Variability in sonographic anterior drawer test measurements of the ankle: Experienced versus beginner examiners.

BACKGROUND: This study aimed to clarify the variability in the measurements of stress sonography of the ankle and determine the effects of examiner experience on the measurements. METHODS: Twenty examiners (10 experienced and 10 beginners) were included in the study. Each examiner performed stress ultrasonography on a patient with a chronic anterior talofibular ligament injury and a patient with an intact ligament using the reverse anterior drawer method. Changes in ligament length before versus after stress were determined. The same 20 examiners performed ultrasonography on two other patients with an injured or intact ATFL using the anterior drawer method. The length change values and variance were compared between the groups using t-tests and F-tests. RESULTS: Using the reverse anterior drawer method, the change in the anterior talofibular ligament length was 3.3 mm (range, 2.2-4.8 mm) in the experienced group and 2.7 mm (0.0-4.1 mm) in the beginner group for the ligament injured patient. The length changes for the patient with intact anterior talofibular ligament were 0.5 mm (0.1-0.9 mm) and 0.4 mm (-0.1-1.5 mm) in the experienced and beginner groups, respectively. There were no significant intergroup differences in measurement amount (P = 0.37) or variance (P = 0.72). Similarly, using the anterior drawer method, no significant differences between the groups were found in measurement amount or variance. CONCLUSION: The quantitative evaluation of stress sonography of the ankle was variable regardless of examiner experience or stress method, particularly in patients with an anterior talofibular ligament injury. The amount of variability appeared to be unacceptably large for clinical application. Our study results highlight the need for technical standardization.

TNFR1 Mediated Apoptosis Is Protective against Mycobacterium avium in Mice.

Mycobacterium avium is an intracellular proliferating pathogen that causes chronic refractory respiratory infection. Although apoptosis induced by M. avium has been reported in vitro, the role of apoptosis against M. avium infection in vivo remains unclear. Here, we investigated the role of apoptosis in mouse models of M. avium infection. Tumor necrosis factor receptor-1 knockout mice (TNFR1-KO) andTNFR2-KO micewere used. M. avium (1 × 107 cfu/body) was administered intratracheally to mice. Apoptosis in lungs was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling and lung histology as well as cell death detection kits using BAL fluids. TNFR1-KO mice were susceptible to M. avium infection compared with TNFR2-KO and wild type mice based on the bacterial number and lung histology. Higher numbers of apoptotic cells were detected in the lungs of TNFR2-KO and wild-type mice were compared with TNFR1-KO mice. The inhalation of Z-VAD-FMK deteriorated M. avium infection compared with vehicle-inhaled controls. Overexpression of Iκ-B alpha by adenovirus vector attenuated M. avium infection. Our study showed apoptosis had an important role in innate immunity against M. avium in mice. The induction of apoptosis in M. avium-infected cells might be a new strategy to control M. avium infection.

Identification of Genomic Alterations Acquired During Treatment With EGFR-TKIs in Non-small Cell Lung Cancer.

BACKGROUND/AIM: Patients with non-small cell lung cancer (NSCLC) treated with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) eventually develop resistance to these drugs. Although various mechanisms of such resistance have been identified, the mechanism in many cases remains unknown. MATERIALS AND METHODS: Whole-exome sequencing was performed for tumor tissue from 15 patients with NSCLC who developed EGFR-TKI resistance. Tumor specimens obtained before EGFR-TKI treatment were also analyzed for four patients and normal white blood cell samples for six patients in order to detect genomic alterations that occurred during treatment. RESULTS: The mutational signature and mutational load acquired during EGFR-TKI treatment varied among patients, with common EGFR-TKI resistance mechanisms including the T790M secondary mutation of EGFR and MET amplification being acquired together with many other genomic alterations. Our results provide insight into the mutational landscape acquired during the development of EGFR-TKI resistance in NSCLC.

Acquisition of the T790M resistance mutation during afatinib treatment in EGFR tyrosine kinase inhibitor-naïve patients with non-small cell lung cancer harboring EGFR mutations.

