Proximal and Distal Bronchioles Contribute to the Pathogenesis of Non-Cystic Fibrosis Bronchiectasis.

Rationale: Non-cystic fibrosis bronchiectasis (NCFB) may originate in bronchiolar regions of the lung. Accordingly, there is a need to characterize the morphology and molecular characteristics of NCFB bronchioles. Objectives: Test the hypothesis that NCFB exhibits a major component of bronchiolar disease manifest by mucus plugging and ectasia. Methods: Morphologic criteria and region-specific epithelial gene expression, measured histologically and by RNA in situ hybridization and immunohistochemistry, identified proximal and distal bronchioles in excised NCFB lungs. RNA in situ hybridization and immunohistochemistry assessed bronchiolar mucus accumulation and mucin gene expression. CRISPR-Cas9-mediated IL-1R1 knockout in human bronchial epithelial cultures tested IL-1α and IL-1ß contributions to mucin production. Spatial transcriptional profiling characterized NCFB distal bronchiolar gene expression. Measurements and Main Results: Bronchiolar perimeters and lumen areas per section area were increased in proximal, but not distal, bronchioles in NCFB versus control lungs, suggesting proximal bronchiolectasis. In NCFB, mucus plugging was observed in ectatic proximal bronchioles and associated nonectatic distal bronchioles in sections with disease. MUC5AC and MUC5B mucins were upregulated in NCFB proximal bronchioles, whereas MUC5B was selectively upregulated in distal bronchioles. Bronchiolar mucus plugs were populated by IL-1ß-expressing macrophages. NCFB sterile sputum supernatants induced human bronchial epithelial MUC5B and MUC5AC expression that was >80% blocked by IL-1R1 ablation. Spatial transcriptional profiling identified upregulation of genes associated with secretory cells, hypoxia, interleukin pathways, and IL-1ß-producing macrophages in mucus plugs and downregulation of epithelial ciliogenesis genes. Conclusions: NCFB exhibits distinctive proximal and distal bronchiolar disease. Both bronchiolar regions exhibit bronchiolar secretory cell features and mucus plugging but differ in mucin gene regulation and ectasia.

Waning cellular immune responses and predictive factors in maintaining cellular immunity against SARS-CoV-2 six months after BNT162b2 mRNA vaccination.

Several clinical trials have shown that the humoral response produced by anti-spike antibodies elicited by coronavirus disease 2019 (COVID-19) vaccines gradually declines. The kinetics, durability and influence of epidemiological and clinical factors on cellular immunity have not been fully elucidated. We analyzed cellular immune responses elicited by BNT162b2 mRNA vaccines in 321 health care workers using whole blood interferon-gamma (IFN-γ) release assays. IFN-γ, induced by CD4 + and CD8 + T cells stimulated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike epitopes (Ag2), levels were highest at 3 weeks after the second vaccination (6 W) and decreased by 37.4% at 3 months (4 M) and 60.0% at 6 months (7 M), the decline of which seemed slower than that of anti-spike antibody levels. Multiple regression analysis revealed that the levels of IFN-γ induced by Ag2 at 7 M were significantly correlated with age, dyslipidemia, focal adverse reactions to full vaccination, lymphocyte and monocyte counts in whole blood, Ag2 levels before the second vaccination, and Ag2 levels at 6 W. We clarified the dynamics and predictive factors for the long-lasting effects of cellular immune responses. The results emphasize the need for a booster vaccine from the perspective of SARS-CoV-2 vaccine-elicited cellular immunity.

Interpretations of SARS-CoV-2 IgM and IgG antibody titers in the seroepidemiological study of asymptomatic healthy volunteers.

