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Rationale: The clinical features and prognosis of nontuberculous mycobacterial (NTM) pleuritis and pleural effusion combined with NTM lung disease remain unclear. Objectives: To investigate the cli...

Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection., 「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.

To elucidate the host genetic loci affecting severity of SARS-CoV-2 infection, or Coronavirus disease 2019 (COVID-19), is an emerging issue in the face of the current devastating pandemic. Here, we report a genome-wide association study (GWAS) of COVID-19 in a Japanese population led by the Japan COVID-19 Task Force, as one of the initial discovery GWAS studies performed on a non-European population. Enrolling a total of 2,393 cases and 3,289 controls, we not only replicated previously reported COVID-19 risk variants (e.g., LZTFL1, FOXP4, ABO, and IFNAR2), but also found a variant on 5p35 (rs60200309-A at DOCK2) that was significantly associated with severe COVID-19 in younger (-8 (odds ratio = 2.01, 95% confidence interval = 1.58-2.55). This risk allele was prevalent in East Asians, including Japanese (minor allele frequency [MAF] = 0.097), but rarely found in Europeans. Cross-population Mendelian randomization analysis made a causal inference of a number of complex human traits on COVID-19. In particular, obesity had a significant impact on severe COVID-19. The presence of the population-specific risk allele underscores the need of non-European studies of COVID-19 host genetics.

Objectives To determine the usefulness of hsa-miR-346, a potential biomarker enhancing the activity of non-tuberculous mycobacterial diseases, as a biomarker of tuberculosis activity. Methods We investigated whether hsa-miR-346 is secreted by human macrophages infected with Mycobacterium tuberculosis (M. tuberculosis) in an in vitro study. In addition, a cross-sectional study was conducted first to evaluate whether serum hsa-miR-346 is elevated in patients with tuberculosis compared with that in healthy individuals. Second, we conducted a retrospective study to evaluate whether anti-tuberculosis treatment reduces serum hsa-miR-346 levels. Results Log hsa-miR-346 levels were significantly elevated in the supernatant of human macrophages infected with M. tuberculosis in a dose-dependent manner. The mean serum log hsa-miR-346 levels were −15.48 (−15.76 to −15.21) in patients with tuberculosis and −16.12 (−16.29 to −15.95) in healthy volunteers, which significantly differed. In addition, hsa-miR-346 significantly decreased at 2 months from starting an anti-tuberculosis treatment. Conclusions We consider hsa-miR-346 as a potential biomarker enhancing the tuberculosis activity.

IntroductionThe rapid spread of COVID-19 posed a global burden. Substantial number of people died of the disease in the acute phase of infection. In addition, a significant proportion of patients have been reported to suffer from post-acute phase symptoms, sequelae of COVID-19, which may negatively influence the quality of daily living and/or socioeconomic circumstances of the patients. However, no previous study has comprehensively and objectively assessed the quality of life of patients by using existing international scales. Further, evidence of socioeconomic consequences among patients with COVID-19 is scarce. To address the multidimensional issues from sequelae of COVID-19, evidence from comprehensive surveys beyond clinical perspectives is critical that investigates health, and social determinants of disease progression as well as socioeconomic consequences at a large scale.Methods and analysisIn this study, we plan to conduct a nationwide and comprehensive survey for the sequelae of COVID-19 in a total of 1000 patients diagnosed at 27 hospitals throughout Japan. This study will evaluate not only the health-related status of patients from clinical perspectives but also the Health-related Quality of Life (HRQoL) scores, socioeconomic status and consequences to discuss the sequelae of the disease and the related risk factors. The primary endpoint is the frequency of long-term complications of COVID-19 infection. The secondary endpoints are risk factors for progression to sequelae of COVID-19 infection. The study will provide robust and important evidence as a resource to tackle the issues from the sequelae of COVID-19 from the multi-dimensional perspectives.Ethics and disseminationThis trial was approved by the Keio University School of Medicine Ethics Committee (20200243, UMIN000042299). The results of this study will be reported at a society meeting or published in a peer-reviewed journal.

Background The chronic obstructive pulmonary disease (COPD) Assessment Test (CAT) is a subjective measure of quality of life. The aim of this study was to examine the characteristics of COPD patients with increasing CAT scores within 3 years. Methods Keio University and its affiliate hospitals conducted an observational COPD cohort study over 3 years. St. George's Respiratory Questionnaire (SGRQ) and CAT were completed at baseline and annually thereafter. Patients who had at least 3 CAT scores were included (n = 315). The ΔCAT score/year and ΔSGRQ score/year were calculated by the slope between each of the measures and the date of measurement. Results The median ΔCAT score/year was 0.4, and ΔCAT score/year was significantly correlated with ΔSGRQ total score/year. Using an annual cut-off CAT score of +2 points, patients who deteriorated (n = 79) were older, had lower %FEV 1 , and more severe emphysema on computed tomography scan at baseline than patients who did not deteriorate. The baseline value was not a determinant of subsequent changes in the CAT score. Longitudinal changes in the CAT score were positively correlated with those in the SGRQ score. Conclusions Old age and severe COPD, not the CAT score at one time point, predicted worsening quality of life.

