Transcriptome Reveals Cathepsin K in Periodontal Ligament Differentiation.

Periodontal ligaments (PDLs) play an important role in remodeling the alveolar bond and cementum. Characterization of the periodontal tissue transcriptome remains incomplete, and an improved understanding of PDL features could aid in developing new regenerative therapies. Here, we aimed to generate and analyze a large human PDL transcriptome. We obtained PDLs from orthodontic treatment patients, isolated the RNA, and used a vector-capping method to make a complementary DNA library from >20,000 clones. Our results revealed that 58% of the sequences were full length. Furthermore, our analysis showed that genes expressed at the highest frequencies included those for collagen type I, collagen type III, and proteases. We also found 5 genes whose expressions have not been previously reported in human PDL. To access which of the highly expressed genes might be important for PDL cell differentiation, we used real-time polymerase chain reaction to measure their expression in differentiating cells. Among the genes tested, the cysteine protease cathepsin K had the highest upregulation, so we measured its relative expression in several tissues, as well as in osteoclasts, which are known to express high levels of cathepsin K. Our results revealed that PDL cells express cathepsin K at similar levels as osteoclasts, which are both expressed at higher levels than those of the other tissues tested. We also measured cathepsin K protein expression and enzyme activity during cell differentiation and found that both increased during this process. Immunocytochemistry experiments revealed that cathepsin K localizes to the interior of lysosomes. Last, we examined the effect of inhibiting cathepsin K during cell differentiation and found that cathepsin K inhibition stimulated calcified nodule formation and increased the levels of collagen type I and osteocalcin gene expression. Based on these results, cathepsin K seems to regulate collagen fiber accumulation during human PDL cell differentiation into hard tissue-forming cells.

PLAP-1/Asporin Regulates TLR2- and TLR4-induced Inflammatory Responses.

Periodontal ligament-associated protein 1 (PLAP-1)/asporin is an extracellular matrix protein preferentially expressed in periodontal ligaments. PLAP-1/asporin inhibits the cytodifferentiation and mineralization of periodontal ligament cells and has important roles in the maintenance of periodontal tissue homeostasis. However, the involvement of PLAP-1/asporin in inflammatory responses during periodontitis is poorly understood. This study hypothesized that PLAP-1/asporin might affect the pathogenesis of periodontitis by regulating periodontopathic bacteria-induced inflammatory responses. Proinflammatory cytokine expression induced by Toll-like receptor 2 (TLR2) and TLR4 was significantly downregulated when PLAP-1/asporin was overexpressed in periodontal ligament cells. Similarly, recombinant PLAP-1/asporin inhibited TLR2- and TLR4-induced proinflammatory cytokine expression in macrophages. We also confirmed that NF-κB activity induced by TLR2 and TLR4 signaling was suppressed by the addition of recombinant PLAP-1/asporin. Furthermore, IκB kinase α degradation induced by TLR4 was reduced by PLAP-1/asporin. Immunoprecipitation assays demonstrated the binding abilities of PLAP-1/asporin to both TLR2 and TLR4. Taken together, PLAP-1/asporin negatively regulates TLR2- and TLR4-induced inflammatory responses through direct molecular interactions. These findings indicate that PLAP-1/asporin has a defensive role in periodontitis lesions by suppressing pathophysiologic TLR signaling and that the modulating effects of PLAP-1/asporin might be useful for periodontal treatments.

PLAP-1/Asporin Positively Regulates FGF-2 Activity.

PLAP-1 is an extracellular matrix protein that is predominantly expressed in the periodontal ligament within periodontal tissue. It was previously revealed that PLAP-1 negatively regulates bone morphogenetic protein 2 and transforming growth factor ß activity through direct interactions. However, the interaction between PLAP-1 and other growth factors has not been defined. Here, we revealed that PLAP-1 positively regulates the activity of fibroblast growth factor 2 (FGF-2), a critical growth factor in tissue homeostasis and repair. In this study, we isolated mouse embryonic fibroblasts (MEFs) from Plap-1(-/-) mice generated in our laboratory. Interestingly, Plap-1(-/-) MEFs exhibited enhanced responses to bone morphogenetic protein 2 but defective responses to FGF-2, and Plap-1 transfection into Plap-1(-/-) MEFs rescued these defective responses. In addition, binding assays revealed that PLAP-1 promotes FGF-2-FGF receptor 1 (FGFR1) complex formation by direct binding to FGF-2. Immunocytochemistry analyses revealed colocalization of PLAP-1 and FGF-2 in wild-type MEFs and reduced colocalization of FGF-2 and FGFR1 in Plap-1(-/-) MEFs compared with wild-type MEFs. Taken together, PLAP-1 positively regulates FGF-2 activity through a direct interaction. Extracellular matrix-growth factor interactions have considerable effects; thus, this approach may be useful in several regenerative medicine applications.

