Background: The influence of hospitalization owing to pneumonia on changes in body composition has not been specifically reported. We conducted a prospective cohort study of patients with community-acquired pneumonia (CAP) requiring hospitalization to test the hypothesis that hospitalization affects body composition. Methods: Sixty-four consecutive patients with CAP were recruited. Body composition was measured within 24 h of admission and 24 h before discharge using bioelectrical impedance analysis. The association between changes in body composition and variables obtained at admission was investigated. Index values were calculated as weight divided by height squared. Results: The mean age of the patients was 76.0 ± 8.7 years (78.1% males). The median length of hospitalization was 12.0 days. Weight, body mass index (BMI), skeletal muscle (SM), SM index, fat-free mass (FFM), and FFM index significantly decreased (p < 0.001 for each), but fat mass (FM) and FM index did not. The serum total protein level was the only independent predictor of the lowest quartile of change in SM index (<-0.4) after adjusting for age and sex (p = 0.004). Conclusions: In summary, weight and BMI significantly decreased during hospitalization in patients with CAP, which was attributed to SM reduction. Patients with low serum total protein levels on admission were at risk of an accelerated decrease in the SM index. Nutritional intervention and rehabilitation are important for these patients.

A 54-year-old woman diagnosed with eosinophilic granulomatosis accompanied by polyangiitis (EGPA) was in remission with prednisolone (PSL) and mepolizumab treatment. During the disease course, vasculitis recurrence was not apparent, but her asthma became uncontrollable and was accompanied by elevated antineutrophil cytoplasmic antibody (ANCA) levels. Asthma control did not improve after switching from mepolizumab to another biological agent, benralizumab. Consequently, the treatment with benralizumab was changed to tezepelumab, after which her symptoms immediately improved and her ANCA levels decreased, enabling reduction in the PSL dosage. This case highlights the potential of tezepelumab to reduce ANCA levels in EGPA-related severe asthma.

BACKGROUND: Asthma is a heterogeneous disease with variable response to treatment. Genetic backgrounds are involved in the severity of type 2 asthma, but their effects on responses to biologics remain unknown. This study aimed to clarify the role of genetic factors in response to biologics in patients with severe asthma. METHODS: Adults with severe asthma receiving biologics were enrolled in this multicentre, observational, real-world study. The responses to biologics were evaluated using Physicians' Global Evaluation of Treatment Effectiveness (GETE). Optimal biologic for each patient was also determined based on the best GETE score for the biologic used or currently used biologic. Three single nucleotide polymorphisms (IL1RL1, rs1420101; IL4RA, rs8832; and TSLP rs1837253) were examined. RESULTS: Among the 113 patients analysed, 53 (46.9%) had an excellent GETE score for at least one biologic. These patients with an excellent GETE score for at least one biologic, particularly for benralizumab, had the risk genotype of rs1420101 more frequently than the remaining patients, independent of the clinical demographics. Regarding the optimal biologic for each patient, anti-IL-5 drugs were optimal for patients with the rs1420101 TT or rs8832 GG genotype. Furthermore, dupilumab was similarly effective, regardless of the risk genotypes examined in this study. CONCLUSION: IL1RL1 rs1420101 TT genotype and/or IL4RA rs8832 GG genotype may predict an excellent or optimal response to biologic therapy in each patient, particularly to anti-interleukin-5 targeted therapy. The elucidation of genetic predisposition may improve the management of severe asthma in the era of biologics.

BACKGROUND: Pulmonary arterial hypertension (PAH)-specific therapies are generally ineffective in patients with pulmonary hypertension associated with lung disease (PH-LD). The aim of this preliminary study was to evaluate the potential efficacy of selexipag, titrated according to individual tolerance, in patients with PH-LD. METHODS: Consecutive patients diagnosed with PH-LD between October 2016 and March 2019, who received selexipag treatment, were retrospectively evaluated. Specific parameters, including changes in hemodynamic parameters, 6-min walk distance (6MWD), and partial pressure of atrial oxygen/fraction of inspiratory oxygen (PaO2/FiO2) were evaluated. Patients whose 6MWD improved ≥20 m were defined as responders. RESULTS: Eight patients with PH-LD were included, comprising four with chronic obstructive pulmonary disease (COPD), two with interstitial lung disease (ILD) related to rheumatoid arthritis, one with ILD related to systemic sclerosis, and one with pulmonary Langerhans cell histiocytosis. No statistically significant improvements in hemodynamic parameters and 6MWD were noted following selexipag treatment. However, four patients showed improvements in 6MWD ≥20 m at follow-up and were considered responders. They had a higher body mass index (BMI) and lower PaO2/FiO2 at baseline than non-responders (p = 0.02 and p = 0.04, respectively). No Grade 3 or 4 adverse events were observed. CONCLUSIONS: Selexipag was effective in half of the PH-LD cases, emphasizing higher BMI and lower PaO2/FiO2 as possible indicators for favorable response. Since selexipag starting at a low dose with subsequent titration may reduce the risk of early adverse events, it can be considered a treatment option for PH-LD. Further large-scale studies are warranted to confirm these findings.

BACKGROUND: Acute exacerbations (AEs) of fibrotic idiopathic interstitial pneumonia (fIIP) that require hospitalization occur in some patients. During hospitalization, these patients can develop hospital-acquired pneumonia (HAP), a common hospital-acquired infection with a high mortality rate. However, the characteristics of HAP in AE-fIIP remain unknown. The purpose of this study was to determine the incidence, causative pathogens, and outcomes of HAP in patients with AE-fIIP. METHODS: The medical records of consecutive patients who were hospitalized with AE-fIIP from January 2008 to December 2019 were analyzed for the incidence, causative pathogen, and survival of HAP. The records of patients with an obvious infection-triggered AE were excluded from analysis. RESULTS: There were 128 patients with AE-fIIP (89 with idiopathic pulmonary fibrosis [IPF] and 39 with non-IPF fIIP) who were hospitalized a total of 155 times (111 with IPF and 44 with non-IPF fIIP). HAP occurred in 49 patients (40 with IPF and 9 with non-IPF fIIP). The incidence and the in-hospital mortality rates of HAP in patients with AE-fIIP were high, at 32.2% and 48.9%, respectively. Corynebacterium spp. was the most common causative pathogen, which was followed by human cytomegalovirus (HCMV). CONCLUSIONS: The incidence and the in-hospital mortality rates of HAP in patients with AE-fIIP are high. To improve their survival, patients with fIIP who had AEs and HAP should receive prompt empirical treatment for possible infections with Corynebacterium spp. and testing for HCMV.

BACKGROUND: Obstructive sleep apnea (OSA) is one of the major co-morbidities and aggravating factors of asthma. In OSA-complicated asthma, obesity, visceral fat, and systemic inflammation are associated with its severity, but the role of bronchial hyperresponsiveness (BHR) is unclear. We investigated the involvement of BHR and mediastinal fat width, as a measure of visceral fat, with OSA severity in patients with OSA and asthma-like symptoms. METHODS: Patients with OSA who underwent BHR test and chest computed tomography scan for asthma-like symptoms were retrospectively enrolled. We evaluated the relationship between apnea-hypopnea index (AHI) and PC20 or anterior mediastinal fat width, stratified by the presence or absence of BHR. RESULTS: OSA patients with BHR (n = 29) showed more obstructive airways and frequent low arousal threshold and lower mediastinal fat width, and tended to show fewer AHI than those without BHR (n = 25). In the overall analysis, mediastinal fat width was significantly positively correlated with AHI, which was significant even after adjustment with age and gender. This was especially significant in patients without BHR, while in OSA patients with BHR, there were significant negative associations between apnea index and airflow limitation, and hypopnea index and PC20. CONCLUSIONS: Risk factors for greater AHI differed depending on the presence or absence of BHR in OSA patients with asthma-like symptoms. In the presence of BHR, severity of asthma may determine the severity of concomitant OSA.