Introduction: Nontuberculous mycobacterial (NTM) pleuritis, pleural effusion combined with NTM lung disease (NTM-LD), has not been previously described as a common manifestation of NTM infections. Aims: This study aimed to investigate the clinical features and prognosis of NTM pleuritis. Methods: A retrospective observational study was performed including patients with NTM pleuritis fromJanuary 2001 to December 2017 at 8 hospitals. NTM pleuritis was defined as a positive culture of NTM from pleural effusion. Furthermore, we matched patients with NTM-LD but without pleuritis by sex and age (within a 10-year range) to obtain comparative data for individuals. We investigated the association between clinical parameters and prognosis. Results: We identified 58 patients with NTM pleuritis (median age, 72 years; 35 females; Mycobacterium avium complex [MAC], 48 patients). The median follow-up period was 16 months and 26 patients died. Patients with NTM pleuritis had significantly worse survival than sex- and age-matched patients presenting NTM-LD without pleuritis (P Conclusions: Our results suggest that prognosis of NTM pleuritis is worse than NTM-LD without pleuritis. Moreover, lower BMI, UPD, and no treatment history after pleuritis are associated with poor prognosis in patients with NTM pleuritis.

MicroRNA (miRNA) has been recently recognized as a biomarker of various diseases; however, there are no known miRNAs associated with Mycobacterium avium complex (MAC) pulmonary disease. In addition, there are no known biomarkers to precisely reflect disease activity after the diagnosis of MAC pulmonary disease. Thus, we sought to identify a miRNA which is a candidate biomarker of MAC pulmonary disease activity. Serum hsa-miR-346 concentrations of 16 patients with M. avium pulmonary disease were significantly higher than those of 16 healthy controls (p = 0.047). The secretion of hsa-miR-346 increased in a multiplicity of infection-dependent manner in M. avium-infected macrophages. Serum hsa-miR-346 levels of 5 patients with bacterial conversion at the end of follow-up were significantly lower than those at the beginning of the follow-up (p = 0.043). In addition, the longitudinal change in serum hsa-miR-346 concentration correlated with bacterial load in 2 patients with M. avium pulmonary disease. Based on our results, it is supposed that MAC-infected macrophages in pulmonary lesions produce hsa-miR-346, which is then secreted into the bloodstream. The magnitude of this process could be quantitatively controlled by the bacterial load, suggesting that serum hsa-miR-346 is a potentially useful biomarker of MAC pulmonary disease activity.

A 60-year-old male presented with cough and fever (case 1), and a 77-year-old female patient with hemoptysis (case 2) were admitted to IUHW Shioya Hospital. Interferon gamma release assay (IGRA: QFT-3G and T-SPOT. TB) were both performed on each patient; the QFT-3G was positive and T-SPOT.TB was negative in both cases despite different final diagnosis. While case 1 was determined to have active tuberculosis, in case 2 active tuberculosis was eliminated as the diagnosis. The contradictory results in these patients should serve as a reminder to clinicians that they should not rely on the results of IGRA testing for the diagnosis of active tuberculosis. Keywords: Interferon Gamma Release Assay; Negative; Tuberculosis; T-SPOT; QFT

A 27-year-old man with a 4-month history of treatment for miliary tuberculosis at another hospital was admitted to our hospital for continued treatment. Computed tomography showed new lesions in the S8 area of the liver and spleen, despite resolution of chest radiographic findings. Because these new lesions were still present after 8 months of treatment, we performed laparoscopic drainage of the liver abscess. Purulent material drained from the lesion revealed positive polymerase chain reaction results for Mycobacterium tuberculosis, and identification of granuloma with infiltrating lymphocytes and plasma cells confirmed the diagnosis of tubercular liver abscess. Pathological changes in the spleen over the clinical course were also regarded as representing tubercular abscess. Postoperative course was good, and tuberculosis treatment ended after 12 months. Tubercular liver abscess subsequently showed prominent reduction, and the tubercular splenic abscess disappeared on abdominal ultrasonography. Tubercular hepatosplenic abscesses appearing during tubercular treatment are rare. We report this valuable case in which laparoscopic drainage of a liver abscess proved useful for diagnosis and treatment.

Although the endothelial expression of various adhesion molecules substantially differs between pulmonary microvessels, their importance for neutrophil and lymphocyte sequestration in ventilator-induced lung injury (VILI) has not been systematically analyzed. We investigated the kinetics of polymorphonuclear cells (PMN) and mononuclear cells (MN) in the acinar microcirculation of the isolated rat lung with VILI by real-time confocal laser fluorescence microscopy, with or without inhibition of ICAM-1, VCAM-1, or P-selectin by monoclonal antibodies (MAb). Adhesion molecules in each microvessel were estimated by intravital fluorescence microscopy or immunohistochemical staining. In high tidal volume-ventilated lungs, 1) ICAM-1, VCAM-1, and P-selectin were differently upregulated in venules, arterioles, and capillaries; 2) venular PMN rolling was improved by inhibition of ICAM-1, VCAM-1, or P-selectin, whereas arteriolar PMN rolling was improved by ICAM-1 or VCAM-1 inhibition; 3) capillary PMN entrapment was ameliorated only by anti-ICAM-1 MAb; and 4) MN rolling in venules and arterioles and MN entrapment in capillaries were improved by ICAM-1 and VCAM-1 inhibition. In conclusion, the contribution of endothelial adhesion molecules to abnormal leukocyte behavior in VILI-injured microcirculation is microvessel and leukocyte specific. ICAM-1- and VCAM-1-dependent, but P-selectin-independent, arteriolar PMN rolling, which is expected to reflect the initial stage of tissue injury, should be taken as a phenomenon unique to ventilator-associated lung injury.