The T790M secondary mutation of the epidermal growth factor receptor (EGFR) gene accounts for 50% to 60% of cases of resistance to the first-generation EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib. The prevalence of T790M in EGFR mutation-positive patients who acquire resistance to the irreversible, second-generation EGFR-TKI afatinib has remained unclear, however. We here determined the frequency of T790M acquisition at diagnosis of progressive disease in patients with EGFR-mutated non-small cell lung cancer (NSCLC) treated with afatinib as first-line EGFR-TKI. Among 56 enrolled patients, 37 individuals underwent molecular analysis at rebiopsy. Of these 37 patients, 16 individuals (43.2%) had acquired T790M, including 11/21 patients (52.4%) with an exon 19 deletion of EGFR and 5/13 patients (38.5%) with L858R. None of three patients with an uncommon EGFR mutation harbored T790M. T790M was detected in 14/29 patients (48.3%) with a partial response to afatinib, 1/4 patients (25%) with stable disease, and 1/4 patients (25%) with progressive disease as the best response. Median progression-free survival after initiation of afatinib treatment was significantly (P = 0.043) longer in patients who acquired T790M (11.9 months; 95% confidence interval, 8.7-15.1) than in those who did not (4.5 months; 95% confidence interval, 2.0-7.0). Together, our results show that EGFR-mutated NSCLC patients treated with afatinib as first-line EGFR-TKI acquire T790M at the time of progression at a frequency similar to that for patients treated with gefitinib or erlotinib. They further underline the importance of rebiopsy for detection of T790M in afatinib-treated patients.

Critical role of tumor necrosis factor receptor 1 in the pathogenesis of pulmonary emphysema in mice.

COPD is a major cause of chronic morbidity and mortality throughout the world. Although tumor necrosis factor-α (TNF-α) has a critical role in the development of COPD, the role of different TNF receptors (TNFRs) in pulmonary emphysema has not been resolved. We aimed to clarify the role of TNFRs in the development of pulmonary emphysema. TNF-α transgenic mice, a murine model of COPD in which the mice spontaneously develop emphysema with a large increase in lung volume and pulmonary hypertension, were crossed with either TNFR1-deficient mice or TNFR2-deficient mice. After 6 months, the gross appearance of the lung, lung histology, and pulmonary and cardiac physiology were determined. In addition, the relationship between apoptosis and emphysema was investigated. Pulmonary emphysema-like changes disappeared with deletion of TNFR1. However, slight improvements were attained with deletion of TNFR2. Apoptotic cells in the interstitium of the lung were observed in TNF-α transgenic mice. The apoptotic signals through TNFR1 appear critical for the pathogenesis of pulmonary emphysema. In contrast, the inflammatory process has a less important role for the development of emphysema.

The clinical efficacy and safety of micafungin-itraconazole combination therapy in patients with pulmonary aspergilloma.

In spite of recent advances in chemotherapy, the treatment of pulmonary aspergilloma remains unsatisfactory. To evaluate the clinical efficacy and safety of combination therapy for pulmonary aspergilloma, we conducted a multi-institutional prospective study using micafungin (MCFG) and itraconazole (ITCZ). Adult patients who fulfilled the criteria for pulmonary aspergilloma were enrolled in this study. After patient consent had been obtained, intravenous MCFG 150 mg/day and an oral capsule of ITCZ 200 mg/day were administered for at least 1 month. The primary endpoint was the response assessed using an algorithm that incorporated the levels of improvement by evaluating clinical symptoms and signs, mycological and serological tests, and diagnostic imaging. A total of 17 patients were enrolled from three facilities. The response rate to the combination therapy was 58.8% (10/17). The long-term control in the group of patients who responded to treatment was better than that in the group of patients who did not respond. Adverse events occurred in 6 of the 17 patients (35.3%), but there were no severe adverse events. MCFG-ITCZ combination therapy appeared to be relatively safe and effective in patients with pulmonary aspergilloma.

The clinical efficacy and safety of a fluoroquinolone-containing regimen for pulmonary MAC disease.