The coronavirus disease 2019 (COVID-19) pandemic has had a great influence on medical practice in Japan. In this study, an online questionnaire-based survey was conducted among doctors routinely involved in the treatment of asthma. The questions included in the survey pertained to their thoughts on asthma treatment amidst COVID-19, changes in their clinical approach toward patients with asthma, and the behavioral changes in patients in the pandemic era. The results revealed a significant impact of the pandemic on asthma treatment. Regardless of whether or not they were directly involved in the treatment of patients with COVID-19, the doctors had avoided using nebulizers in outpatient wards/clinics and routine pulmonary function testing. An increase in canceled appointments and inappropriate/non-adherence to treatment among their patients were noticeable. Furthermore, the survey revealed an extensive impact of the pandemic on the doctors engaged in asthma treatment irrespective of the differences in their medical backgrounds.

There is a concern that persons with underlying respiratory disease may have increased susceptibility to COVID-19 and/or increased severity/mortality if infected. However, information regarding such patients during the first wave of the epidemic is lacking in Japan. We surveyed chest physicians nationwide, and collected anonymous data concerning 1444 patients. Among COVID-19 patients, the prevalence of asthma, chronic obstructive pulmonary disease (COPD), and interstitial lung diseases (ILD) was 3.4%, 4.8%, and 1.5%, respectively. Among COVID-19 patients with these 3 comorbidities, exacerbation of the comorbidity occurred in 12.2%, 18.8%, and 36.4%, respectively, and mortality (6.2% overall) was 4.1%, 13.0%, and 31.8%, respectively. The prevalence of asthma among COVID-19 patients was not higher than that for the general population, and mortality in COVID-19 patients with asthma was not higher than mortality in COVID-19 patients without underlying respiratory disease. COVID-19 patients having COPD or ILD had relatively high mortality, especially for ILD.

BACKGROUND: The utility of bronchoscopy for patients with suspected immune checkpoint inhibitor (ICI)-related pneumonitis is currently debatable. The purpose of this study was to examine the findings of bronchoalveolar lavage (BAL) analysis and transbronchial lung biopsy (TBLB) in non-small cell lung cancer (NSCLC) patients with ICI-related pneumonitis, and to elucidate the clinical significance of bronchoscopy for this health condition. PATIENTS AND METHODS: Consecutive NSCLC patients treated with ICIs, diagnosed with ICI-related pneumonitis after undergoing bronchoscopy between October 2015 and March 2019 were retrospectively screened. Findings of BAL fluid analysis and/or TBLB specimen histology were reviewed. RESULTS: Twelve patients underwent bronchoscopy for the diagnosis of ICI-related pneumonitis, ten of whom underwent BAL. An increase in the proportion of lymphocytes higher than 20% was observed in all ten patients. An increase in the proportion of neutrophils (> 10%) and eosinophils (> 10%) was observed in two and one patient, respectively. TBLB specimens were analyzed for eight patients. Major histologic findings included alveolitis in seven (87.5%) and organizing pneumonia (OP) in five (62.5%) patients. Other findings included acute lung injury and fibrosis. All twelve patients demonstrated favorable outcomes. CONCLUSION: A major characteristic of BAL analysis in ICI-related pneumonitis with NSCLC was an increased proportion of lymphocytes. The histologic features of lung tissue included alveolitis and/or OP. Acute lung injury and fibrosis were observed. Although the necessity of bronchoscopy should be determined on a case-by-case basis, it is necessary to assess these parameters when proper differential diagnosis is needed.

Phosphatidylinositol-specific phospholipase C (PI-PLC) and cytosolic phospholipase A2 (cPLA2) regulate both eosinophil degranulation and leukotriene (LT) synthesis via PI-PLC-mediated calcium influx and cPLA2 activation. Phosphatidylcholine-specific phospholipase C (PC-PLC) likely plays a key role in cellular signaling, including the eosinophilic allergic inflammatory response. This study examined the role of PC-PLC in eosinophil LT synthesis and degranulation using tricyclodecan-9-yl-xanthogenate (D609), a PC-specific PLC inhibitor. D609 inhibited N-formyl-met-leu-phe + cytochalasin B (fMLP/B)-induced arachidonic acid (AA) release and leukotriene C4 (LTC4) secretion. However, at concentrations that blocked both AA release and LTC4 secretion, D609 had no significant inhibitory effect on stimulated cPLA2 activity. D609 also partially blocked fMLP/B-induced calcium influx, indicating that inhibition of AA release and LTC4 secretion by D609 is due to inhibition of calcium-mediated cPLA2 translocation to intracellular membranes, not inhibition of cPLA2 activity. In addition, D609 inhibited fMLP/B-stimulated eosinophil peroxidase release, indicating that PC-PLC regulates fMLP/B-induced eosinophil degranulation by increasing the intracellular calcium concentration ([Ca2+]i). Overall, our results showed that PC-PLC is critical for fMLP/B-stimulated eosinophil LT synthesis and degranulation. In addition, degranulation requires calcium influx, while PC-PLC regulates LTC4 synthesis through calcium-mediated cPLA2 activation.

Pulmonary rehabilitation (PR) is recommended as an effective treatment for patients with chronic obstructive pulmonary disease (COPD). Previous meta-analyses showed that PR improves exercise capacity and health-related quality of life (HRQOL). However, they did not evaluate the effect of PR on the sensation of dyspnea. We searched six databases in May 2019 for randomized controlled trials (RCTs) that examined PR, including supervised lower limb endurance training as a minimal essential component that was continued for 4-12 weeks, in patients with stable COPD, with changes from baseline dyspnea as a primary outcome. Secondary outcomes were changes in exercise capacity, HRQOL, activity of daily life (ADL), physical activity (PA), and adverse events. We calculated the pooled weighted mean difference (MD) using a random effects model. We identified 42 studies with 2150 participants. Compared with the control, PR improved dyspnea, as shown using the British Medical Research Council (MRC) questionnaire (MD, -0.64; 95% CI, -0.99 to -0.30; p = 0.0003), transitional dyspnea index (MD, 1.95; 95% CI, 1.09 to 2.81; p = 0.0001), modified Borg score during exercise (MD, -0.62; 95% CI, -1.10 to -0.14; p = 0.01), and Chronic Respiratory Questionnaire (CRQ) dyspnea score (MD, 0.91; 95% CI, 0.39 to 1.44; p = 0.0007). PR significantly increased exercise capacity measured by the 6 min walking distance time, peak workload, and peak VO2. It improved HRQOL measured by the St. George's Respiratory Questionnaire and CRQ, but not on PA or ADL. These results indicated that PR programs including lower limb endurance training improve dyspnea, HRQOL, and exercise capacity in patients with stable COPD.

