice were injected intraperitoneally with anti-very late antigen-4 (VLA-4), anti-leukocyte function-associated antigen-1 (LFA-1), or a control Ab every other day from Day 5 to Day 13 post-immunization. The eyes and spleens were harvested on Day 14 or 28. The eyes were used for histologic/cytokine mRNA expression analyses. The spleens were used for Ag-recall cytokine production assays and intracellular cytokine assays. Treatment with both Abs led to a profoundly significant reduction in severity of uveitis and cytokine mRNA expression in the eye. However, cytokine production by splenocytes was significantly upregulated. Discontinuation of Ab treatment led to an increase in uveitis severity and cytokine mRNA expression in the eye, but led to a decrease in cytokine production and intracellular IFN-γ(+) and IL-17A(+)cytokine profile by splenocytes. Thus, blockade of these molecules using specific Abs may be a therapeutic option for patients with uveitis; however, such treatment must be continued.

Behavior of bone marrow-derived cells following in vivo transplantation: differentiation into stromal cells with roles in organ maintenance.

After injection of green fluorescent protein-positive (GFP(+)) bone marrow (BM) cells into lethally irradiated wild-type mice, the organs of the recipient mice [BM transplantation (BMT) mice] were regenerated; however, irradiation of the cecum or spleen (only) blocked their regeneration with loss of injected BM cells. These results suggest that the donor cells first enter the BM and then migrate to the peripheral organs. The maintenance of epithelial structure and function is controlled by interactions between stromal cells and the epithelia; the organ is stable only if the stroma is functioning normally. In BMT mice, intestinal GFP(+) stromal cells were regenerated fairly rapidly although GFP(+) cells were observed only rarely in the intestinal epithelium even if it passes several weeks or months post BMT, indicating that BM-derived stromal cells play a pivotal role in epithelial renewal and are crucial for maintaining organ structure and function. BM-derived cells in the periphery possess a special key to return to the BM and then to migrate to various organs to become resident cells.

Radiofrequency ablation for ground-glass opacity-dominant lung adenocarcinoma.

PURPOSE: To evaluate retrospectively the clinical utility of lung radiofrequency (RF) ablation for the treatment of ground-glass opacity (GGO)-dominant lung adenocarcinoma. MATERIALS AND METHODS: From August 2004 through May 2012, 33 consecutive patients (14 men and 19 women; mean age, 71.1 y; age range, 46-84 y) with 42 lung tumors having ≥ 50% GGO component received lung RF ablation. The mean maximum tumor diameter was 1.6 cm ± 0.9 (range, 0.7-4.0 cm). Feasibility, safety, local tumor progression, and survival were evaluated. RESULTS: For the 42 RF sessions, after RF electrodes were placed in each target tumor, planned ablation protocols were completed in all sessions (100%; 42 of 42). No deaths related to the RF procedure occurred. Major and minor complication rates were 4.8% and 23.8%, respectively. Local tumor progression developed in 6 tumors (14.3%; 6 of 42) during a mean follow-up of 42 months ± 23 (range, 5-92 mo). Four of six tumors with local progression were controlled by repeated RF ablation. No evidence of disease was achieved in 31 of 33 patients (93.9%) at the end of the follow-up period. All but one patient (who died of brain hemorrhage) are alive today. Overall and cancer-specific survival rates were 100% and 100% at 1 year, 96.4% (95% confidence interval [CI], 77.5%-99.5%) and 100% at 3 years, and 96.4% (95% CI, 77.5%-99.5%) and 100% at 5 years, respectively. CONCLUSIONS: Lung RF ablation is a feasible, safe, and useful therapeutic option to control GGO-dominant lung adenocarcinoma.

[First case of lung abscess due to Yersinia pseudotuberculosis in Japan].

A 63-year-old previously healthy man was admitted to our hospital with diarrhea that had lasted for about 4 weeks, high fever and dyspnea. Chest computed tomography showed consolidation with a low-density area in the right middle lobe and small nodules with feeding vessels in the right upper lobe. On Day 8, a cavity was observed in the consolidation, and the lymph nodes in the mediastinum became necrotic. Yersinia pseudotuberculosis (serotype 4b) was cultured from blood, bronchial washing fluid, and lung tissue specimens. We diagnosed the lung lesions as septic pulmonary embolism caused by enterocolitis. We started treatment with tazobactam/piperacillin. It has been reported that high-dose ceftriaxone (CTRX) is effective, but CTRX at normal doses and other beta-lactams are less effective or even ineffective. Therefore, we changed to CTRX (4g/day) on Day 5, CTRX (2g/day) on Day 8, and oral cefditoren pivoxil (600 mg/day; a third-generation cephalosporin) on Day 18. Antibiotic therapy resulted in a favorable response. The patient was discharged from our hospital on day 25 in good health. To the best of our knowledge, this is the first case of a lung abscess caused by Y. pseudotuberculosis reported in Japan.