Despite recent advances in chemotherapy, the treatment of pulmonary Mycobacterium avium complex (MAC) disease remains unsatisfactory. Judging from its MIC, fluoroquinolones including gatifloxacin (GFLX) are expected to demonstrate efficacy against MAC disease. However, there have been few clinical studies using fluoroquinolones. Therefore, a prospective study to evaluate the clinical efficacy and safety of a fluoroquinolone-containing regimen for the treatment of pulmonary MAC disease was conducted. In this trial, patients with pulmonary MAC disease received protocol-guided combined chemotherapy with rifampin (RFP) and ethambutol (EB) plus either GFLX or clarithromycin (CAM). Adult patients who fulfilled the criteria of the ATS definition of pulmonary MAC disease were enrolled in this study. The patients provided their informed consent, and treatments were administered for 1 year. Of 27 patients enrolled from three facilities, 14 patients were treated with the CAM-containing regimen and 13 patients were treated with the GFLX-containing regime. Four patients did not complete the 1-year treatment because of adverse events. Nine patients (64.3%) in the CAM group and 11 patients (84.6%) in the GFLX group achieved eradication of pathogens. Adverse events were observed more frequently in the GFLX group than in the CAM group. However, there were no severe adverse events in either group. The long-term results showed a similar relapse rate between the CAM and GFLX groups. The fluoroquinolone-containing regimen demonstrated both high efficacy and relative safety for pulmonary MAC disease that was similar to that of the CAM-containing regimen, which is considered to be the standard regimen.

Clinical efficacy and safety of cefepime in febrile neutropenic patients with lung cancer.

Fever often occurs along with chemotherapy-induced neutropenia. This condition is referred to as febrile neutropenia (FN). Excellent guidelines for FN treatment have recently been published; however, there has so far been insufficient research concerning FN associated with solid tumors, especially in Japan. A multi-institution prospective study of cefepime for the treatment of FN in lung cancer patients was conducted. The objective of this study was to determine the efficacy and safety of cefepime for FN in lung cancer patients. Cefepime (2 g x 2/day) was administered to patients with FN after treatment for lung cancer. The therapeutic response rate, the effect of the drug on pathogen populations, and the incidence of adverse effects were statistically analyzed. Twenty-one patients with FN were registered for this study. One case was excluded because of protocol violation; therefore, a total of 20 cases were analyzed. Three days after the administration of cefepime, improvement was evident in 15 cases. The response rate was 75%, 95% CI: 53.1-88.8. After 7 days, 17 patients experienced improvement in their condition (85%, 95% CI: 64.0-94.8). Carbapenem was eventually substituted for cefepime in three cases, and all cases finally displayed improvement. There was no mortality. Pathogens for FN were detected in three cases and they disappeared in one case. Four patients experienced adverse side effects, including skin eruption, serum bilirubin elevation, neutrophil depletion, and anterior chest pain. There were no severe adverse events. In this study, cefepime demonstrated a high degree of clinical efficacy and safety in the treatment of FN. Empiric monotherapy using cefepime is a recommended regimen for FN in patients with lung cancer in Japan.

A case of pulmonary epithelioid hemangioendothelioma surviving 10 years without treatment.

A 19-year-old woman was referred to our hospital because of bilateral multiple nodular shadows on the chest radiograph. She complained of no symptoms. The pulmonary lesions were diagnosed pathologically as epithelioid hemangioendothelioma. She has been followed without treatment for more than 10 years. Among all lesions, only two pulmonary nodules enlarged slightly, and it is interesting that one showed significant uptake in a fluorodeoxyglucose positron emission tomography (FDG-PET) scan. The current case suggests the clinical usefulness of an FDG-PET scan in a pulmonary epithelioid hemangioendothelioma (PEH) patient.

[A case of secondary pulmonary alveolar proteinosis associated with myelodysplastic syndrome, complicated with disseminated M. abscessus infection].

A 27-year-old man was admitted to our hospital complaining of a persistent high fever since August 2007. Chest radiography showed infiltration shadows in the right lower lung field. Chest CT revealed scattered small nodular shadows and patchy consolidations in the right lower lobe. He was diagnosed as secondary pulmonary alveolar proteinosis (sPAP) associated with myelodysplastic syndrome (MDS), confirmed by video-assisted thoracic surgery (VATS) and bone marrow aspiration. Sera were negative for anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibody. He developed a subcutaneous abscess and meningitis caused by M. absessus after VATS. After a long-course of antibiotic therapy, an allogenic peripheral blood stem cell transplantation was performed. But he died of graft versus host disease and M. abscessus sepsis 87 days after transplantation.