BACKGROUND: We obtain summary estimates of the accuracy of additional objective tests for the diagnosis of adult asthma using systematic review and meta-analysis of diagnostic test accuracy studies. METHODS: Medline, Embase, and other relevant electronic databases were searched for papers published between January 1989 and December 2016. Studies were included if they evaluated the diagnostic accuracy of objective tests, including airway reversibility (AR), airway hyperresponsiveness (AHR), and fractionated exhaled nitric oxide (FeNO) for the diagnosis of adult asthma in patients with symptoms suggestive of asthma. If papers were assessed appropriate using the adapted QUADAS-2 tool, meta-analysis was conducted using the hierarchical bivariate model. This hierarchical model accounts for both within and between study variability. RESULTS: Sixteen studies reported the performance of the evaluated objective tests at presentation. For diagnosis of adult asthma, overall sensitivity and specificity for AR were 0.39 (95% confidence interval [CI] 0.18 to 0.66) and 0.95 (95% CI 0.86 to 1.00); for AHR, 0.86 (95% CI 0.61 to 1.00) and 0.95 (95% CI 0.77 to 1.00); for FeNO, 0.65 (95% CI 0.53 to 0.77) and 0.83 (95% CI 0.75 to 0.90). Comprehensive comparison of three diagnostic tools for adult asthma using the back-calculated likelihood rate (LR) showed that AR and AHR corresponded to a higher LR+, and AHR gave a lower LR-. CONCLUSIONS: In the current situation of no gold standard for diagnosis of adult asthma, AR and AHR are appropriate for ruling-in the true diagnosis, and AHR is superior for ruling-out a diagnosis. Since each objective test had a specific characteristic, it should be chosen depending on the situation, such as the capacity of the institution and the conditions of patients.

A 52-year-old woman was referred to our hospital presenting with epigastric pain and weight loss. A contrast- enhanced abdominal computed tomography (CT) scan showed a low-density mass in the body of the pancreas, indicative of a malignancy. Endoscopic ultrasound-guided fine needle aspiration of the pancreatic mass was performed three times and showed no specific findings. A distal pancreatectomy was performed, and a pathological examination revealed epitheli- oid cell granulomas and necrosis. Ziehl-Neelsen staining did not reveal acid-fast bacilli in the pancreatic mass. A diagnosis of tuberculosis or sarcoidosis of the pancreas was con- sidered; however, the patient chose to undergo a follow-up examination without therapeutic intervention because the pancreatic mass had been removed completely and she had recovered well. Four months after the operation, the patient was readmitted to our hospital for insulin therapy for pancreatic diabetes. She presented with a fever and a productive cough, and a chest CT scan showed multiple nodules in both upper lobes. A bronchoscopy was performed and bronchoalveolar lavage fluid cultures for Mycobacterium tuberculosis were positive. The patient received antitubercular quadri-therapy and showed symptomatic and radiologic improvement. At the initial examination, we had been unable to establish the correct diagnosis; however, the detection of pulmonary lesions led to the time-delayed diagnosis of pancreatic tuber- culosis. Owing to its rarity, it is difficult to diagnose pancreatic tuberculosis using clinical symptoms and radiological imaging modalities; thus, pathologic and bacteriologic confirmation is essential. To avoid performing an unnecessary laparotomy in patients with pancreatic tuberculosis, increased vigilance and an accurate diagnostic approach are required.

AIM: To evaluate the influence of comorbidities and aging on pulmonary rehabilitation (PR) efficacy in patients with chronic obstructive pulmonary disease (COPD). METHODS: This was a retrospective cohort study of patients with COPD attending an outpatient PR program. Comorbidity information was collected with the Charlson Index, BODE index and COPD-specific comorbidity test, and also included other common conditions not included in these indexes. The efficacy of PR was defined as a 54-m increase in 6-min walk distance or a four-point decrease in St. George's Respiratory Questionnaire score. Patients were divided into two age groups according to the median age of 72 years. RESULTS: A total of 21 of 52 patients (40%) showed a clinically significant benefit by the 6-min walk distance, and 29 patients (55.8%) by the St. George's Respiratory Questionnaire score. PR efficacy was not different between the elderly group and the younger group by either parameter. A total of 98% of the patients had at least one chronic comorbidity. Hypertension was the most frequently reported comorbidity (28.5%). Higher body mass index, Hospital Anxiety and Depression Scale anxiety score and St. George's Respiratory Questionnaire total score were associated with a good response to PR by the 6-min walk distance. None of the individual comorbidities or indexes were correlated with the efficacy of PR. Multiple logistic regression analysis showed that body mass index was independently associated with the response to PR. CONCLUSIONS: PR is equally effective in elderly and younger patients with COPD, with efficacy influenced by body mass index and anxiety. Geriatr Gerontol Int 2016; 16: 934-941.

BACKGROUND: The characteristics of phenotypes of elderly patients with asthma are unknown. The aim of this study was to classify these phenotypes using lung function tests and images from high-resolution computed tomography (HRCT), and to identify associations between clinical characteristics and phenotypes. METHODS: A cross-sectional study was conducted in 165 elderly patients (>65 years of age) who underwent a multidimensional assessment of clinical and functional status and comorbidity. The patients were divided into three phenotypes: (1) asthma-predominant, (2) asthma-obstructive airway disease (OAD) overlap without emphysema, and (3) asthma-OAD overlap with emphysema (asthma-emphysema overlap) based on chest HRCT. A receiver operating characteristic (ROC) curve was constructed to evaluate the cutoff for differentiating between the two OAD phenotypes. Multivariate analysis was also used to distinguish between these two phenotypes. RESULTS: The phenotypes were asthma-predominant in 48 patients (29%), asthma-OAD without emphysema in 36 (22%), and asthma-emphysema in 81 (49%). Patients with asthma-emphysema were more frequent smokers. In multivariate analysis, smoking status (odds ratio 2.92: 95% CI 1.21-7.00, P = 0.03) and % predicted FEV1 ≤70% (odds ratio 3.18: 95% CI 1.13-8.92, P = 0.03) differed significantly between the asthma-emphysema and asthma-OAD without emphysema phenotypes. CONCLUSIONS: Half of elderly patients with asthma are characterized by asthma-emphysema overlap. Our results showed that elderly patients with asthma who are smokers and have moderate or severe OAD are also likely to have emphysema.

Emphysema on high-resolution computed tomography of the chest is the recent focus in the general practice in idiopathic pulmonary fibrosis (IPF). However, adequate attention has not been paid to obstructive disorder. Therefore, we retrospectively evaluated the association between the degree of airway obstruction and longevity in IPF subjects, with a hypothesis that lower forced expiratory volume in 1 second (FEV1)/forced vital capacity (FVC) has an impact on prognosis. One hundred and fourteen consecutive IPF subjects who had been diagnosed with IPF and had undergone evaluation including pulmonary function test from January 2008 to May 2013 were included in the study. The relationship between baseline data and survival was examined. FEV1/FVC was widely distributed, ranging from 48.6% to 100%. On both univariate and multivariate Cox's regression analyses, lower FEV1/FVC was significantly associated with better survival (hazard ratio of 1.07 and 1.04 and 95% confidential interval of 1.03-1.10 and 1.01-1.08, respectively). Even on analysis with backward selection, FEV1/FVC remained a significant prognostic factor. FEV1/FVC is widely distributed and negatively predicts survival in IPF. A FEV1/FVC should be assessed in "real-world" general practice. Also, the effect of smoking on the clinical course of IPF should be investigated further.