Asthma is characterized by acute and chronic airway inflammation, and the severity of the airway hyperreactivity correlates with the degree of inflammation. Many of the features of lung inflammation observed in human asthma are reproduced in OVA-sensitized/challenged mice. T lymphocytes, particularly Th2 cells, are critically involved in the genesis of the allergic response to inhaled Ag. In addition to antiapoptotic effects, broad-spectrum caspase inhibitors inhibit T cell activation in vitro. We investigated the effect of the broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), on airway inflammation in OVA-sensitized/challenged mice. OVA-sensitized mice treated with z-VAD-fmk immediately before allergen challenge showed marked reduction in inflammatory cell infiltration in the airways and pulmonary blood vessels, mucus production, and Th2 cytokine production. We hypothesized that the caspase inhibitor prevented T cell activation, resulting in the reduction of cytokine production and eosinophil infiltration. Treatment with z-VAD-fmk in vivo prevented subsequent T cell activation ex vivo. We propose that caspase inhibitors may offer a novel therapeutic approach to T cell-dependent inflammatory airway diseases.

To assess the importance of endothelial intercellular adhesion molecule-1 (ICAM-1) in microvascular leukocyte kinetics in diseased lungs, we investigated the transitional changes in ICAM-1 expression, vascular diameter, and leukocyte behavior in rat pulmonary microcirculation during the development of acute lung injury (ALI) and chronic fibrosis (FIB) evoked by bleomycin (BLM). Observations were made in the isolated perfused lung with a real-time confocal laser luminescence microscope. Microvascular cell kinetics were evaluated by measuring the behavior of fluorescence- labeled leukocytes and erythrocytes in the presence or absence of anti-ICAM-1 monoclonal antibody (1A29). Arteriolar ICAM-1 showed little change at any time after BLM treatment. Venular ICAM-1 was first enhanced at the initial phase of ALI followed by the second upregulation at the early phase of FIB. Capillary ICAM-1 showed a sustained increase at both ALI and FIB. Arteriolar and venular diameters were not altered but capillary diameter decreased during ALI and early FIB stages. Although firm adherence of leukocytes to arteriolar and venular walls was not observed, rolling leukocytes were increased in venules both at the initial phase of ALI and at the early phase of FIB. The leukocyte rolling in venules correlated well with transitional changes in ICAM-1 and was inhibited by 1A29. Sustained entrapment of leukocytes in capillaries was attributed to changes in vascular diameter as well as augmented ICAM-1. In conclusion, ICAM-1 plays an important role in microvascular leukocyte recruitment in both ALI and FIB in the BLM-injured lung.

Intercellular adhesion molecule-1 (ICAM-1) of the vascular endothelium plays a key role in the development of pulmonary oxygen toxicity. We studied the effect of steroid on hyperoxia-induced ICAM-1 expression using cultured endothelial cells in vitro. Human pulmonary artery endothelial cells (HPAECs) were cultured to confluence, and then the monolayers were exposed to either control (21% O2-5% CO2) or hyperoxic (90% O2-5% CO2) conditions with and without a synthetic glucocorticoid, methylprednisolone (MP). MP reduced hyperoxia-induced ICAM-1 and ICAM-1 mRNA expression in a dose-dependent manner. Neutrophil adhesion to hyperoxia-exposed endothelial cells was also inhibited by MP treatment. In addition, MP attenuated hyperoxia-induced H2O2production in HPAECs as assessed by flow cytometry. An electrophoretic mobility shift assay demonstrated that hyperoxia activated nuclear factor-κB (NF-κB) but not activator protein-1 (AP-1) and that MP attenuated hyperoxia-induced NF-κB activation dose dependently. With Western immunoblot analysis, IκB-α expression was decreased by hyperoxia and increased by MP treatment. These results suggest that MP downregulates hyperoxia-induced ICAM-1 expression by inhibiting NF-κB activation via increased IκB-α expression.

We analyzed the effects of hypoxic vasoconstriction and total flow on the distribution of pulmonary perfusion in 38 isolated left rabbit lungs perfused under zone 3 conditions. Lungs were suspended in an upright position, oriented to the apicobasal line. Distributions of regional perfusion rates (RPR) along the vertical and horizontal axes were determined using nonradioactive microspheres labeled with heavy metal elements, which were detectable with X-ray fluorescence spectrometry. Changing the O2 concentration of a respirator and an extracorporeal membrane oxygenator independently, respective influences of active vasoconstriction induced by alveolar hypoxia and pulmonary artery hypoxia (PA hypoxia) on the RPR distribution were examined at a flow rate of 0.4 ml ⋅ min−1 ⋅ g wet lung tissue−1. To analyze the effects of changes in total flow, we investigated the RPR distribution at a perfusion rate of 1.2 ml ⋅ min−1 ⋅ g wet lung tissue−1. The RPR distribution in the absence of hypoxia was inhomogeneous and was augmented in the lower lung fields, whereas alveolar hypoxia shifted the RPR upward and significantly diminished the RPR in the lung base. RPR distributions along the horizontal axes under alveolar hypoxia conditions demonstrated that remarkable hypoxic pulmonary vasoconstriction (HPV) takes place in medial regions at the lung base. PA hypoxia altered the RPR distribution in qualitatively the same manner as alveolar hypoxia. Increased flow rate augmented the RPR in the lung, except in the dorsobasal region. These results suggest that the occurrence of HPV and the vascular conductance are not uniform throughout the lung.