INTRODUCTION: The usefulness of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody tests in asymptomatic individuals has not been well validated, although they have satisfied sensitivity and specificity in symptomatic patients. In this study, we investigated the significance of IgM and IgG antibody titers against SARS-CoV-2 in the serum of asymptomatic healthy subjects. METHODS: From June 2020, we recruited 10,039 participants to the project named the University of Tokyo COVID-19 Antibody Titer Survey (UT-CATS), and measured iFlash-SARS-CoV-2 IgM and IgG (YHLO IgM and IgG) titers in the collected serum. For the samples with increased IgM or IgG titers, we performed additional measurements using Elecsys Anti-SARS-CoV-2 Ig (Roche total Ig) and Architect SARS-CoV-2 IgG (Abbott IgG) and investigated the reactivity to N, S1, and receptor binding domain (RBD) proteins. RESULTS: After setting the cutoff value at 5 AU/mL, 61 (0.61%) were positive for YHLO IgM and 104 (1.04%) for YHLO IgG. Few samples with elevated YHLO IgM showed reactivity to S1 or RBD proteins, and IgG titers did not increase during the follow-up in any samples. The samples with elevated YHLO IgG consisted of two groups: one reacted to S1 or RBD proteins and the other did not, which was reflected in the results of Roche total Ig. CONCLUSIONS: In SARS-CoV-2 seroepidemiological studies of asymptomatic participants, sufficient attention should be given to the interpretation of the results of YHLO IgM and IgG, and the combined use of YHLO IgG and Roche total Ig might be more reliable.

Measurement of chest wall motion using a motion capture system with the one-pitch phase analysis method.

Spirometry is a standard method for assessing lung function. However, its use is challenging in some patients, and it has limitations such as risk of infection and inability to assess regional chest wall motion. A three-dimensional motion capture system using the one-pitch phase analysis (MCO) method can facilitate high precision measurement of moving objects in real-time in a non-contacting manner. In this study, the MCO method was applied to examine thoraco-abdominal (TA) wall motion for assessing pulmonary function. We recruited 48 male participants, and all underwent spirometry and chest wall motion measurement with the MCO method. A significant positive correlation was observed between the vital capacity (Spearman's ρ = 0.68, p < 0.0001), forced vital capacity (Spearman's ρ = 0.62, p < 0.0001), and tidal volume (Spearman's ρ = 0.61, p < 0.0001) of spirometry and the counterpart parameters of MCO method. Moreover, the MCO method could detect regional rib cage and abdomen compartment contributions and could assess TA asynchrony, indicating almost complete synchronous movement (phase angle for each compartment: - 5.05° to 3.86°). These findings suggest that this technique could examine chest wall motion, and may be effective in analyzing chest wall volume changes and pulmonary function.

Epidemiological study using IgM and IgG antibody titers against SARS-CoV-2 in The University of Tokyo, Japan (UT-CATS).

INTRODUCTION: The worldwide pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continued to date. Given that some of the patients with coronavirus disease 2019 (COVID-19) are asymptomatic, antibody tests are useful to determine whether there is a previous infection with SARS-CoV-2. In this study, we measured IgM and IgG antibody titers against SARS-CoV-2 in the serum of asymptomatic healthy subjects in The University of Tokyo, Japan. METHODS: From June 2020, we recruited participants, who were students, staff, and faculty members of The University of Tokyo in the project named The University of Tokyo COVID-19 Antibody Titer Survey (UT-CATS). Following blood sample collection, participants were required to answer an online questionnaire about their social and health information. We measured IgG and IgM titers against SARS-CoV-2 using iFlash-SARS-CoV-2 IgM and IgG detection kit which applies a chemiluminescent immunoassay (CLIA) for the qualitative detection. RESULTS: There were 6609 volunteers in this study. After setting the cutoff value at 10 AU/mL, 32 (0.48%) were positive for IgG and 16 (0.24%) for IgM. Of six participants with a history of COVID-19, five were positive for IgG, whereas all were negative for IgM. The median titer of IgG was 0.40 AU/mL and 0.39 AU/mL for IgM. Both IgG and IgM titers were affected by gender, age, smoking status, and comorbidities. CONCLUSIONS: Positive rates of IgG and IgM titers were relatively low in our university. Serum levels of these antibodies were affected by several factors, which might affect the clinical course of COVID-19.