This study was designed to investigate the association of perceived dyspnoea intensity with cortical oxygenation and cortical activation during exercise in patients with chronic obstructive pulmonary disease (COPD) and exertional hypoxaemia.Low-intensity exercise was performed at a constant work rate by patients with COPD and exertional hypoxaemia (n=11) or no hypoxaemia (n=16), and in control participants (n=11). Cortical oxyhaemoglobin (oxy-Hb) and deoxyhaemoglobin (deoxy-Hb) concentrations were measured by multichannel near-infrared spectroscopy. Increased deoxy-Hb is assumed to reflect impaired oxygenation, whereas decreased deoxy-Hb signifies cortical activation.Exercise decreased cortical deoxy-Hb in control and nonhypoxaemic patients. Deoxy-Hb was increased in hypoxaemic patients and oxygen supplementation improved cortical oxygenation. Decreased deoxy-Hb in the pre-motor cortex (PMA) was significantly correlated with exertional dyspnoea in control participants and patients with COPD without hypoxaemia. In contrast, increased cortical deoxy-Hb concentration was correlated with dyspnoea in patients with COPD and hypoxaemia. With the administration of oxygen supplementation, exertional dyspnoea was correlated with decreased deoxy-Hb in the PMA of COPD patients with hypoxaemia.During exercise, cortical oxygenation was impaired in patients with COPD and hypoxaemia compared with control and nonhypoxaemic patients; this difference was ameliorated with oxygen supplementation. Exertional dyspnoea was related to activation of the pre-motor cortex in COPD patients.

The ecological mechanisms underlying the diversification of autotrophic species into endosymbiotic lifestyles and the ways in which the evolution of endosymbiotic species is ecologically and evolutionarily affected by sister lineages/lines that are adapted to extra-host environments remain unclear. In this paper, we investigated a differentiation process of algal species in which an endosymbiotic type was differentiated phenotypically from a free-living ancestral clone, by using an experimental model called the CET microcosm, which contains a green alga (Micractinium sp.), a bacterium (Escherichia coli), and a ciliate (Tetrahymena thermophila) cultured together without an external resource supply for over 5 years. We then analyzed the algal diversification process by comparing algal phenotypic properties regarding cell-aggregate formation and their effects on the survival of Tetrahymena (using a clone isolated on day 2668) in the absence of bacteria. We examined 13 Micractinium clones, including both ancestral and derived clones isolated from long-term (day 1819-1847) CET microcosm cultures. The results revealed that the free-living ancestral algal strain diversified in sympatry into an aggregate-forming type that associates with E. coli, and a non-aggregate-forming type that associates with Tetrahymena. Furthermore, a competition experiment revealed that the endosymbiotic (non-aggregate-forming) type was less adapted to the extracellular environment than the aggregate-forming type. This result suggests that severe competition with a nonendosymbiotic sister line in the extra-host environment favors the host-benefiting phenotype in an endosymbiotic line, because such symbionts can enhance the longevity of the host, thereby enabling the survival and reproduction of the symbiont within the host.

Large-scale production of fully human IgG (hIgG) or human polyclonal antibodies (hpAbs) by transgenic animals could be useful for human therapy. However, production level of hpAbs in transgenic animals is generally very low, probably due to the fact that evolutionarily unique interspecies-incompatible genomic sequences between human and non-human host species may impede high production of fully hIgG in the non-human environment. To address this issue, we performed species-specific human artificial chromosome (HAC) engineering and tested these engineered HAC in cattle. Our previous study has demonstrated that site-specific genomic chimerization of pre-B cell receptor/B cell receptor (pre-BCR/BCR) components on HAC vectors significantly improves human IgG expression in cattle where the endogenous bovine immunoglobulin genes were knocked out. In this report, hIgG1 class switch regulatory elements were subjected to site-specific genomic chimerization on HAC vectors to further enhance hIgG expression and improve hIgG subclass distribution in cattle. These species-specific modifications in a chromosome scale resulted in much higher production levels of fully hIgG of up to 15 g/L in sera or plasma, the highest ever reported for a transgenic animal system. Transchromosomic (Tc) cattle containing engineered HAC vectors generated hpAbs with high titers against human-origin antigens following immunization. This study clearly demonstrates that species-specific sequence differences in pre-BCR/BCR components and IgG1 class switch regulatory elements between human and bovine are indeed functionally distinct across the two species, and therefore, are responsible for low production of fully hIgG in our early versions of Tc cattle. The high production levels of fully hIgG with hIgG1 subclass dominancy in a large farm animal species achieved here is an important milestone towards broad therapeutic applications of hpAbs.

Towards the goal of producing fully human polyclonal antibodies (hpAbs or hIgGs) in transchromosomic (Tc) cattle, we previously reported that Tc cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin (Ig) heavy-chain (hIGH), kappa-chain (hIGK), and lambda-chain (hIGL) germline loci produced physiological levels of hIgGs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, were homozygously inactivated (bIGHM-/-, bIGHML1-/-; double knockouts or DKO). However, because endogenous bovine immunoglobulin light chain loci are still intact, the light chains are produced both from the hIGK and hIGL genomic loci on the HAC and from the endogenous bovine kappa-chain (bIGK) and lambda-chain (bIGL) genomic loci, resulting in the production of fully hIgGs (both Ig heavy-chains and light-chains are of human origin: hIgG/hIgκ or hIgG/hIgλ) and chimeric hIgGs (Ig heavy-chains are of human origin while the Ig light-chains are of bovine origin: hIgG/bIgκ or hIgG/bIgλ). To improve fully hIgG production in Tc cattle, we here report the deletion of the entire bIGL joining (J) and constant (C) gene cluster (bIGLJ1-IGLC1 to bIGLJ5-IGLC5) by employing Cre/loxP mediated site-specific chromosome recombination and the production of triple knockout (bIGHM-/-, bIGHML1-/- and bIGL-/-; TKO) Tc cattle. We further demonstrate that bIGL cluster deletion greatly improves fully hIgGs production in the sera of TKO Tc cattle, with 51.3% fully hIgGs (hIgG/hIgκ plus hIgG/hIgλ).

A 12-year-old boy admitted to a local hospital with fever, migratory arthralgia, and periosteal reaction on X Ray. He was transferred to our hospital because magnetic resonance imaging scan of his whole body showed multiple abnormal signals in bones. Laboratory findings on admission showed the increased erythrocyte sedimentation rate, uric acid, lactate dehydrogenase, alkaline phosphatase, C-reactive protein, immunoglobulin G, hemolytic complement activity and soluble interleukin-2 receptor. Peripheral blood and bone marrow examination did not show any abnormality. The clinical appearance of his condition suggested the diagnosis of chronic recurrent multifocal osteomyelitis (CRMO). He was treated with steroid, however his fever and bone pain continued. A bone and bone marrow biopsy was performed and the results of histopathology showed precursor-B acute leukemia/lymphoma. His bone pain relapsed after the chemotherapy for ALL. Finally, blast cells resembling L3 morphology were detected in the peripheral blood. The reevaluated bone marrow was predominantly replaced with Burkitt like lymphoblasts. He was diagnosed with Burkitt lymphoma by further specific examination.