To investigate the pathogenesis of pulmonary oxygen toxicity, we examined the effect of hyperoxia on adhesion molecule expression in cultured human pulmonary artery endothelial cells (HPAEC) and human umbilical vein endothelial cells (HUVEC). Endothelial cell monolayers were exposed to either hyperoxic (90% O(2)-5% CO(2)) or normoxic (21% O(2)-5% CO(2)) conditions for various periods. The level of intercellular adhesion molecule (ICAM)-1 expression had increased in hyperoxia-exposed HPAEC and HUVEC at 48 h (194 +/- 38 and 233 +/- 56%, respectively; P < 0.001) and at 72 h (200 +/- 43 and 223 +/- 52%, respectively; P < 0.001) compared with normoxic conditions. These hyperoxia-induced ICAM-1 expressions were dose dependently attenuated by a protein kinase C inhibitor (H-7). In contrast, the levels of P-selectin and E-selectin expression in HPAEC and HUVEC were unchanged. The levels of ICAM-1 mRNA and the numbers of adherent neutrophils were increased in HPAEC and HUVEC at 48 and 72 h of hyperoxia. On the other hand, hyperoxia caused neutrophil H(2)O(2) production without affecting the level of CD11/CD18 expression. These results suggest that increased ICAM-1 expression in endothelial cells plays an important role in neutrophil accumulation during hyperoxia.

A 49-year-old male who had been treated for pulmonary tuberculosis and tuberculous pleurisy in 2007 was referred to our hospital with the complaint of dyspnea on exertion in Nov. 2009. Chest X-ray showed increased pleural effusion compared with that remaining after the previous treatment of pleurisy in 2008. A chest CT revealed that fluid collection was surrounded by thickened pleura. Thoracocentesis was performed, and yellow milky liquid was obtained. The pleural effusion contained few cells. The triglyceride concentration was 83 mg/dl, and the cholesterol level was very high at 628 mg/dl. Based on these findings we diagnosed this case as chyliform pleural effusion. Both smear of acid-fast bacilli and PCR-TB test of the pleural effusion were positive, but culture was negative for mycobacterium, suggesting that this chyliform pleural effusion was produced by the former episode of tuberculous pleurisy, not by the recent reactivation of tuberculous pleurisy. The ADA concentration in the pleural effusion was high at 91.7 IU/l. No increase in the amount of pleural effusion was observed after thoracocentesis without any anti-tuberculosis therapy.

A 24-year-old man who had been treated 3 months for tuberculous pleurisy presented with thoracic back pain. Chest CT showed a new lesion abutting the pleura, despite the disappearance of pleural effusion. Two weeks later, the mass abutting the pleura progressed to form a new intrapulmonary infiltrative shadow. A transbronchial lung biopsy was performed and the histopathologic examination of the specimen from this lesion revealed granulomatous inflammation without caseous necrosis or acid-fast bacilli. No acid-fast bacilli were cultured from the bronchoalveolar lavage fluid. Anti-tuberculosis medication was continued without change, and the lesions finally resolved. More than 3 years have passed since the completion of anti-tuberculosis chemotherapy, and no recurrence has been observed. We believe that these lesions were pulmonary tuberculomas and transient intra-pulmonary infiltration due to non-specific inflammation, caused secondarily by an excessive immune response, as in paradoxical worsening.

Emphasis in treating patients with infectious pulmonary tuberculosis has come to be laid on the execution of reliable standard chemotherapy. As a result, hospitalization for a prolonged period has become unnecessary any more. However, few attempts have been made so far on the determination of discharging criteria.We performed a questionnaire survey to hospitals with wards for tuberculosis in Kanto area and asked questions on the current status of discharging criteria.The effective response rate to the survey was 63.0 %. Sputum smear examination carried out mainly by Ziehl-Neelsen method and fluorescence method in 17.2% and 72.4 % of the hospitals, respectively. Sputum culture examination was carried out using mainly Ogawa medium and a liquid medium in 62.1% and 27.6% of the hospitals, respectively. Discharging criteria were standardized in 79.3% of hospitals. Negative sputum smear was used as the criterion for determining discharge in 11 sets of criteria. Negative sputum culture was used as the criterion for determining discharge in 17 sets of criteria. In the remaining one hospital, patients were to be discharged after 2-month treatment. There was no consistency in the procedure and the frequency of sputum examinations, how many negative results are needed to confirm negative status and the criteria for judgment.These results suggest that further evaluation must be made on the treatment outcome at each hospital, and the standard discharging criteria should be worked out taking into account the capacity of each hospital and the care situation of local community.