Thrombomodulin modulates dendritic cells via both antagonism of high mobility group protein B1 and an independent mechanism.

BACKGROUND: Thrombomodulin treatment modulates the properties of dendritic cells (DCs) converting them from immunogenic to tolerogenic and inducing its own expression on DCs. Thrombomodulin binds to the inflammatory mediator, high mobility group protein B1 (HMGB1), antagonizing signalling through its receptor, receptor for advanced glycation end products (RAGE). METHODS: To test if soluble thrombomodulin could antagonize HMGB1 signaling via RAGE on DCs. DCs were prepared from mouse bone marrow cells or human monocytes. In some experiments dendritic cells were sorted into thrombomodulin+ and thrombomodulin- populations. Expression of surface maturation markers was determined by flow cytometry following treatment with thrombomodulin in the presence or absence of HMGB1. RESULTS: Thrombomodulin+ dendritic cells secrete less HMGB1 into the medium. HMGB1 reduces the effects of thrombomodulin on expression of DC maturation markers. Treatment with thrombomodulin reduces the expression of maturation markers such as CD80 and CD86 and increases the expression of thrombomodulin on the DC surface. Treatment of DCs with neutralizing anti-HMGB1 antibody acted synergistically with thrombomodulin in increasing thrombomodulin expression on DCs. Treatment with thrombomodulin can still reduce the expression of surface markers on DCs derived from mice that are deficient in RAGE showing that thrombomodulin can affect DCs by an alternative mechanism. CONCLUSIONS: The results of this study show that thrombomodulin modulates DCs both by antagonizing the interaction of HMGB1 with RAGE and by an independent mechanism.

Physical Activity Using a Wearable Device as an Alternative to Performance Status in Patients With Advanced Lung Cancer.

Importance: The Eastern Cooperative Oncology Group Performance Status (ECOG PS) is extensively used to guide treatment decisions in patients with advanced lung cancer. However, its assessment is subjective, potentially leading to discordance among observers. Objective: To investigate the association between measured physical activity and ECOG PS, as well as the potential prognostic value of physical activity measurements in patients with advanced lung cancer. Design, Setting, and Participants: This single-institution, prospective observational study enrolled 119 patients with advanced lung cancer scheduled to receive systemic therapy as outpatients at Matsusaka Municipal Hospital in Mie, Japan. Participants wore the wearable device amuelink (Sony) for up to 14 days to measure physical activity, including metabolic equivalent tasks, distance walked, and number of steps taken. ECOG PS was assessed at enrollment, which took place from December 2021 to August 2022. Main Outcomes And Measures: The primary end point was estimating the area under the curve (AUC) for classification into ECOG PS of 2 or higher using physical activity measurements. An analysis of the association with survival was also conducted. Results: Among the 119 patients (median [range] age, 72 (32-88) years; 71 [59.7%] male), mean distance walked (MDW) had the highest diagnostic value for classifying an ECOG PS of 2 or greater, with an AUC of 0.818 (95% CI, 0.703-0.934). Moreover, MDW was also associated with 6-month survival, with an AUC of 0.806 (95% CI, 0.694-0.918). Survival curves significantly diverged based on the MDW threshold, indicating a potential association with survival outcome (hazard ratio, 0.17; 95% CI, 0.05-0.57). Conclusions and Relevance: The cohort study suggests that MDW, as measured by a wearable device, was associated with ECOG PS and may serve as a predictor of health status alongside ECOG PS categories. It demonstrates the potential of objectively measured physical activity in complementing subjective ECOG PS assessments in patients with advanced lung cancer. Further research is needed to confirm the prognostic value of physical activity measurements.

The Current Achievements of Multi-Gene Panel Tests in Clinical Settings for Patients with Non-Small-Cell Lung Cancer.

Some multi-gene panel tests have been implemented in clinical settings to guide targeted therapy in non-small-cell lung cancer (NSCLC) in Japan. The current performance of multi-gene panel tests under the condition that the Oncomine Dx Target Test (ODxTT) and Amoy Dx® Pan Lung Cancer PCR panel (AmoyDx-multi) are available remains relatively unknown. We retrospectively reviewed consecutive patients with NSCLC, whose FFPE samples were considered for genetic testing. We assessed the submission rates, the success rates, and the driver oncogene detection rates of multi-gene panel tests. A total of 225 patients were histologically newly diagnosed with NSCLC or diagnosed with a recurrence of NSCLC without a previous multi-gene panel test at our institution. Among the 225 patients, the FFPE samples of 212 patients (94.2%) were submitted for multi-gene panel testing, including 191 samples (84.9%) for the ODxTT and 21 samples (9.3%) for the AmoyDx-multi. Among the 212 samples submitted to multi-gene panel tests, the success rate was 99.5% (211/212). The detection rate of driver oncogene alterations for all histologies was 52.4% (111/212), and that for adenocarcinoma was 69.7% (106/152). A favorable submission rate and success rate of multi-gene panel tests were shown, along with a favorable detection rate in recent clinical settings.