Clinical characteristics classified by the serum KL-6 level in patients with organizing pneumonia.

BACKGROUND: The serum Krebs von der Lungen-6 (KL-6) level is a useful marker correlated with the severity of various interstitial lung diseases. There have been few reports about the clinical characteristics of organizing pneumonia (OP) associated with the serum KL-6 levels. OBJECTIVE: This study was performed to determine whether the serum KL-6 levels can help determine the optimal treatment for OP. DESIGNS: Patients diagnosed with OP by clinical, radiological and histopathological findings were retrospectively reviewed. The OP patients were classified into two groups based on their serum KL-6 levels: normal KL-6 and high KL-6 groups. The two groups were compared with regard to their clinical and radiological data and therapeutic response one month after the start of treatment. RESULTS: The clinical records of twenty-two patients diagnosed with OP were reviewed. The serum KL-6 level was elevated in 11 of the 22 patients. There were no obvious differences in the clinical data between the two groups, although patients in the normal KL-6 group tended to have a fever. There were no significant differences in the chest X-ray (CXR) score or computed tomography (CT) score between the two groups. The CXR scores were correlated with the serum KL-6 levels. At 1 month after the diagnosis, 11 patients who needed treatment with prednisolone were included in the high KL-6 group. CONCLUSIONS: Patients with normal KL-6 levels showed lower CXR and CT scores. The serum KL-6 level on admission is a useful marker to judge the need for corticosteroid treatment in OP patients.

Octet spin fractions and the proton spin problem.

The relatively small fraction of the spin of the proton carried by its quarks presents a major challenge to our understanding of the strong interaction. Traditional efforts to explore this problem have involved new and imaginative experiments and QCD based studies of the nucleon. We propose a new approach to the problem that exploits recent advances in lattice QCD. In particular, we extract values for the spin carried by the quarks in other members of the baryon octet in order to see whether the suppression observed for the proton is a general property or depends significantly on the baryon structure. We compare these results with the values for the spin fractions calculated within a model that includes the effects of confinement, relativity, gluon exchange currents, and the meson cloud required by chiral symmetry, finding a very satisfactory level of agreement given the precision currently attainable.

Potential bioavailability of phosphorus in citrus orchard soil to Microcystis aeruginosa.

Algal blooms in eutrophic water bodies are controlled by inputs of phosphorus (P) as the growth-limiting nutrient. Runoff particulate P associated with soil from fields often predominates among P fractions. Here, an algal bioassay to investigate the potential bioavailability of particulate P in soil collected from a citrus orchard was conducted. Microcystis aeruginosa was cultured in medium containing soil as the sole source of P. The P in the soil was not notably solubilized after autoclaving. Analyses of chlorophyll-a, suspended solids, particulate organic carbon, and particulate organic nitrogen showed that M. aeruginosa could utilize some of the P present in the soil, perhaps that in particulate form, but this form of P was not sufficient to maintain optimum growth.

Pulmonary involvement of autoimmune pancreatitis.

BACKGROUND: A wide variety of systemic lesions have been seen in patients with autoimmune pancreatitis. The pulmonary involvement of autoimmune pancreatitis was analysed to clarify the clinicopathological features of pulmonary lesions in comparison with pulmonary sarcoidosis. MATERIALS AND METHODS: Nineteen patients had autoimmune pancreatitis and eight had pulmonary sarcoidosis. The symptoms, laboratory data, chest computed tomography, Gallium-67 scintigraphy, pulmonary function testing and bronchoscopy findings, including the histological IgG4-immunostaining and IgG subclasses in the bronchoalveolar lavage in autoimmune pancreatitis, were collected to compare them with pulmonary sarcoidosis. RESULTS: The serum total protein, IgG and IgG4 levels were found to be significantly elevated in comparison with pulmonary sarcoidosis. In autoimmune pancreatitis, 17 patients showed bilateral hilar lymphadenopathy, while eight showed pulmonary nodules on chest computed tomography. Eighteen of 19 patients on Gallium-67 scintigraphy showed accumulation spots in either the hilar or mediastinal lymph nodes. Six patients with pulmonary nodules demonstrated accumulation spots in the corresponding lesions on chest computed tomography. All eight patients with pulmonary sarcoidosis showed accumulation spots in either the hilar or mediastinal lymph nodes. Bronchoalveolar lavage IgG4 in autoimmune pancreatitis showed a significant increase in comparison with pulmonary sarcoidosis. The histological findings obtained by a transbronchial lung biopsy showed the infiltration of lymphocytes and plasma cells in the thickened interstitum and alveoli with IgG4-positive plasma cell infiltration in patients with autoimmune pancreatitis. CONCLUSION: IgG4 in the bronchoalveolar lavage was seen at remarkably increased levels and IgG4-positive plasma cells were identified in the pulmonary lesions of patients with autoimmune pancreatitis.