Although c-Jun NH2-terminal kinase (JNK) has been implicated in the pathogenesis of transplantation-induced ischemia/reperfusion (I/R) injury in various organs, its significance in lung transplantation has not been conclusively elucidated. We therefore attempted to measure the transitional changes in JNK and AP-1 activities in I/R-injured lungs. Subsequently, we assessed the effects of JNK inhibition by the three agents including SP600125 on the degree of lung injury assessed by means of various biological markers in bronchoalveolar lavage fluid and histological examination including detection of apoptosis. In addition, we evaluated the changes in p38, extracellular signal-regulated kinase, and NF-κB-DNA binding activity. I/R injury was established in the isolated rat lung preserved in modified Euro-Collins solution at 4°C for 4 h followed by reperfusion at 37°C for 3 h. We found that AP-1 was transiently activated during ischemia but showed sustained activation during reperfusion, leading to significant lung injury and apoptosis. The change in AP-1 was generally in parallel with that of JNK, which was activated in epithelial cells (bronchial and alveolar), alveolar macrophages, and smooth muscle cells (bronchial and vascular) on immunohistochemical examination. The change in NF-κB qualitatively differed from that of AP-1. Protein leakage, release of lactate dehydrogenase and TNF-α into bronchoalveolar lavage fluid, and lung injury were improved, and apoptosis was suppressed by JNK inhibition. In conclusion, JNK plays a pivotal role in mediating lung injury caused by I/R. Therefore, inhibition of JNK activity has potential as an effective therapeutic strategy for preventing I/R injury during lung transplantation.

Although permissive hypercapnia improves the prognosis of patients with acute respiratory distress syndrome, it has not been conclusively determined whether hypercapnic acidosis (HA) is harmful or beneficial to sustained inflammation of the lung. The present study was designed to explore the molecular mechanism of HA in modifying lipopolysaccharide (LPS)-associated signals in pulmonary endothelial cells. LPS elicited degradation of inhibitory protein B( IB)-, but not IB-, resulting in activation of nuclear factor (NF)-B in human pulmonary artery endothelial cells. Exposure to HA significantly attenuated LPS-induced NF-B activation through suppressing IB- degradation. Isocapnic acidosis and buffered hypercapnia showed qualitatively similar but quantitatively smaller effects. HA did not attenuate the LPS-enhanced activation of activator protein-1. Following the reduced NF-B activation, HA suppressed the mRNA and protein levels of intercellular adhesion molecule-1 and interleukin-8, resulting in a decrease in both lactate dehydrogenase release into the medium and neutrophil adherence to LPS-activated human pulmonary artery endothelial cells. In contrast, HA did not inhibit LPS-enhanced neutrophil expression of integrin, Mac-1. Based on these findings, we concluded that hypercapnic acidosis would have anti-inflammatory effects essentially through a mechanism inhibiting NF-B activation, leading to downregulation of intercellular adhesion molecule-1 and interleukin-8, which in turn inhibits neutrophil adherence to pulmonary endothelial cells. The mortality of acute respiratory distress syndrome (ARDS) remains unacceptably high at 40–60% (1). Most patients with ARDS undergo mechanical ventilation, and one of the most important concepts in the recent treatment of patients with ARDS is the recognition that a classical setting of mechanical ventilation with a relatively large tidal volume can worsen or even cause new lung injury (2). To

The hydrogen ion is an important factor in the alteration of vascular tone in pulmonary circulation. Endothelial cells modulate vascular tone by producing vasoactive substances such as prostacyclin (PGI2) through a process depending on intracellular Ca2+concentration ([Ca2+]i). We studied the influence of CO2-related pH changes on [Ca2+]iand PGI2production in human pulmonary artery endothelial cells (HPAECs). Hypercapnic acidosis appreciably increased [Ca2+]ifrom 112 ± 24 to 157 ± 38 nmol/l. Intracellular acidification at a normal extracellular pH increased [Ca2+]icomparable to that observed during hypercapnic acidosis. The hypercapnia-induced increase in [Ca2+]iwas unchanged by the removal of Ca2+from the extracellular medium or by the depletion of thapsigargin-sensitive intracellular Ca2+stores. Hypercapnic acidosis may thus release Ca2+from pH-sensitive but thapsigargin-insensitive intracellular Ca2+stores. Hypocapnic alkalosis caused a fivefold increase in [Ca2+]icompared with hypercapnic acidosis. Intracellular alkalinization at a normal extracellular pH did not affect [Ca2+]i. The hypocapnia-evoked increase in [Ca2+]iwas decreased from 242 ± 56 to 50 ± 32 nmol/l by the removal of extracellular Ca2+. The main mechanism affecting the hypocapnia-dependent [Ca2+]iincrease was thought to be the augmented influx of extracellular Ca2+mediated by extracellular alkalosis. Hypercapnic acidosis caused little change in PGI2production, but hypocapnic alkalosis increased it markedly. In conclusion, both hypercapnic acidosis and hypocapnic alkalosis increase [Ca2+]iin HPAECs, but the mechanisms and pathophysiological significance of these increases may differ qualitatively.