The Japanese Herbal (Kampo) Medicine Hochuekkito Attenuates Lung Inflammation in Lung Emphysema.

Chronic obstructive pulmonary disease (COPD) is a systemic inflammatory disorder. It often causes weight loss, which is considered a poor prognostic factor. A Japanese herbal Kampo medicine, Hochuekkito (TJ-41), has been reported to prevent systemic inflammation and weight loss in COPD patients, but the underlying biological mechanisms remain unknown. In the present study, we investigated the role of TJ-41 in vivo using a mouse model of lung emphysema. We used lung epithelium-specific Taz conditional knockout mice (Taz CKO mice) as the lung emphysema model mimicking the chronic pulmonary inflammation in COPD. Acute inflammation was induced by intratracheal lipopolysaccharide administration, simulating COPD exacerbation. Mice were fed a diet containing 2% TJ-41 or a control diet. Taz CKO mice showed increased numbers of inflammatory cells in the bronchoalveolar lavage fluid compared to control mice. This effect was reduced by TJ-41 treatment. In the acute exacerbation model, TJ-41 mitigated the increased numbers of inflammatory cells in the bronchoalveolar lavage fluid and attenuated lung inflammation in histopathological studies. Additional in vitro experiments using the human macrophage cell line U-937 demonstrated that lipopolysaccharide-induced tumor necrosis factor-alpha expression was significantly downregulated by TJ-41. These results suggest that TJ-41 has anti-inflammatory effects in lung emphysema both in the chronic phase and during an acute exacerbation. In conclusion, our study sheds light on the anti-inflammatory effects of TJ-41 in lung emphysema. This establishes its potential as a new anti-inflammatory therapy and a preventive medicine for exacerbations during the long-time maintenance of COPD patients.

FOXL1 Regulates Lung Fibroblast Function via Multiple Mechanisms.

Fibroblasts provide a structural framework for multiple organs and are essential for wound repair and fibrotic processes. Here, we demonstrate functional roles of FOXL1 (forkhead box L1), a transcription factor that characterizes the pulmonary origin of lung fibroblasts. We detected high FOXL1 transcripts associated with DNA hypomethylation and super-enhancer formation in lung fibroblasts, which is in contrast with fibroblasts derived from other organs. RNA in situ hybridization and immunohistochemistry in normal lung tissue indicated that FOXL1 mRNA and protein are expressed in submucosal interstitial cells together with airway epithelial cells. Transcriptome analysis revealed that FOXL1 could control a broad array of genes that potentiate fibroblast function, including TAZ (transcriptional coactivator with PDZ-binding motif)/YAP (Yes-associated protein) signature genes and PDGFRα (platelet-derived growth factor receptor-α). FOXL1 silencing in lung fibroblasts attenuated cell growth and collagen gel contraction capacity, underscoring the functional importance of FOXL1 in fibroproliferative reactions. Of clinical importance, increased FOXL1 mRNA expression was found in fibroblasts of idiopathic pulmonary fibrosis lung tissue. Our observations suggest that FOXL1 regulates multiple functional aspects of lung fibroblasts as a key transcription factor and is involved in idiopathic pulmonary fibrosis pathogenesis.

Active mTOR in Lung Epithelium Promotes Epithelial-Mesenchymal Transition and Enhances Lung Fibrosis.