BACKGROUND: To predict the presence of asthma in adult patients with respiratory symptoms, we developed a scoring algorithm using clinical parameters. METHODS: We prospectively analysed 566 adult outpatients who visited Kinki University Hospital for the first time with complaints of nonspecific respiratory symptoms. Asthma was comprehensively diagnosed by specialists using symptoms, signs, and objective tools including bronchodilator reversibility and/or the assessment of bronchial hyperresponsiveness (BHR). Multiple logistic regression analysis was performed to categorise patients and determine the accuracy of diagnosing asthma. RESULTS: A scoring algorithm using the symptom-sign score was developed, based on diurnal variation of symptoms (1 point), recurrent episodes (2 points), medical history of allergic diseases (1 point), and wheeze sound (2 points). A score of >3 had 35% sensitivity and 97% specificity for discriminating between patients with and without asthma and assigned a high probability of having asthma (accuracy 90%). A score of 1 or 2 points assigned intermediate probability (accuracy 68%). After providing additional data of forced expiratory volume in 1 second/forced vital capacity (FEV(1)/FVC) ratio <0.7, the post-test probability of having asthma was increased to 93%. A score of 0 points assigned low probability (accuracy 31%). After providing additional data of positive reversibility, the post-test probability of having asthma was increased to 88%. CONCLUSIONS: This pragmatic diagnostic algorithm is useful for predicting the presence of adult asthma and for determining the appropriate time for consultation with a pulmonologist.

Therapeutic human polyclonal antibodies (hpAbs) derived from pooled plasma from human donors are Food and Drug Administration approved biologics used in the treatment of a variety of human diseases. Powered by the natural diversity of immune response, hpAbs are effective in treating diseases caused by complex or quickly-evolving antigens such as viruses. We previously showed that transchromosomic (Tc) cattle carrying a human artificial chromosome (HAC) comprising the entire unrearranged human immunoglobulin heavy-chain (hIGH) and kappa-chain (hIGK) germline loci (named as κHAC) are capable of producing functional hpAbs when both of the bovine immunoglobulin mu heavy-chains, bIGHM and bIGHML1, are homozygously inactivated (double knockouts or DKO). However, B lymphocyte development in these Tc cattle is compromised, and the overall production of hpAbs is low. Here, we report the construction of an improved HAC, designated as cKSL-HACΔ, by incorporating all of the human immunoglobulin germline loci into the HAC. Furthermore, for avoiding the possible human-bovine interspecies incompatibility between the human immunoglobulin mu chain protein (hIgM) and bovine transmembrane α and β immunoglobulins (bIgα and bIgβ) in the pre-B cell receptor (pre-BCR) complex, we partially replaced (bovinized) the hIgM constant domain with the counterpart of bovine IgM (bIgM) that is involved in the interaction between bIgM and bIgα/Igβ; human IgM bovinization would also improve the functionality of hIgM in supporting B cell activation and proliferation. We also report the successful production of DKO Tc cattle carrying the cKSL-HACΔ (cKSL-HACΔ/DKO), the dramatic improvement of B cell development in these cattle and the high level production of hpAbs (as measured for the human IgG isotype) in the plasma. We further demonstrate that, upon immunization by tumor immunogens, high titer tumor immunogen-specific human IgG (hIgG) can be produced from such Tc cattle.

A single bout of prolonged endurance exercise stimulates glucose transport in skeletal muscles, leading to post-exercise muscle glycogen supercompensation if sufficient carbohydrate is provided after the cessation of exercise. Although we recently found that short-term sprint interval exercise also stimulates muscle glucose transport, the effect of this type of exercise on glycogen supercompensation is uncertain. Therefore, we compared the extent of muscle glycogen accumulation in response to carbohydrate feeding following sprint interval exercise with that following endurance exercise. In this study, 16-h-fasted rats underwent a bout of high-intensity intermittent swimming (HIS) as a model of sprint interval exercise or low-intensity prolonged swimming (LIS) as a model of endurance exercise. During HIS, the rats swam for eight 20-s sessions while burdened with a weight equal to 18% of their body weight. The LIS rats swam with no load for 3 h. The exercised rats were then refed for 4, 8, 12, or 16 h. Glycogen levels were almost depleted in the epitrochlearis muscles of HIS- or LIS-exercised rats immediately after the cessation of exercise. A rapid increase in muscle glycogen levels occurred during 4 h of refeeding, and glycogen levels had peaked at the end of 8 h of refeeding in each group of exercised refed rats. The peak glycogen levels during refeeding were not different between HIS- and LIS-exercised refed rats. Furthermore, although a large accumulation of muscle glycogen in response to carbohydrate refeeding is known to be associated with decreased insulin responsiveness of glucose transport, and despite the fact that muscle glycogen supercompensation was observed in the muscles of our exercised rats at the end of 4 h of refeeding, insulin responsiveness was not decreased in the muscles of either HIS- or LIS-exercised refed rats compared with non-exercised fasted control rats at this time point. These results suggest that sprint interval exercise enhances muscle glycogen supercompensation in response to carbohydrate refeeding as well as prolonged endurance exercise does. Furthermore, in this study, both HIS and LIS exercise prevented insulin resistance of glucose transport in glycogen supercompensated muscle during the early phase of carbohydrate refeeding. This probably led to the enhanced muscle glycogen supercompensation after exercise.

The use of non-integrating human artificial chromosomes (HACs) in gene therapy possibly allows for safe and reliable genetic modification of human cells without insertional mutagenesis and/or unexpected oncogene activations. Although we previously demonstrated that the HAC provides long-term therapeutic erythropoietin (EPO) production in normal human primary fibroblasts (hPFs), the expression level of EPO was too low to provide medical benefits for human therapy. Thus, the next challenge for the application of this system in therapeutic purposes is to improve the transgene expression on HACs. Here, we newly constructed chromosome 14-based HACs and examined the effects of the telomere and promoter regions on the expression level of the tansgene in hPFs. We showed that the use of natural telomere/sub-telomere and enhancers within the 5' untranslated region of the human ubiquitin C gene greatly increased (over 1000-fold) the EPO production in hPFs. Furthermore, we demonstrated the reprogramming of mouse embryonic fibroblasts by HAC-mediated introduction of four transcription factors, and established induced pluripotent stem cells with no trace of the HACs carrying multiple expression cassettes with large genome fragments. These results indicate that this HAC system could allow us to manipulate multiple transgenes efficiently in human primary cells, providing a promising tool not only for gene therapy but also for investigating genome functions in drug discoveries.

In our continuing effort to generate transgenic chickens, sonoporation was chosen to insert an exogenous gene into the chicken genome. An EGFP expression vector (pCAG-EGFPac) and microbubbles were injected into the central disc of stage-X blastoderm or the germinal crescent of stage-4 embryos, followed by ultrasonic vibration. Nineteen chicks out of 108 treated embryos hatched, six females and six males out of these 19 chicks grew to sexual maturity and two females and three males lived for 3 years. Genomic DNA from 17 out of 35 gonads from embryos and chicks that died before sexual maturity was EGFP-positive by PCR. No EGFP sequence was detected in the genomic DNA of 322 embryos from six sexually mature females and the semen from four sexually mature males by PCR. When genomic DNA was obtained from various tissues of five 3-year-old chickens, the EGFP sequence was amplified from the genomic DNA of the breast muscle of a female (No. 85). The above sequence was subjected to DNA sequencing and verified to be the EGFP sequence. These results showed that sonoporation is an effective tool for the transduction of exogenous genes into chicken embryos for the generation of transgenic chickens.