The issue of whether the acinar microvessel response to alveolar hypoxia and hypercapnia is impaired in injured lungs has not been vigorously addressed, despite the importance of knowing whether it is or not when treating patients with serious lung injury in terms of permissive hypercapnia. Applying a real-time laser confocal luminescence microscope, we studied hypoxia- and hypercapnia-induced changes in the diameter of the intra-acinar arterioles, venules, and capillaries of isolated rat lungs harvested from animals exposed for 48 h to 21% O2 ( group N) or 90% O2 ( group H). Measurements were made with and without inhibition of nitric oxide (NO) synthase (NOS) by N ω-nitro-l-arginine methyl ester or of cyclooxygenase (COX) by indomethacin at different basal vascular tones evoked by thromboxane A2(TXA2) analog. Hypoxia in the absence of TXA2 contracted arterioles in group N but not in group H. Attenuated hypoxia-induced arteriole constriction was restored almost fully by inhibiting NOS and partially by inhibiting COX. Hypercapnia induced venule dilation in group N, but did not dilate venules in group H, irrespective of TXA2. NOS inhibition in hypercapnia unexpectedly enhanced venule and arteriole dilation in group H. These responses no longer occurred when NOS and COX were inhibited simultaneously. In conclusion, microvessel reactions to hypoxia and hypercapnia are abnormal in hyperoxia-injured acini, in which NO directly attenuates hypoxia-induced arteriole constriction, whereas COX inhibited by excessive NO impedes hypercapnia-induced microvessel dilation.

Although ventilator-associated pulmonary injury, such as oxygen toxicity or barotrauma, has given rise to critical problems for mechanically ventilated patients with serious lung diseases, mechanical ventilation is an essential procedure to maintain appropriate tissue oxygenation, pH, and Pco2 in patients with respiratory distress (Dreyfuss and Saumon, 1998). In order to reduce the possibility of ventilator-associated pulmonary barotrauma, permissive hypercapnia has been applied as appropriate ventilatory support to acute respiratory distress syndrome (ARDS) (Feihl and Perret, 1994). In spite of the fact that there are some favorable prospective trials reporting that permissive hypercapnia improved mortality in patients with ARDS induced by heterogeneous background disorders (Hickling et al., 1994, Amato et al., 1998), efficacy and indication of this newly developed ventilator strategy are still controversial (Hudson, 1998). Permissive hypercapnia requires higher inspired oxygen concentration to maintain arterial oxygen tension than conventional mechanical ventilation mode and induces systemic hypercapnic acidosis for at least 48 hours (Carvahlo et al., 1997), thus arising the question whether hyperoxia and hypercapnic acidosis would exert a significant influence on sustained inflammation including ARDS.

To assess the effects of exposure of the lung to hyperoxic conditions on reactivity of pulmonary microcirculation to hypoxic stimulation, we measured hypoxia-elicited overall pulmonary pressor changes (HPV) and microvascular diameter changes in intraacinar arterioles, venules, and capillaries in isolated perfused rat lungs exposed to a hyperoxic environment (90% O2). To estimate the importance of vasoactive prostaglandins and nitric oxide (NO) for HPV modification, we examined the roles of constitutive and inducible forms of cyclooxygenase (COX-1 and COX-2) and those of NO synthase (eNOS and iNOS). Indomethacin was used for inhibiting both COX-1 and COX-2, while NS-398 was used as a selective inhibitor of COX-2. Both eNOS and iNOS were suppressed by L-NAME, whereas iNOS alone was inhibited by aminoguanidine. Microvascular diameter was measured with a real-time confocal laser scanning luminescence microscope. We found that (1) exposure to hyperoxia caused overall HPV and arteriolar constriction to be attenuated; (2) the blunted HPV was restored by L-NAME but not by aminoguanidine, indomethacin, or NS-398; and (3) arteriolar constriction was improved by either L-NAME, aminoguanidine, or indomethacin but only slightly by NS-398. In conclusion, attenuation of overall HPV in hyperoxia-exposed lungs is explicable mainly by excessive NO generated via eNOS, while impaired arteriolar constriction is caused by NO yielded by eNOS and iNOS as well as by vasodilating prostaglandin(s) produced by COX-1.

Abstract —To elucidate the differential reactivity of pulmonary microvessels in the acini to hypoxia, excessive CO 2 , and increased H + , we investigated changes in the diameter of precapillary arterioles, postcapillary venules, and capillaries in isolated rat lungs on exposure to normocapnic hypoxia (2% O 2 ), normoxic hypercapnia (15% CO 2 ), and isocapnic acidosis (0.01 mol/L HCl). Microvascular diameters were precisely examined using a real-time confocal laser scanning luminescence microscope coupled to a high-sensitivity camera with an image intensifier. Measurements were made under conditions with and without indomethacin or N ω -nitro- l -arginine methyl ester to assess the importance of vasoactive substances produced by cyclooxygenase (COX) or NO synthase (NOS) as it relates to the reactivity of pulmonary microvessels to physiological stimuli. We found that acute hypoxia contracted precapillary arterioles that had diameters of 20 to 30 μm but did not constrict postcapillary venules of similar size. COX- and NOS-related vasoactive substances did not modulate hypoxia-elicited arteriolar constriction. Hypercapnia induced a distinct venular dilatation closely associated with vasodilators produced by COX but not by NOS. Arterioles were appreciably constricted in isocapnic acidosis when NOS, but not COX, was suppressed, whereas venules showed no constrictive response even when both enzymes were inhibited. Capillaries were neither constricted nor dilated under any experimental conditions. These findings suggest that reactivity to hypoxia, CO 2 , and H + is not qualitatively similar among intra-acinar microvessels, in which COX- and NOS-associated vasoactive substances function differently.