The mTOR pathway is one of the key signal cascades in the pathogenesis of idiopathic pulmonary fibrosis. Previous studies have mainly focused on this pathway in the fibroblasts and/or myofibroblasts, but not in the epithelial cells. In this study, we sought to investigate the role of the mTOR pathway in lung epithelial cells in lung fibrosis. Using Sftpc-mTORSL1+IT transgenic mice, in which active mTOR is conditionally expressed in lung epithelial cells, we assessed the effects of chronically activated mTOR in lung epithelial cells on lung phenotypes as well as bleomycin-induced lung fibrosis. Furthermore, we isolated alveolar epithelial cell type 2 from mice and performed RNA sequencing. Sftpc-mTORSL1+IT transgenic mice had no obvious abnormal findings, but, after bleomycin administration, showed more severe fibrotic changes and lower lung compliance than control mice. RNA sequencing revealed Angptl4 (angiopoietin-like protein 4) as a candidate downstream gene of the mTOR pathway. In vitro studies revealed that ANGPTL4, as well as mTOR, promoted tight junction vulnerability and epithelial-mesenchymal transition. mTOR activation in lung epithelial cells promoted lung fibrosis and the expression of ANGPTL4, a novel downstream target of the mTOR pathway, which could be related to the etiology of fibrosis.

Preoperative intervention with long-acting bronchodilators for the reduction of postoperative pulmonary complications in untreated patients with obstructive lung disease.

BACKGROUND: Obstructive lung disease (OLD) is a risk factor for postoperative pulmonary complications (PPC) and is incidentally discovered during preoperative evaluation. The key treatments for OLD are inhaled long-acting bronchodilators (LAB). However, the advantage of preoperative bronchodilator treatment for patients with OLD remains unclear. The aim of this study is to elucidate the effect of preoperative LAB treatment in patients with untreated OLD on postoperative outcomes. METHODS: In this propensity-matched cohort study, we included patients who were referred to the pulmonologists for untreated OLD. The patients were either treated with LAB or left untreated. The primary outcome was the incidence of prolonged oxygen therapy (>3 days) in the postoperative period. We evaluated patients' characteristics with and without the use of LAB using propensity score (PS) matching weight. Subsequently, the outcomes in the two groups were compared. RESULTS: We analysed 614 patients; 132 patients were part of the LAB group and 482 were included in the control group. In the crude analysis, the incidence of prolonged oxygen therapy was higher in the LAB group than in the control group (odds ratio [OR] = 1.35; P = 0.04). However, after PS matching weight, no statistically significant differences in prolonged oxygen therapy (OR = 1.15), incidence of prolonged intensive care unit stay, endotracheal re-intubation postoperatively and in-hospital death between the groups were identified. CONCLUSION: There is a limited benefit of preoperative treatment with inhaled LAB for the reduction of PPC in patients with untreated OLD.

Prevalence of bone pain decreases as lymph node stage increases in nonsmall cell lung cancer patients.

According to lung cancer guidelines, positron emission tomography scan is recommended for initial evaluation of bone metastasis. However, guidelines differ in their recommendations for when it should be used. We investigated the appropriate use of bone imaging in nonsmall cell lung cancer (NSCLC) patients. One hundred seventy-seven consecutive NSCLC patients who had distant metastases at presentation and were admitted between January 2012 and April 2016 were retrospectively reviewed. Among patients with bone metastases, we explored bone pain, number of bone metastases, location of bone metastases, and clinical tumor (T) and lymph node (N) classification. Sixty-three patients had bone metastases. There was a trend toward an increase in prevalence of bone metastases as lymph node stage increased. The prevalence of bone pain significantly decreased as N stage increased (p = 0.017). N0 and N2-3 patients were more likely to have multiple bone metastases (p = 0.038). Compared with patients who had a single bone metastasis, patients with multiple metastases had a significantly higher probability of having at least 1 bone metastasis located in the thorax or upper abdomen. All N0 patients have at least 1 bone metastasis in the thorax or upper abdomen. Clinical N0 NSCLC patients with bone metastasis are likely to have bone pain and have multiple bone metastases. N2-3 patients are more likely to have bone metastases but less likely to have bone pain. If NSCLC patients do not have bone pain, and CT of the chest and upper abdomen does not reveal any lymph node or bone metastasis, further survey for bone metastases may be omitted; bone imaging should be performed in N2 and N3 patients regardless of symptoms.