This study aimed to investigate the ability of single nucleotide polymorphism (SNP) haplotypes in chicken mtDNA for presumption of the origins of chicken meat. We typed five SNPs of the D-loop region in mtDNA by allele-specific PCR (AS-PCR) in 556 hens, that is 233 White Leghorn (WL), 50 Dekalb-TX35 (D-TX), 140 Barred Plymouth Rock (BPR) and 133 Rhode Island Red (RIR) kept in the National Institute of Livestock and Grassland Science (NILGS, Tsukuba, Japan). Five haplotypes were observed among those chickens by AS-PCR. WL, D-TX, BPR and RIR displayed three, two, one and four SNP haplotypes, respectively. By a combination of the haplotypes by AS-PCR and the breeds, these chickens were classified into 10 groups. After the D-loop was sequenced in two chickens from every group (20 individuals), 15 SNP sites (including one insertion) and eight sequence haplotypes were observed. In conclusion, haplotype variation was observed in and among the layer breeds of the NILGS. This study demonstrates that SNP haplotypes in mtDNA should be appropriate for the presumption of the origins of chicken meat.

BACKGROUND: Various factors have been reported to be useful for predicting future exacerbations. OBJECTIVE: This study was intended to determine a usefulness of a combination of a patient-based questionnaire, such as the Asthma Control Test (ACT) score with objective assessments, such as forced expiratory volume in 1 second (FEV(1)) and/or exhaled nitric oxide (FE(NO)), for predicting future exacerbations in adult asthmatics. METHODS: We therefore enrolled 78 subjects with mild to moderate asthma, who were clinically stable for 3 months who all had been regularly receiving inhaled steroid treatment. All subjects underwent a routine assessment of asthma control including the ACT score, spirometry, and FE(NO), and then were followed up until a severe exacerbation occurred. The predictors of an increased risk of severe exacerbation were identified and validated using decision trees based on a classification and regression tree (CART) analysis. The properties of the developed models were the evaluated with the area under the ROC curve (AUC) (95% confidence interval [CI]). RESULTS: The CART analysis automatically selected the variables and cut-off points, the ACT score

Mutations in the Prop1 gene are responsible for murine Ames dwarfism and human combined pituitary hormone deficiency with hypogonadism. Recently, we reported that PROP1 is a possible transcription factor for gonadotropin subunit genes through plural cis-acting sites composed of AT-rich sequences containing a TAAT motif which differs from its consensus binding sequence known as PRDQ9 (TAATTGAATTA). This study aimed to verify the binding specificity and sequence of PROP1 by applying the method of SELEX (Systematic Evolution of Ligands by EXponential enrichment), EMSA (electrophoretic mobility shift assay) and transient transfection assay. SELEX, after 5, 7 and 9 generations of selection using a random sequence library, showed that nucleotides containing one or two TAAT motifs were accumulated and accounted for 98.5% at the 9th generation. Aligned sequences and EMSA demonstrated that PROP1 binds preferentially to 11 nucleotides composed of an inverted TAAT motif separated by 3 nucleotides with variation in the half site of palindromic TAAT motifs and with preferential requirement of T at the nucleotide number 5 immediately 3' to a TAAT motif. Transient transfection assay demonstrated first that dimeric binding of PROP1 to an inverted TAAT motif and its cognates resulted in transcriptional activation, whereas monomeric binding of PROP1 to a single TAAT motif and an inverted ATTA motif did not mediate activation. Thus, this study demonstrated that dimeric binding of PROP1 is able to recognize diverse palindromic TAAT sequences separated by 3 nucleotides and to exhibit its transcriptional activity.

We cloned a porcine ortholog of homeodomain transcription factor Msx1 from the porcine pituitary cDNA library. The amino acid sequence of Msx1 shows high conservation among mammalian species. RT-PCR for porcine fetal and postnatal pituitaries showed that Msx1 is already expressed at early fetal day 40, decreases to a low level before birth and then remarkably decreases after birth. On the other hand, Msx1 expression was observed in all pituitary-derived cell line tested, with most in a gonadotrope lineage LbetaT4. Transfection assay demonstrated that Msx1 markedly repressed the basal Cga and Fshb gene expression, while Lhb expression was affected slightly. Taken together, Msx1 may play a role in repressing gene expression in the fetal and postnatal periods.

[抄録]胸水貯留は胸腔内に過剰な液体が貯留する病態であり，胸水の性状より漏出性および滲出性胸水に分類される.胸水貯留の原因疾患に伴い治療方針が異なるため，鑑別が重要となる.今回我々は胸水の貯留を認め，その原因疾患の診断に苦慮した1例を経験したため，鑑別診断を中心に文献的考察を加えて報告する.

We investigate an ecological mechanism by which endosymbiotic associations evolve, with a particular focus on the relationship between the evolution of endosymbiosis between auto- and heterotrophic organisms, and the stages of ecosystem development. For this purpose we conducted a long-term microcosm culture composed of three species, a green alga (Chlorella vulgaris), a bacterium (Escherichia coli), and a ciliated protozoan (Tetrahymena thermophila) for 3 years. During this culture T. thermophila cells harboring Chlorella cells emerged by phagocytotic uptake, and increased in frequency, reaching ca. 80-90%. This level was maintained in the late stage of ecosystem dynamics. Analysis of the ecosystem dynamics in the microcosm revealed that a complex causal process through direct/indirect interactions among ecosystem components led to reduction in dissolved O2 and food (E. coli) available to the T. thermophila, which gave a selective advantage to the organisms in the endosymbiotic association. This result suggests that the endosymbiosis evolves in a mature stage of ecosystem development, where reproduction and survival of prospective partner organisms is highly resource-limited and density-dependent, favoring efficient matter/energy transfers among participating organisms due to physical proximity. Consequently, a complex web of interactions and pathways of matter/energy flow in ecosystem evolves from an initially simple one.

Structure-based drug design was used to systematically synthesize PU3-dimers. The cytotoxicity of PU3 dimers 6 against breast cancer cell lines was evaluated, and their potency increased as the length of the bridging linker increased. Among the compounds tested, 6e with a C-20 linker was the most potent and exhibited a 20- to 30-fold increase in activity compared with that of the parent compound 5. Western blot analyses of the cell lysates treated with 6c revealed that 6c resulted in the concentration-dependent degradation of the Hsp90 client protein Her2, which is consistent with other Hsp90 inhibitors.

A bout of prolonged aerobic exercise can enhance the sensitivity of muscle glucose uptake to insulin, and this may be mediated by activation of 5'-adenosine monophosphate-activated protein kinase (AMPK). The aim of this study was to examine whether high-intensity short-term exercise resulting in a significantly greater increase in the activation of AMPK is more effective in enhancing muscle insulin sensitivity compared with low-intensity prolonged aerobic exercise. We measured insulin sensitivity after high-intensity intermittent swimming (HIS) or low-intensity continuous swimming (LIS) exercise in rat epitrochlearis muscle. During HIS, the rats underwent eight 20-second bouts of swimming with a weight equal to 18% of body weight. The LIS rats swam with no load for 3 hours. High-intensity intermittent swimming increased (P < .05) 2-deoxyglucose uptake approximately 8-fold, whereas LIS increased it (P < .05) approximately 2-fold immediately after exercise compared with rested muscle. This response was associated with an increase (P < .05) in phosphorylation of AMPK Thr(172) and its downstream target acetyl-coenzyme A carboxylase (ACC) Ser(79) in HIS (13- and 6-fold, respectively) and LIS (2.8- and 2-fold, respectively) immediately after exercise. In contrast, submaximal (30 microU/mL) insulin-stimulated 2-deoxyglucose uptake measured 4 hours after exercise was 73% and 46% higher (P < .05) in LIS and HIS, respectively, compared with rest. The HIS exercise resulted in a greater activation of AMPK compared with LIS, but insulin sensitivity was higher after LIS compared with HIS. The results suggest that HIS is not more effective in enhancing insulin sensitivity than LIS. Thus, AMPK activation immediately after exercise may not be the only factor that determines the magnitude of the exercise-induced increase in insulin sensitivity in rat epitrochlearis muscle.