Thrombin-activatable fibrinolysis inhibitor protects against acute lung injury by inhibiting the complement system.

Acute lung injury (ALI) is a devastating disease with an overall mortality rate of 30 to 40%. The coagulation/fibrinolysis system is implicated in the pathogenesis of ALI. Thrombin-activatable fibronolysis inhibitor (TAFI) is an important component of the fibrinolysis system. Recent studies have shown that the active form of TAFI can also regulate inflammatory responses by its ability to inhibit complement C3a, C5a, and osteopontin. We hypothesized that TAFI might have a protective role in ALI. To demonstrate this hypothesis, the development of ALI was compared between wild-type (WT) and TAFI-deficient mice. ALI was induced by intratracheal instillation of LPS. Control mice were treated with saline. Animals were killed 24 hours after LPS. The number of inflammatory cells and the concentration of total protein and inflammatory cytokines were significantly increased in bronchoalveolar lavage fluid from LPS-treated, TAFI-deficient mice compared with their WT counterparts. Significantly higher concentrations of C5a were found in bronchoalveolar lavage fluid and plasma in LPS-treated TAFI knockout mice compared with WT mice. Pretreatment with inhaled C5a receptor antagonist blocked the detrimental effects of TAFI deficiency to levels found in WT mice. Our results show that TAFI protects against ALI, at least in part, by inhibiting the complement system.

A new strategy for healthcare-associated pneumonia: a 2-year prospective multicenter cohort study using risk factors for multidrug-resistant pathogens to select initial empiric therapy.

BACKGROUND: Optimal empiric therapy for hospitalized patients with healthcare-associated pneumonia (HCAP) is uncertain. METHODS: We prospectively applied a therapeutic algorithm, based on the presence of risk factors for multidrug-resistant (MDR) pathogens in a multicenter cohort study of 445 pneumonia patients, including both community-acquired pneumonia (CAP; n = 124) and HCAP (n = 321). RESULTS: MDR pathogens were more common (15.3% vs 0.8%, P < .001) in HCAP patients than in CAP patients, including Staphylococcus aureus (11.5% vs 0.8%, P < .001); methicillin-resistant S. aureus (6.9% vs 0%, P = .003); Enterobacteriaceae (7.8% vs 2.4%, P = .037); and Pseudomonas aeruginosa (6.9% vs 0.8%, P = .01). Using the proposed algorithm, HCAP patients with ≥2 MDR risk factors, one of which was severity of illness (n = 170), vs HCAP patients with 0-1 risk factor (n = 151) had a significantly higher frequency of MDR pathogens (27.1% vs 2%, P < .001). In total, 93.1% of HCAP patients were treated according to the therapy algorithm, with only 53% receiving broad-spectrum empiric therapy, yet 92.9% received appropriate therapy for the identified pathogen. Thirty-day mortality was significantly higher for HCAP than for CAP (13.7% vs 5.6%, P = .017), but among HCAP patients with 0-1 MDR risk factor, mortality was lower than with ≥2 MDR risk factors (8.6% vs 18.2%, P = .012). In multivariate analysis, initial treatment failure, but not inappropriate empiric antibiotic therapy, was a mortality risk factor (odds ratio, 72.0). CONCLUSIONS: Basing empiric HCAP therapy on its severity and the presence of risk factors for MDR pathogens is a potentially useful approach that achieves good outcomes without excessive use of broad-spectrum antibiotic therapy. CLINICAL TRIALS REGISTRATION: Japan Medical Association Center for Clinical Trials, JMA-IIA00054.

Promotion and maintenance of leukemia by ERG.

The Ets-related gene (ERG) located on human chromosome 21 encodes a transcription factor and is thought to be causally related to Down syndrome-associated acute megakaryocytic leukemia in childhood. In clinical adult leukemia, however, increased expression of ERG is indicative of poor prognosis in T-cell acute lymphoblastic leukemia and cytogenetically normal acute myeloid leukemia, although the involvement of ERG in the development of adult leukemia remains elusive. Here, we show that forced expression of ERG in adult BM cells alters differentiation and induces expansion of T and erythroid cells and increases frequencies of myeloid progenitors in mouse BM transplantation models. The expanded T cells then develop T-cell acute lymphoblastic leukemia after acquisition of mutations in the Notch1 gene. Targeted expression of ERG into B cells also altered differentiation and promoted growth of precursor B cells. Overall, these findings suggest a general role of ERG in promoting growth of adult hematopoietic cells in various lineages. In line with this, shRNA-mediated silencing of ERG expression attenuated growth of human leukemia cell lines of various lineages. Thus, ERG is capable of promoting the development of leukemia and is crucial for its maintenance.