Exosomal MicroRNA Expression Profiling in Patients with Lung Adenocarcinoma-associated Malignant Pleural Effusion.

BACKGROUND/AIM: Cytological analysis for diagnosing malignant pleural effusion associated with lung adenocarcinoma (Ad-MPE) shows limited sensitivity and novel diagnostic biomarkers are needed. The aim of this study was to evaluate the profile of four microRNAs (miRNAs) in exosomes in Ad-MPE and benign (non-neoplastic) pleural effusion (BPE). MATERIALS AND METHODS: A total of 56 pleural effusion samples from patients with lung adenocarcinoma and benign diseases were collected. Exosomal miRNA expressions were evaluated using real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The expression levels of miR-182 and miR-210 were significantly higher in the Ad-MPE than in the BPE samples. The receiver operating characteristics curve analyses of miR-182 and miR-210 for diagnosis of Ad-MPE yielded areas under the receiver operating characteristic curves of 0.87 and 0.81, respectively. CONCLUSION: These miRNA signatures may have a diagnostic potential for differentiating Ad-MPE from BPE.

Early stage non-small cell lung cancer patients need brain imaging regardless of symptoms.

BACKGROUND: Japanese Lung Cancer Society and ESMO guideline recommends screening for brain metastasis in all patients with non-small cell lung cancer (NSCLC), while NCCN/ACCP guidelines do not recommend screening patients who are asymptomatic and with clinical stage I NSCLC. However, brain metastasis sometimes occurs in early stage NSCLC patients without any neurological symptoms. METHODS: We retrospectively reviewed medical records of 124 patients admitted to the University of Tokyo Hospital with stage IV NSCLC from January 2012 to April 2016. We analyzed clinical stage, the presence of the central nervous system manifestations and the number of brain metastases. RESULTS: Forty-six out of 124 cases had brain metastasis at presentation. The brain metastasis group had larger number of female, never smokers and patients with EGFR mutation compared with extracranial metastasis group. Twenty-one of 35 adenocarcinoma cases with brain metastasis had EGFR mutations. Out of 46 brain metastasis patients, 29 patients (63%) were asymptomatic and patients with EGFR mutations were significantly less likely to have neurological symptoms (4/21 vs. 7/14, p = 0.049). Six out of 46 cases with brain metastasis (13%) were clinical T1-2aN0. In clinical T1-2aN0 cases, only one patient had neurological symptoms at presentation. CONCLUSION: In clinical T1-2aN0 lung cancer patients with brain metastasis, almost all patients were asymptomatic. Patients with EGFR mutations and brain metastasis were likely to be asymptomatic. Regardless of central nervous system symptoms, routine brain imaging seems warranted in all NSCLC patients, especially in areas where patients have a higher frequency of EGFR mutations.

Sudden Death in a Patient with Pulmonary Veno-occlusive Disease (PVOD) and Severe Pulmonary Hypertension.

A 58-year-old woman was referred to our hospital with a chief complaint of exertional dyspnea. Bronchoscopy failed to establish a diagnosis, and the patient subsequently died suddenly due to respiratory insufficiency because of advanced pulmonary hypertension (PH). The pathological diagnosis at autopsy was pulmonary veno-occlusive disease (PVOD). PVOD is difficult to diagnose antemortem and has a poor prognosis. Lung transplantation is the only curative treatment for PVOD.

Reversible Monolayer/Spheroid Cell Culture Switching by UCST-Type Thermoresponsive Ureido Polymers.