TNF receptor 1 and 2 contribute in different ways to resistance to Legionella pneumophila-induced mortality in mice.

Legionella pneumophila is one of the most important pathogens which cause community-acquired pneumonia. Although TNF-alpha is considered to play an important role in response to bacteria, the role of the TNF-alpha receptor on L. pneumophila infection remains to be elucidated. To investigate this, we infected TNF receptor deficient mice with L. pneumophila. L. pneumophila was inoculated intranasally into TNF receptor (TNFR)-1-knock-out mice or TNFR2-knock-out mice. The mortality rate, histology of the lung, bacterial growth in the lung, and bronchoalveolar lavage (BAL) fluids were investigated. The bacterial growth of L. pneumophila in the macrophages was also studied. Almost all the mice survived after an intranasal inoculation of 1x10(6)CFU/head of L. pneumophila, but more than 90% mice were killed after inoculation of 1x10(8)CFU/head of L. pneumophila. In the case of TNFR1-knock-out mice and TNFR2-knock-out mice, a high mortality rate was observed after inoculation of 1x10(7)CFU/head of L. pneumophila in comparison to wild-type mice. The lung histology from both the TNFR1-knock-out mice documented severe lung injury at day 3 after inoculation. The clearance of L. pneumophila in the lung of the TNFR1-knock-out mice was slower than those from both the TNFR2-knock-out mice and the wild-type mice. Moreover, L. pneumophila growth in the peritoneal macrophages from the TNFR1-knock-out mice was observed. Interestingly, a lack of neutrophils accumulation in the BAL fluids and a dysregulation of cytokines (IFN-gamma, interleukin-12, and TNF-alpha) were observed in the TNFR1-knock-out mice. On the contrary, large accumulation of neutrophils in BAL fluids was observed in TNFR2-knock-out mice. These data suggested that a TNFR1 deficiency led to a compromise of the innate immunity against L. pneumophila, while a TNFR2 deficiency induced an excessive inflammatory response and resulted in death. The present study confirmed that TNFR1 and TNFR2 play a crucial, but different role in the control of L. pneumophila-induced mortality.

Biphasic effects of free radical scavengers against bleomycin-induced pulmonary fibrosis.

Oxidative stress plays a critical role in the development of pulmonary fibrosis. However, the effects of treatment with anti-oxidant agents against pulmonary fibrosis have not yet been thoroughly investigated. In this study, the effect of MCI-186, a novel free radical scavenger, on bleomycin-induced pulmonary fibrosis was investigated. Bleomycin (0.05units/mouse) was administered intratracheally into C57Bl/6 mice. MCI-186 was given to bleomycin-treated mice intraperitoneally from (i) day -3 to day 7, or from (ii) day 10 to day 28 after bleomycin administration in successive days. At 28 days after bleomycin administration, pulmonary fibrosis was then assessed by lung histology and hydroxyproline. MCI-186 inhibited H(2)O(2)-induced DNA damage in bronchial epithelium in vitro. MCI-186 decreased the lipid peroxide content, a marker for DNA damage, in the lung and reduced 8-OHdG positive cells in the lung in vivo. During the early period (day -3 to day 7) administration, MCI-186 partially attenuated bleomycin-induced pulmonary fibrosis. However, during the late period (day 10 to day 28) MCI-186 exacerbated pulmonary fibrosis, based on the histology and hydroxyproline content. In this condition, MCI-186 in the late period decreased the number of apoptosis cells induced by bleomycin, and therefore it might contribute to the deterioration of pulmonary fibrosis. These data indicate that MCI-186, radical scavenger, has a biphasic effect on bleomycin-induced pulmonary fibrosis in mice. Careful attention should be paid before clinical application of new remedies for pulmonary fibrosis.

Attenuation of pulmonary hypertension, but not emphysematous change, by breeding emphysema model mice at sea level.