Telomerase-mediated life-span extension enables the expansion of normal cells without malignant transformation, and thus has been thought to be useful in cell therapies. Currently, integrating vectors including the retrovirus are used for human telomerase reverse transcriptase (hTERT)-mediated expansion of normal cells; however, the use of these vectors potentially causes unexpected insertional mutagenesis and/or activation of oncogenes. Here, we established normal human fibroblast (hPF) clones retaining non-integrating human artificial chromosome (HAC) vectors harboring the hTERT expression cassette. In hTERT-HAC/hPF clones, we observed the telomerase activity and the suppression of senescent-associated SA-beta-galactosidase activity. Furthermore, the hTERT-HAC/hPF clones continued growing beyond 120days after cloning, whereas the hPF clones retaining the silent hTERT-HAC senesced within 70days. Thus, hTERT-HAC-mediated episomal expression of hTERT allows the extension of the life-span of human primary cells, implying that gene delivery by non-integrating HAC vectors can be used to control cellular proliferative capacity of primary cultured cells.

Human telomerase reverse transcriptase (hTERT) and epidermal growth factor receptor (EGFR) play an important role in many cancers including gynecological cancers. We previously reported the usefulness of a quantitative highly sensitive detection method for hTERT mRNA in the serum of cancer patients. By this method, we attempted to elucidate the diagnostic evaluation of serum hTERT mRNA for gynecologic malignancies. In 174 female patients with gynecological lesions (47 with ovarian lesions, 63 with uterine lesions, 2 with malignancies in other gynecological lesions, and 62 benign lesions) and 20 healthy individuals, we measured serum hTERT mRNA and EGFR mRNA by using the newly developed real-time quantitative RT-PCR. We examined their sensitivity and specificity in cancer diagnosis, clinical significance in comparison with conventional tumor markers, and their correlations with the clinical parameters by using multivariate analyses. Serum hTERT mRNA showed higher values in patients with gynecologic cancers than in those with benign diseases and healthy individuals. The hTERT mRNA level independently correlated with the presence of cancers (P=0.004 for both ovarian and uterine cancer) and clinical stage (P<0.001). The sensitivity and specificity of hTERT mRNA in cancer diagnosis was 74.4% and 74.1%, respectively. The hTERT mRNA level showed a significant correlation with CA125 by Pearson's relative test (P=0.035) and with histological findings in ovarian cancer by the Friedman test (P<0.004). EGFR mRNA did not display any differences between the diseases. hTERT mRNA is useful for diagnosing gynecologic cancer and is superior to conventional tumor markers. Therefore, serum hTERT mRNA is a novel and available biomarker for gynecologic malignancies.

Using a newly developed assay of telomerase reverse transcriptase (hTERT) mRNA in serum by real-time RT-PCR, we previously reported this assay to be superior to other tumor markers for hepatoma. In this study, we aimed to clarify its clinical significance as a biomarker for lung cancer. In 112 patients with lung tumor and 80 individuals without cancer, we measured serum hTERT mRNA and epidermal growth factor receptor (EGFR) mRNA levels, using a quantitative one-step real-time RT-PCR assay. We examined its sensitivity and specificity in lung cancer diagnosis, its clinical significance in comparison with other tumor markers, and its correlation with the clinical parameters using multivariate analyses and correlation relative tests. The copy number of serum hTERT mRNA was independently correlated with tumor size, tumor number, presence of metastasis and recurrence, and smoking (all P < 0.05). EGFR mRNA correlated with tumor number and clinical stage (both P < 0.05). The sensitivity and specificity in lung cancer diagnosis were 89.0% and 72.7% for hTERT mRNA, and 71.3% and 80.0% for EGFR mRNA, respectively. hTERT mRNA was superior to other tumor markers in lung cancer diagnosis. For both mRNAs, serum levels were significantly correlated with levels in lung cancer tissues (both P < 0.05). The copy number of hTERT mRNA significantly decreased after the surgical treatment. The data suggest that hTERT mRNA, especially when combined with EGFR mRNA, is a novel and excellent biomarker for pulmonary malignancies to diagnose and assess the clinical stage.

It is very important for immunotherapy to release Th2-dominated immunological conditions in patients with malignant diseases. In the present study, we assessed the intracellular cytokine profiles of CD4 positive cells in peripheral blood in patients with colorectal cancer using a flow cytometric analysis and we investigated whether Th2-dominated immunological conditions could be released by PSK. Peripheral blood samples were collected preoperatively from 57 patients with colorectal cancer before and after the oral administration of PSK (3g/day x 1 week). After the PSK treatment, CD4(+)IL-10(+)T-cell percentages decreased significantly, whereas no significant change occurred in proportions of CD4(+)IL-6(+)T-cells. In the after/ before PSK treatment percentages, the ratio of CD4(+)IL-10(+)T-cells were significantly lower in non-recurrent patients compared with recurrent patients, whereas no significant difference was seen in the ratio of CD4(+)IL-6(+) T-cells. These results suggest that the after/before percentage ratio of CD4(+)IL-10(+)T-cells may be useful predicting parameters for the selection of responders.

We examined the electrophysiological effects of trapidil on the ionic currents influencing the repolarization and on the transient inward current (ITi) that can cause triggered arrhythmia using the whole-cell patch-clamp technique in guinea pig ventricular myocytes. Trapidil shortened the action potential duration (APD) and increased the delayed rectifier potassium current (IK) in a concentration-dependent manner. The effect of trapidil on the rapidly and slowly activating components of IK (IKr and IKs, respectively) was studied by the envelope of tails test. Trapidil failed to affect IKr and selectively enhanced IKs. Trapidil increased the amplitude of the L-type Ca2+ current (ICa,L), with an acceleration of its inactivation, whereas isoproterenol, a beta-adrenoceptor agonist, increased the amplitude of the ICa,L in a different manner. Isoproterenol activated ITi; however, trapidil not only failed to facilitate ITi but also suppressed isoproterenol-induced ITi. The inhibitory effect of trapidil on isoproterenol-induced ITi is at least partly via a reduction of Ca2+ overload through an acceleration of ICa,L inactivation and/or a sarcoplasmic reticulum (SR) Ca channel modulation. These results suggest that trapidil does not prolong the QT interval and has an antiarrhythmic effect on arrhythmias elicited by triggered activity secondary to Ca2+ overload at much higher concentrations than clinical concentration.