Multicellular spheroids have been studied in the fields of oncology, stem cell biology, and tissue engineering. In this study, we found a new polymer material for thermo-controlled spheroid/monolayer cell culture switching. The polymers that have pendant ureido groups (ureido polymers) exhibited upper critical solution temperature-type phase separation behavior. Cells in monolayer culture were converted to spheroids by the addition of ureido polymers below phase separation temperature (Tp). Time-lapse observations indicated that cells began to migrate and aggregate to form the spheroids to avoid contact with phase-separated polymer (coacervates) on the surface of the culture dish. We supposed that the coacervates seemingly suppressed interaction between cell and the dish surface or extracellular matrices. By increasing culture temperature above Tp, the spheroids began to collapse into a monolayer of cells due to dissolution of the coacervates. These results indicated that cell morphology could be repeatedly switched by changing the culture temperature in the presence of ureido polymers.

Analysis of drug treatment outcome in clarithromycin-resistant Mycobacterium avium complex lung disease.

BACKGROUND: Although the isolation of clarithromycin (CAM)-resistant Mycobacterium avium complex (MAC) indicates a poor treatment outcome and increased mortality, there have been only a few reports on drug treatment for CAM-resistant MAC lung disease. We aimed to reveal the effectiveness of the continuation of a macrolide and the use of a multidrug regimen in the treatment of CAM-resistant MAC lung disease. METHODS: Among patients with MAC pulmonary disease as defined by the 2007 criteria of the American Thoracic Society and the Infectious Diseases Society of America statement, those with CAM-resistant MAC (minimum inhibitory concentration ≥32 µg/ml) isolated, newly diagnosed and treated from January 2009 to June 2013 were analysed in this study. Effectiveness was measured based on culture conversion rate and improvement of radiological findings. RESULTS: Thirty-three HIV-negative patients were analysed in this study. Twenty-six were treated with a regimen containing CAM or azithromycin (AZM), and 21 patients were treated with three or more drugs except macrolide. The median duration to be evaluated was 10.4 months after beginning the treatment regimen. Sputum conversion (including cases of inability to expectorate sputum) was achieved in 12 (36%) patients. Radiological effectiveness improved in 4 (12%) patients, was unchanged in 11 (33%) patients and worsened in 18 (55%) patients. In the multivariate analysis, CRP <1.0 mg/dl (p = 0.017, odds ratio 12, 95% confidence interval (CI) 1.6-95) was found to be the only significant risk factor for radiological non-deterioration, and no significant risk factors for microbiological improvement were found. CONCLUSIONS: Our results suggested that continuation of macrolides or the addition of a new quinolone or injectable aminoglycoside to therapy with rifampicin and ethambutol would not improve clinical outcome after the emergence of CAM-resistant MAC. However, further prospective study is required to evaluate the precise clinical efficacy and effectiveness of these drugs.

[Retrospective Study of Clinical Features and Prognosis of Pneumococcal Pneumonia Among Patients Under 65 Years of Age].

The 23-valent pneumococcal polysaccharide vaccine (PPSV23) for elderly people has been included in the National Immunization Program (NIP) of Japan since October 2014. Targets for PPSV23 were restricted to persons ≥65 years of age and persons 60 to 64 years of age with an underlying severe physical disability (expressed as 1st grade in Japan). In this study, the clinical courses of non-target persons <65 years of age were compared between those with non-severe underlying diseases (A group) and those without underlying diseases (B group), and the need to expand the targets for PPSV23 within the NIP was investigated. Persons with pneumococcal pneumonia who were diagnosed based on a positive sputum or blood culture result were enrolled between January 2004 and April 2014. As a result, the number of subjects in A group was 2.6 times larger than that in B group, and this difference was especially pronounced (4.2 times) among subjects between the age of 60 to 64 years. These findings suggest that persons with underlying disease without a 1st grade physical disability might also be susceptible to pneumococcal pneumonia. No significant differences in the severity of pneumonia, the length of treatment, or the rates of admission were seen between A group and B group. The severity of pneumonia and the rates of admission among targets of the NIP were significantly higher than those of A group. In conclusion, our study suggests that A group should also be included among the targets of the NIP and that all targets eligible to receive the pneumococcal vaccine within NIP should be inoculated.

Imported case of acute respiratory tract infection associated with a member of species nelson bay orthoreovirus.