Tumor necrosis factor (TNF)-alpha is a key pro-inflammatory cytokine, thought to be important in the pathogenesis of pulmonary emphysema. TNF-alpha overexpression in the lung leads to the phenotypic features of pulmonary emphysema, pulmonary hypertension, and right ventricular hypertrophy in mice bred in Denver, 5240 feet/1600 m of altitude. This study hypothesized that the altitude could affect the development of pulmonary emphysema as well as pulmonary hypertension. To investigate the effect of the altitude, TNF-alpha transgenic mice were bred at sea level, Fukuoka, Japan. The pulmonary physiology and histology demonstrated similar development of pulmonary emphysema, compared to the mice bred in Denver. With respect to pulmonary hypertension, right ventricular hypertrophy was attenuated. Interestingly, mortality rate was significant lower in the mice bred at sea level. In contrast with the results in Denver, a significant decrease of vascular endothelial growth factor (VEGF) and its receptors expression was not found. From these data, we consider that the altitude affects development of pulmonary hypertension through the expression of VEGF and its receptors. In contrast, the effect of altitude was not clear regarding the development of pulmonary emphysema.

The role of collagenases in experimental pulmonary fibrosis.

Matrix metalloproteinases (MMPs) expression plays a critical role in extracellular matrix deposition. Although several pieces of evidence have so far indicated that gelatinase contributes to the development of pulmonary fibrosis, the role of collagenase remains uncertain. In this study, we attempted to determine the role of collagenase using a bleomycin-induced pulmonary fibrosis model. Bleomycin was instilled into mice intratracheally. Bronchoalveolar lavage fluid (BAL) specimens were analyzed for gelatin and casein zymography, as well as by immunoblotting. The histology of the lungs and hydroxyproline contents were also assessed. MMPs inhibitor, CGS27023A, was simultaneously orally administered. Collagenases were induced in BAL fluids after bleomycin administration based on the data of zymography and immunohistochemistry. The co-administration of MMPs inhibitor, CGS27023A, with bleomycin resulted in worsening pulmonary fibrosis with inhibition of collagenase. The worsening of pulmonary fibrosis was mainly induced by CGS27023A administration in the late phase of bleomycin-induced pulmonary fibrosis development, but not in the early phase. The present data indicated that collagenase plays an anti-fibrotic role in the bleomycin-induced pulmonary fibrosis model. Collagenase has a greater effect on fibrosis phase than inflammatory phase in the bleomycin-induced pulmonary fibrosis in the mice.

Successful treatment of refractory chronic necrotizing pulmonary aspergillosis with micafungin.

A 63-year-old man was admitted to our hospital because he complained of fever and productive cough; this was associated with cavitary infiltrates on his chest X-ray. Although several antibiotics were given, his symptoms did not improve. Bronchofiberscope investigation yielded Aspergillus fumigatus; thus, he was diagnosed with chronic necrotizing pulmonary aspergillosis. Itraconazole, 200 mg/day, was given, and his symptoms and infiltrates on chest X-ray gradually improved. After 2 months of treatment, new infiltrates appeared on a chest X-ray. Antibacterial agents had also shown no effect, and voriconazole was substituted for itraconazole. However, the infiltrates progressed in spite of the voriconazole administration. We added micafungin to the voriconazole treatment. Both his symptoms and the infiltrates on chest X-rays improved. Because voriconazole is thought to be the most effective agent against Aspergillus spp., it is difficult to treat cases that are refractory to voriconazole. The treatment of this case provides invaluable information on how to treat pulmonary aspergillosis related to diseases other than hematologic malignancies.

Doxycycline attenuated lung injury by its biological effect apart from its antimicrobial function.

Antibiotics can have a biological effect apart from their anti-bacterial effect. We hypothesized that doxycycline could attenuate acute lung injury through its biological effect. Lipopolysaccharide or doxycycline-resistant Streptococcus pneumoniae was administered intratracheally into mice with the co-administration of doxycycline. Thereafter, the lung pathology, intraalveolar inflammatory cells, bacterial number, and matrix metalloproteinases were investigated. Matrix metalloproteinases, neutrophil migration, and alveolar destruction were induced by lipopolysaccharide. Doxycycline was thus found to improve all of these symptoms. In addition, an inhibitor of matrix metalloproteinases, CGS27023A, attenuated lipopolysaccharide-induced lung injury. Doxycycline also attenuated the lung injury induced by doxycycline-resistant S. pneumoniae and improved the mortality rate although the bacterial number in the lung did not change. Our data indicated that doxycycline could attenuate acute lung injury through a biological effect that was different from its antibiotic effect.