Central airway stenosis in a patient with autoimmune pancreatitis.

Autoimmune pancreatitis is a unique form of chronic pancreatitis characterised by a high-serum immunoglobulin (Ig)G4 concentration involving various extra pancreatic lesions. A 63-yr-old female with autoimmune pancreatitis complained of cough. Chest computed tomography revealed an irregular stenosis of the central airway, lung hilar and mediastinal lymph node swelling, and a marked thickness of the bronchovascular bundle. Bronchoscopic examination revealed an irregular tracheobronchial stenosis accompanied with an oedematous mucosa and engorged vessels. Lung hilar and mediastinal lymph node swelling, central airway stenosis and bronchoscopic findings remarkably resembled those of sarcoidosis. Bronchial biopsy specimens demonstrated diffuse infiltrations of plasma cells, lymphocytes and eosinophils with fibrosis. Immunostaining showed infiltration of several IgG4-positive plasma cells. The patient was treated with oral prednisolone at 1 mg x kg(-1) x day(-1) for pancreatic lesions. A month later, the lung lesions, including central airway stenosis, lung hilar and mediastinal lymph node swelling, and bronchovascular bundle thickness, had dramatically improved along with improvement of pancreatitis, thus indicating a close association between the two conditions. This is the first report of a patient with autoimmune pancreatitis showing central airway stenosis similar to that of sarcoidosis.

Modulation of mucus production by interleukin-13 receptor alpha2 in the human airway epithelium.

BACKGROUND: IL-13 induces goblet cell hyperplasia and mucus overproduction in airway epithelial cells. IL-13 receptor alpha2 (IL-13Ralpha(2)) has been suggested to act as a 'decoy receptor' in the airway epithelium by inhibiting the IL-13 signal. However, the regulatory mechanisms for mucus production by IL-13Ralpha(2) remain unclear. OBJECTIVE: The aim of this study was to examine the role of IL-13Ralpha(2) in goblet cell hyperplasia and mucus overproduction by IL-13. METHODS: Bronchi were obtained from patients who underwent a lung resection due to lung cancer or benign lung tumours. Normal human bronchial epithelial cells (NHBECs) were isolated and cultured using an air-liquid interface (ALI) method. RESULTS: The number of periodic acid-Schiff's (PAS)-positive cells, goblet cells and MUC5AC-positive cells increased after adding IL-13 into NHBECs. The concentrations of MUC5AC protein in the supernatant and the mRNA expression of MUC5AC significantly increased after adding IL-13, and returned to control levels at 21 days. The mRNA expression of IL-13Ralpha(2) significantly increased at 7 days and then continuously increased up to 21 days. The protein of a soluble form of IL-13Ralpha(2) in the supernatants significantly increased at 14 and 21 days. Anti-IL-13Ralpha(1) antibody and recombinant IL-13Ralpha(2) reduced the number of PAS-positive cells, goblet cells and MUC5AC-positive cells, and MUC5AC mRNA, while the anti-IL-13Ralpha(2) antibody increased the number of these cells and MUC5AC mRNA. The concentration of MUC5AC protein in the supernatant induced by IL-13 was reduced by anti- IL-13Ralpha(1) antibody and recombinant IL-13Ralpha(2). IL-13-induced signal transducer and activator of transcription (STAT) activation was inhibited by anti-IL-13Ralpha(1) antibody and recombinant IL-13Ralpha(2). In contrast, the IL-4-induced mucus production, mucus secretion and STAT activation were not inhibited by recombinant IL-13Ralpha(2). CONCLUSION: The soluble form of IL-13Ralpha(2) may therefore modulate mucus overproduction by IL-13 through the pathway including IL-13Ralpha(1) in NHBECs.

Bronchoscopy-guided radiofrequency ablation as a potential novel therapeutic tool.

The aim of the current study was to assess the safety of bronchoscopy-guided radiofrequency ablation (RFA) and compare the effectiveness between new internal cooled-RFA and standard noncooled-RFA. Normal lungs from sheep were used (n=6). Internal cooled-RFA and standard noncooled-RFA were set to assess the most suitable RFA conditions, such as power output, flow rate and ablation time. Internal cooled-RFA was then applied under the most optimal conditions of power output and flow rate for 15, 30, 60 and 120 s, and two water temperatures either room temperature (RT) water or cold water. Criteria for the most appropriate conditions were set over 15 s of ablation time and 50 degrees C of the tip's temperature. Internal cooled-RFA had no complications. Standard noncooled-RFA was complicated with bronchial bleeding after RFA. On the basis of the histological findings, average temperature and average output, the most appropriate conditions of the cooled-RFA were a power output of 30 W and flow rate of 30 or 40 mL.min(-1). The cooled-RFA using cold water caused a smaller, more discrete, lesion compared with that using RT water. Bronchoscopy-guided internal cooled-radiofrequency ablation was an effective, safe and feasible procedure that could become a potential therapeutic tool in managing lung pathology.