A Japanese man suffered from acute respiratory tract infection after returning to Japan from Bali, Indonesia in 2007. Miyazaki-Bali/2007, a strain of the species of Nelson Bay orthoreovirus, was isolated from the patient's throat swab using Vero cells, in which syncytium formation was observed. This is the sixth report describing a patient with respiratory tract infection caused by an orthoreovirus classified to the species of Nelson Bay orthoreovirus. Given the possibility that all of the patients were infected in Malaysia and Indonesia, prospective surveillance on orthoreovirus infections should be carried out in Southeast Asia. Furthermore, contact surveillance study suggests that the risk of human-to-human infection of the species of Nelson Bay orthoreovirus would seem to be low.

Development and clinical application of a new technique for detecting 'sleep blood pressure surges' in sleep apnea patients based on a variable desaturation threshold.

Obstructive sleep apnea (OSA) places an enormous pressure load on the cardiovascular system by inducing a temporary blood pressure (BP) surge (sleep BP surge (SLBPS)), often resulting in target organ damage and cardiovascular events, such as left ventricular hypertrophy, sudden death, myocardial infarction and stroke. Accurate measurement of SLBPS would be valuable for the risk stratification of OSA patients. We developed a new oxygen-triggered BP monitoring system based on a variable SpO(2) threshold (VT algorithm) to selectively detect severe SLBPS, which are associated with morbidity, and evaluated its performance in comparison with a previous technique based on a fixed SpO(2) threshold (FT algorithm). In 23 OSA patients, the correlation between individual minimum SpO(2) values and SLBPS was not significant when the FT algorithm was used alone (r=0.400, P=0.058) but became significant (r=0.725, P<0.0001) when the VT algorithm was additionally used. In another 13 OSA patients, when the FT algorithm was eliminated from the FT+VT algorithm, the number of BP readings was drastically reduced (36±22.7 vs. 61±55.0 times, P=0.004) with a similar correlation between minimum SpO(2) and SLBPS. The correlation between the apnea hypopnea index and SLBPS was significant when measured with the present method, but not when assessed with ambulatory BP monitors (ABPM) simulation (r=0.519, P=0.001 vs. r=0.149, P=0.385). In conclusion, oxygen-triggered BP monitoring with a variable threshold is able to detect severe OSA-related BP surges more specifically and reduce the number of BP readings required during sleep compared with detection using a fixed threshold or the conventional ABPM method.

Nuclear localization and antisense effect of PNA internalized by ASGP-R-mediated endocytosis with protein/DNA conjugates.

In order for peptide nucleic acids (PNAs) to be effective as therapeutic agents, methods for cellular delivery must be developed. Here we demonstrate spontaneous nuclear localization and antisense effects of peptide nucleic acids (PNAs) delivered to hepatic cells through asialoglycoprotein receptor-mediated endocytosis. Asialofetuin conjugates with DNA oligonucleotides (AF/DNA) complementary to the PNA of interest were designed as cell-specific delivery vectors. PNAs hybridized to the asialofetuin-oligonucleotide conjugates were internalized into murine primary hepatocytes and human HepG2 hepatocarcinoma cells effectively through receptor-mediated endocytosis in vitro. After a 4-h incubation, PNAs were largely localized in the nuclei of the cells; the mechanisms involved are still unclear. More than 70% inhibition of telomerase activity was observed when PNAs complementary to the RNA template of human telomerase were delivered to HepG2 cells using AF/DNA. The PNAs were stably associated with the AF/DNA conjugates in 50% serum at 37°C for at least 3h. The PNAs were spontaneously released from the conjugate through a strand exchange mechanism when complementary nucleic acid was added. The complexation of PNAs with the AF/DNA conjugates resulted in delivery of PNAs to liver after intravenous injection into mice. The present study indicates that conjugation to a natural proteinous ligand can be used as a non-toxic vector for cellular delivery of oligonucleotide analogs.