[A case of pulmonary nocardiosis diagnosed five years after hemoptysis].

A 73-year-old man complaining of bloody sputum was admitted to our hospital in August 2000. His giant left lower lung field bulla had been removed in 1997, at which time atelectasis in left S10 was pointed out. Production of bloody sputum was stopped by the emergency bronchial artery embolization, and Nocardia species was found in the sputum. Because of both spontaneous disappearance of Nocardia species and no evidence of Nocardia infection, he was followed carefully by chest radiography every few months. Consolidation appeared in the left lower lung field and right upper lung field in 2005. Nocardia asteroides was frequently obtained from his sputa and lavage fluid under bronchoscopy. Therefore, we diagnosed pulmonary nocardiosis. Oral cotrimoxazole (Trimetoprim 15 mg/kg) was started, the dosage was halved because of adverse effects. Six months of treatment with cotrimoxazole resulted in improvement of the Nocardiosis.

Doxycycline attenuated pulmonary fibrosis induced by bleomycin in mice.

The administration of doxycycline prior to bleomycin in mice attenuated pulmonary fibrosis. Bronchoalveolar neutrophil influx and gelatinase activity, but not caseinolytic activity, were attenuated by doxycycline. Established fibrosis was not affected by doxycycline. Thus, doxycycline might be useful for slowing down pulmonary fibrosis by biological activity other than antibacterial activity.

Serum surfactant protein D is increased in acute and chronic inflammation in mice.

Surfactant protein A (SP-A) and surfactant protein D (SP-D) are important components of innate immunity that can modify the inflammatory response. However, alterations and regulation of SP-A and SP-D in acute and chronic inflammation are not well defined. In addition, serum SP-D may serve as a biomarker of lung inflammation. We determined the expression of SP-A and SP-D in murine models. To study acute inflammation, we instilled bleomycin intrabronchially. To study chronic lung inflammation, we used a transgenic mouse that overexpresses tumor necrosis factor (TNF)-alpha under the control of the SP-C promoter. These mice have a chronic mononuclear cell infiltration, airspace enlargement, pulmonary hypertension, and focal pulmonary fibrosis. In acute inflammation model, levels of mRNA for all surfactant proteins were reduced after bleomycin administration. However, serum SP-D was increased from days 7 to 28 after instillation. In chronic inflammation model, SP-D mRNA expression was increased, whereas the expression of SP-A, SP-B and SP-C was reduced. Both serum and lung SP-D concentrations were increased in chronic lung inflammation. These data clarified profile of SP-A and SP-D in acute and chronic inflammation and indicated that serum SP-D can serve as a biomarker of lung inflammation in both acute and chronic lung injury in mice.

Serum CC-10 in inflammatory lung diseases.

BACKGROUND: Although Clara cell secretory protein (CC-10) has been ascribed an anti-inflammatory role in lung diseases, its precise role remains unclear. OBJECTIVE: To further our understanding of the role of CC-10 in inflammatory lung diseases, CC-10 protein levels were measured. METHODS: Sera or bronchoalveolar lavage (BAL) fluids were collected from patients with different inflammatory lung diseases including bronchial asthma, chronic obstructive lung disease (COPD), sarcoidosis, idiopathic interstitial pneumonia (IIP), chronic eosinophilic pneumonia (CEP), pneumonia and lung cancer. Serum CC-10 concentrations were measured by enzyme-linked immunosorbent assay using urinary protein-1 antibody. Then, the relationships between CC-10 concentrations and lung diseases were investigated. Immunohistochemistry was performed using lung biopsy samples. RESULTS: Increased serum CC-10 levels were recognized in IIP patients, while CC-10 levels were decreased in bronchial asthma patients and CEP patients. Immunohistochemistry revealed an aberrant expression in areas of fibrosis in IIP patients. Serum CC-10 concentrations were not associated with severity among IIP, COPD, and sarcoidosis. In contrast, serum CC-10 concentrations were correlated with FEV(1)/FVC in bronchial asthma patients. CONCLUSIONS: Although the number of patients was quite limited, these data provide new insights into the role of CC-10 in lung diseases, and the possibility that the CC-10 concentration in serum could be a new marker indicating the severity of bronchial asthma.