PURPOSE: We previously reported the usefulness of a qualified highly sensitive detection method for human telomerase reverse transcriptase (hTERT) mRNA in serum with 89.7% sensitivity for hepatocellular carcinoma (HCC). In this study, we developed a quantitative detection method for serum hTERT mRNA and examined the clinical significance in HCC diagnosis. EXPERIMENTAL BACKGROUND: In 64 patients with HCC, 20 with liver cirrhosis, 20 with chronic hepatitis, and 50 healthy individuals, we measured serum hTERT mRNA by using the newly developed real-time quantitative reverse transcription-PCR with SYBR Green I. We examined its sensitivity and specificity in HCC diagnosis, clinical significance in comparison with other tumor markers, and its correlations with the clinical variables by using multivariate analyses. RESULTS: Serum hTERT mRNA showed higher values in patients with HCC than those with chronic liver diseases. hTERT mRNA expression was shown to be independently correlated with clinical variables such as tumor size, number, and degree of differentiation (P < 0.001, each). The sensitivity/specificity of hTERT mRNA and alpha-fetoprotein (AFP) mRNA in HCC diagnosis were 88.2%/70.0% for hTERT and 71.6%/67.5% for AFP, respectively. hTERT mRNA proved to be superior to AFP mRNA, AFP, and des-gamma-carboxy prothrombin in HCC diagnosis. Furthermore, hTERT mRNA in serum was associated with that in HCC tissue. CONCLUSIONS: The usefulness of hTERT mRNA expression in HCC diagnosis and its superiority to conventional tumor markers were shown. Therefore, serum hTERT mRNA is a novel and available marker for HCC diagnosis.

Although superselective continuous intra-arterial infusion has advantages for cancer therapy, intra-arterial chemotherapy is often interrupted by arterial damage due to arteritis. Therefore, an animal model must be developed to elucidate the mechanism of arteritis associated with continuous anti-cancer drug infusion. We developed a new rat model with which to investigate the causal mechanism(s) of vascular damage associated with continuous catheterization chemotherapy. Chemotherapeutic agents (fluorouracil (5-FU) or peplomycin (PEP)) were continuously administered for 7 days into the abdominal aorta of male Sprague-Dawley rats through a catheter fixed in situ. We found that the incidence of apoptotic endothelial cells of the aorta was higher nearer the tip of the catheter. The incidence of apoptosis was higher in the group treated with 5-FU than with PEP. This animal model will be useful to improve arterial damage among patients undergoing chemotherapy using continuous catheterization.

気管支喘息の治療には、キサンチン製剤は気管支拡張作用ならびに抗炎症作用を有し、優れた薬剤である。今回は気管平滑筋収縮に対して、キサンチン製剤の投与方法の違いによる拡張効果を検討した。

令和5年度は、喘息例での胸部CT所見（気管支拡張・細気管支炎像の有無，進展度）等と遺伝子多型との関係を中心に解析した。候補遺伝子としてびまん性汎細気管支炎や非若齢発症喘息のリスク遺伝子であり、分泌型ムチンをコードするHCG22遺伝子の一塩基多型（SNP）rs2523870に着目した。喘息106例において、rs2523870のgenotypeはTT 37名、CT 45名、CC 24名であった。リスクアレルのホモ接合体であるCC型と他との２群比較で、喘息発症年齢、呼吸機能、mReiff score(気管支拡張像の進展度)との関係はなかったが、CC型で好酸球性副鼻腔炎併存率、過去の血液好酸球数、過去の吸入ステロイド量、過去及び直近の胸部CTで細気管支炎罹患葉数が有意に多かった。IL4RA rs8832、IL13 rs20541についても解析を行ったが、mReiff scoreや細気管支炎の罹患葉数との関連は認めなかった。本研究でCC型での細気管支病変が、好酸球性か否かは確認できなかったが、好酸球性副鼻腔炎の併存率から推察すると一定の割合で好酸球性細気管支炎も存在すると考えられる。喘息の細気管支病変例で、好中球性気道炎症を呈するびまん性汎細気管支炎と同様のリスク多型が確認されたことは、分泌型ムチンの異常、気道クリアランスの障害が、炎症型を問わず細気管支病変の形成に寄与する可能性が推察される（J Allergy Clin Immunol 2023 Epub ahead of print）。

1) The acute effects of Shinbuto, a Kampo medicine on the contraction activity of guinea-pig cardiac myocytes were studied using a motion analyzer. Shinbuto (3-5 mg/ml) tended to increase the contractile amplitude of myocytes by 56-369% in a concentration dependent manner. However, results obtained under different conditions were inconsistent. 2) The effect of long-term Shinbuto administration on heart and renal genes expression in mice. About 50 genes in the heart and about 100 genes expressed in the kidney showed marked changes. One apoptosis-related gene showed increased expression in the heart, but a decrease in the kidney. 3) The effect of long-term Shinbuto administration to diseased animals was examined in hypertensive heart failure model rats, spontaneously hypertensive rats (SHR). Although control animals (SHR) did not show any symptoms of heart failure nor an increase in natriuretic peptide, each of two of the five ingredients of Shinbuto individually tended to suppress the blood pressure in SHR, while the other three did not show any effect on blood pressure. Each ingredient of Shinbuto changed the expressions of genes related to heat shock protein, renin-angiotensin system, potassium ion and calcium ion channels. These genes seem to be related to hypertension. Gene expressions changed markedly following Shinbuto administration to normal mice, but did not show any remarkable change in these diseased rats. These findings suggest that the effects of Shinbuto, a Kampo medicine vary not only with species, but also with pathological conditions.

ヒト好酸球におけるPC-PLC(ホスファチジルコリン特異的ホスホリパーゼC)の細胞内Ca^<2+>濃度変化に及ぼす影響と脱顆粒における役割をPI-PLC(ホスファチジルイノシトール特異的ホスホリパーゼC)と比較して検討した。比較するにあたって、PC-PLC特異的阻害剤であるD609とPI-PLC特異的阻害剤であるET-18-OCH_3を用いた。 1.PI-PLC、PC-PLC阻害剤はともにfMLP刺激によるEPO (Eosinophil peroxidase)放出を抑制した。 2.Amplex redを用いたPC-PLC activity assayにおいてPC-PLC阻害剤はPC-PLCの活性化を抑制したが、PI-PLC阻害剤はPC-PLC活性に影響を与えなかった。 また、PI-PLC阻害剤はIP_3産生を抑制したが、PC-PLC阻害剤はIP_3 (inositol triphosphate)産生に影響を与えなかった。 3.PI-PLC阻害剤は細胞内Ca^<2+>濃度上昇を抑制したが、PC-PLC阻害剤は影響を与えなかった。 4.PKC (Protein kinase C)阻害剤はfMLP刺激によるEPO放出を抑制した。 以上の結果より、PI-PLCはPKC活性化とCa^<2+>influxをダウンレギュレートすることによってヒト好酸球の脱顆粒を抑制している。一方、それとは対照的にPC-PLCは細胞内カルシウム濃度の変化に非依存的に脱顆粒を制御していると考えられた。

Cytosolic phospholipase A_2 (PLA_2) is the rate-limiting enzyme involved in the conversion of membrane phospholipids to arachidonic acid (AA) and lysophospholipids, which are readily metabolized to inflammatory mediators such as leukotrienes, thromboxane and platelet-activation factor. It has been recognized that receptor antagonists of leukotrienes or thronboxane are effective for treatment of bronchial asthma. To inhibit cPLA2 thus might be more effective than using leukotrienes or thronboxane receptor antagonist on treatment of bronchial asthma. We previously reported that cPLA2 regulates adhesion of human eosinophils to ICAM-1 or VCAM-1, and that inhibition of cPLA2 blocked eosinophil migration to lung tissue and antigen-induced airway hyperresponsiveness. In this study we investigated whether activity and quantitatition of cPLA2 in eosinophils from patients with asthma is more excessive than its from healthy donors. Both AA release and cPLA2 activity from patients with asthma by 1□M FMLP were greater than its from healthy donors significantly. Quantitation of cPLA2 is 0.36±0.13 ng/10^6 cells from healthy donors and 0.52±0.17 ng/10^6 cells from patients with asthma(p=0.0285)