To elucidate the differential role of various adhesion molecules in distorting leukocyte behavior in the microvasculature of hyperoxia-exposed rat lungs, we investigated fluorescein-labeled leukocyte and erythrocyte kinetics in isolated lungs taken from the animals exposed to 90% O2 for 48 h under conditions in which endothelial intercellular adhesion molecule-1 (ICAM-1) and P-selectin were inhibited by appropriate monoclonal antibodies (1A29 for ICAM-1 and ARP2-4 for P-selectin), while leukocyte L-selectin was restrained with fucoidin. Measurements of blood cell kinetics were made by a confocal laser luminescence microscope coupled with a high-speed video camera. In addition, we histologically examined leukocyte accumulation within the alveolar septa and ICAM-1 as well as P-selectin expressions in the lung. We found that P-selectin expression was sparsely enhanced only in arterioles, whereas ICAM-1 was significantly induced in both venules and capillaries. Firm adhesion of leukocytes was not identified in arterioles and venules, whereas leukocyte rolling was evident in both the vessels. Arteriolar rolling was regulated via a P-selectin- and ICAM-1-independent but L-selectin-dependent mechanism, whereas venular rolling was mediated via a P-selectin-independent but ICAM-1- and L-selectin-dependent pathway. Leukocyte sequestration within capillaries was augmented by an ICAM-1-related mechanism. These findings may suggest that, in hyperoxia-exposed lungs, induction of adhesion molecules and their obstacles to leukocyte behavior are qualitatively different among arterioles, venules, and capillaries.

Pulmonary oxygen toxicity (POT) is an important clinical problem that occurs in patients on long-term mechanical ventilation requiring a high inspired-oxygen concentration. Although the histological evidence of POT is pulmonary edema with neutrophil infiltration into the lung parenchyma, the pathogenesis of POT is not fully understood. To elucidate the mechanism of the development of POT, we investigated the effect of hyperoxia (90% O2, 5% CO2) on adhesion molecule expression in cultured human endothelial cells. The level of intercellular adhesion molecule-1 (ICAM-1) expression had increased in hyperoxia-exposed endothelial cells at 48h and at 72h as compared with normoxic control (21% O2, 5% CO2). In contrast, the levels of P-selectin and E-selectin expression were unchanged during hyperoxic exposure. These hyperoxia-induced ICAM-1 expressions were dose dependently attenuated by a protein kinase C inhibitor (H-7). The levels of ICAM-1 mRNA and the numbers of adherent neutrophils were increased at 48h and at 72 h of hyperoxia-exposed endothelial cells. These results suggest that increased ICAM-1 expression in endothelial cells plays an important role in neutrophil accumulation during POT.

This study was designed to examine how leukocyte traffic occurs in the pulmonary microcirculation under physiological shear rates. The leukocyte-endothelium interaction was visualized in perfused rat lungs using an intravital highspeed confocal laser video microscope by injecting fluorescence-tagged isolated leukocytes. In the control lung, transient cessation of leukocyte movement was observed in pulmonary capillaries. The percentage of leukocytes displaying such behavior increased in response to prestimulation by chemoattractants. As a consequence, leukocyte velocity decreased and the density of adherent leukocytes in pulmonary microvessels was markedly elevated. In contrast to observations in the mesenteric microcirculation, a major population of the adherent cells was observed in alveolar capillaries rather than in postcapillary venules. These results suggest that the pulmonary microvascular system is characterized by specific adhesive mechanisms for circulating leukocytes distinct from those previously reported in the mesenteric microcirculation.

To resolve the issue whether the O2-sensing mechanism of pulmonary circulation is impaired in lungs exposed to hyperoxia, we prepared isolated perfused lungs from rats exposed to either a normoxic or hyperoxic environment and estimated hypoxia-elicited pressor changes in the pulmonary arteries as well as diameter changes in microvessels in the acini. To assess the important role of vasodilator prostaglandins as well as nitric oxide (NO) in blunting hypoxic pulmonary vasoconstriction (HPV) in hyperoxia-exposed lungs, we examined the effects of inhibition of constitutive and inducible forms of cyclooxygenase (COX-1 and COX-2) and of constitutive and inducible forms of NO synthase (eNOS and iNOS). Indomethacin was used as the inhibitor of both COX-1 and COX-2, while NS-398 was applied as a selective inhibitor of COX-2. Simultaneous restraint of eNOS and iNOS was attained by N ω-nitro-L-arginine methyl ester (L-NAME); restraint of iNOS was achieved by aminoguanidine. By addition of fluorescein isothiocyanate-dextran to the perfusion circuit, the architecture and diameter of the intraacinar microvessel were precisely determined in terms of a real-time confocal laser luminescence microscope. The results derived therefrom are (1) exposure to hyperoxia causes overall HPV and contractility of precapillary arterioles responding to hypoxia to be attenuated; (2) the attenuated overall HPV was significantly restored by adding L-NAME but not by adding aminoguanidine, indomethacin, or NS-398; and (3) hypoxia-induced constriction of precapillary arterioles was ameliorated by either L-NAME, aminoguanidine, or indomethacin but not by NS-398. The extent of recovery of arteriolar constriction in the presence of L-NAME was greater than that in the presence of aminoguanidine. We concluded that overall HPV would be blunted by the enhanced eNOS expression along the arteries rather than those in the acini, while contractility of intraacinar precapillary arterioles would be impaired by augmentation of eNOS and iNOS as well as COX-1 expression.

This study aimed to examine the behavior of stimulated leukocytes in the pulmonary microcirculation. The leukocyte-endothelium interaction was visualized under physiological shear rates in perfused rat lungs using high-speed confocal laser video microscopy. Leukocytes labeled with carboxyfluorescein were stimulated with cytokine-induced neutrophil chemoattractant (CINC/gro), which caused L-selectin shedding and inverse upregulation of CD18. Neither unstimulated nor stimulated leukocytes exhibited rolling in either pulmonary arterioles or venules, whereas both were sequestered in capillaries. Approximately 50% of stimulated leukocytes showed a transient cessation of movement in pulmonary capillaries. The CINC/gro stimulation, which inhibited leukocyte rolling and adhesion to mesenteric venules, reduced leukocyte velocity and increased leukocytes in pulmonary capillaries. Pretreatment with monoclonal antibodies against intercellular adhesion molecule-1 (ICAM-1) or CD18 attenuated these changes. Confocal microfluorography revealed constitutive expression of ICAM-1 not only in venules but also abundantly in capillary networks. These results suggest that selectin-independent, CD18-ICAM-1-dependent capillary sequestration is one of the major mechanisms by which activated leukocytes accumulate in the lungs.

In a model of acute inflammation, neutrophil adhesion and transendothelial migration have been shown to be mediated through the leukocyte adhesion complex, CD11/CD18, and its endothelial ligand, ICAM-1 (7, 17). This extravasation is considered to occur from postcapillary venules. In the pulmonary microcirculation, however, more than 90% of activated neutrophils were observed to migrate from capillaries rather than postcapillary venules (8, 12), and neutrophil deformability (3–6, 21) and shear stress (2, 10) have been proposed to be major factors influencing neutrophil kinetics. Worthen et al. (22) reported that stimulated neutrophils remain in pulmonary capillaries as a result of decreased cellular deformability within the capillary. Vedder et al. (16) demonstrated that blocking of the CDU/CD 18 glycoprotein adherence complex by the anti-CD 18 monoclonal antibody (mAb) inhibited neutrophil accumulation in the gut but did not inhibit the pulmonary sequestration of neutrophils in ischemia and reperfusion injury. In addition, recent data obtained from studies utilizing P-selectin and ICAM-1 double mutant mice showed that neutrophil emigration into the peritoneum required both adhesion molecules, while emigration into the lung required neither (1). There have, however, been studies demonstrating that anti-CD 18 mAb attenuates lung injury induced by tumor necrosis factor (9), gram-negative sepsis (19) and zymosan-activated plasma (11).

The regulating mechanism of hyperoxia-induced ICAM-1 expression has not been elucidated. We studied the effect of antioxidants, including superoxide dismutase (SOD), catalase and N-acetylcysteine (NAC), on hyperoxia-induced ICAM-1 expression in human pulmonary artery endothelial cells (HPAEC) and human umbilical vein endothelial cells (HUVEC). Cells were cultured to confluence and exposed to either hyperoxic or normoxic gas with or without various kinds of antioxidants. The levels of ICAM-1 expression in the endothelial cells and the concentrations of reduced (GSH) and oxidized glutathione (GSSG) in the media were examined by flow cytometry and by spectrophotometry, respectively. After 48-hour exposure to hyperoxia, ICAM-1 expression was increased (HPAEC; 161 ± 21% and HUVEC; 163 ± 16%) and total glutathione concentration in the media was decreased as compared with normoxia. SOD did not change the GSH and GSSG concentrations in the media. Catalase dose-dependently decreased the supernatant GSSG concentration in both HPAEC and HUVEC, while the GSH concentration was nearly constant. NAC dose-dependently increased the supernatant GSH concentrations in both HPAEC and HUVEC. There was no difference in the supernatant GSSG concentrations between the NAC-treated HPAEC and HUVEC. There was no difference in ICAM-1 expression in either HPAEC or HUVEC with SOD treatment. ICAM-1 expressions in 100 U/ml (236 ± 20%) and 1,000 U/ml (315 ± 36%) of catalase were increased in HPAEC, and that in 1,000 U/ml (440 ± 209%) of catalase was increased in HUVEC. Five and 10 U/ml of NAC decreased ICAM-1 expression in HPAEC (141 ± 26% and 113 ± 11%) and HUVEC (119 ± 23% and 106 ± 7%), respectively. These results suggest that extracellular glutathione may play a role in regulating hyperoxia-induced ICAM-1 expression in HPAEC and HUVEC.

The clinical features of pulmonary tuberculosis associated with acquired immunodeficiency syndrome (AIDS) in Japan were surveyed utilizing questionnaires completed by 48 institutes around the Tokyo metropolitan area. We found 11 Japanese and six foreign patients. The average number of patients per institute was 0.37. The Japanese patients had advanced human immunodeficiency virus (HIV) infection. A middle aged man, with fever and cough, nonspecific chest X-ray infiltrates, decreased lymphocyte counts, and a negative tuberculin skin test was the typical presentation of the Japanese patients. The clinical diagnosis was often difficult. The smear positive rate was high among those from whom smears were obtained, suggesting high communicability. None of the isolates indicated multidrug-resistant tuberculosis at the time of diagnosis. In conclusion, sputum smear and culture remain the keys to diagnosing tuberculosis in patients with AIDS, although the clinical picture may be atypical for pulmonary tuberculosis.