HLA class II DPB1, DQA1, DQB1, and DRB1 genotypic associations with occupational allergic cough to Bunashimeji mushroom.

We previously reported that two-third of workers in a Bunashimeji mushroom (Hypsizigus marmoreus) farm complained of respiratory allergic symptoms, but one-third workers did not suffer from such symptoms even when working for a long period. CD4+ T-helper (Th) cells increased, and Th2/Th1 ratio increased in the allergic workers. To address these immunological backgrounds, we have investigated whether there is any relationship between mushroom allergy and human leukocyte antigen (HLA) class II alleles of DPB1, DQA1, DQB1, and DRB1 by using the polymerase chain reaction-restriction fragment length polymorphism (RFLP) and sequencing-based typing methods. We observed that the allele frequencies of DQA1*0103, DQB1*0601, and DRB1*0803 were significantly higher in the workers having no allergic symptoms than allergic workers (DQA1*0103: 57 vs 25%, DQB1*0601: 49 vs 14%, and DRB1*0803: 29 vs 0%). However, this phenomenon was not seen in workers producing another kind of mushroom, Honshimeji (Lyophyllum aggregatum). The HLA-DRB1*0803 allele alone, the DRB1*0803, DQA1*0103, DQB1*0601 haplotype, or both were negatively associated with allergy to Bunashimeji, and these alleles might be involved in the prevention of Bunashimeji mushroom-specific respiratory allergy.

Hypersensitivity pneumonitis induced by spores of Lyophyllum aggregatum.

OBJECTIVES: Lyophyllum aggregatum (LA) is called Shimeji in Japanese and is eaten commonly as a mushroom. Shimeji mushrooms are cultivated in an indoor environment all year round. This study aimed to clarify the clinical features of hypersensitivity pneumonitis (HP) induced by LA. PATIENTS AND SETTING: Ten patients showed mild respiratory symptoms including dry cough, sputum, and low-grade fever. We tried to characterize the clinical features and the findings using chest high-resolution CT (HRCT), pulmonary function tests (PFTs), and BAL fluid (BALF) tests in patients with HP induced by LA. HP was diagnosed from clinical features, HRCT findings, BALF findings, lung histology, and lymphocyte stimulation tests (LSTs) for LA. RESULTS: Laboratory findings showed mean (+/- SD) elevated levels of C-reactive protein (0.78 +/- 1.3 mg/dL), erythrocyte sedimentation rate (48 +/- 23 mm/h), and gamma-globulin (26.9 +/- 7.6%). PFTs revealed a slight decrease in the percentage diffusing capacity of the lung for carbon monoxide, possibly due to the presence of epithelial granulomas in the alveoli. Although 4 of 10 patients showed normal findings on the chest radiograph (CXR), chest HRCT findings of all patients showed centrilobular small nodules and diffuse ground-glass opacities. The BALF testing revealed an increase in total cell counts, showing predominantly activated T lymphocytes. The CD4/CD8 cell ratio was significantly decreased (0.5 +/- 0.3). The results of the LSTs were positive in seven of seven cases. CONCLUSIONS: Since patients with HP induced by LA typically have mild respiratory symptoms and sometimes normal CXR findings, their conditions might remain undiagnosed. However, the chest HRCT images showed the typical subacute phase of HP.

Rhabdomyolysis triggered by an asthmatic attack in a patient with McArdle disease.

We describe a patient with McArdle disease who developed rhabdomyolysis triggered by a bronchial asthmatic attack. A 64-year-old man had chronic pulmonary emphysema with asthma, and an asthmatic attack led to severe rhabdomyolysis that required continuous hemodiafiltration. After 2 years, a physical examination revealed atrophy of the extremities compared with previous examinations, especially of the intercostal muscles. During that time, he suffered two severe bronchial asthmatic attacks. His serum level of creatinine kinase remained between 4,000 and 7,000 IU/l when he did not suffer from asthmatic attacks and rhabdomyolysis had abated. Therefore, we suspected that his recent muscle atrophy was caused by asthmatic attacks, and discussed the possibility of his respiratory muscle weakness due to McArdle disease in relation to his severe bronchial asthmatic attacks as well as chronic obstructive pulmonary disease.

[A case of renal cell carcinoma complicated with interstitial pneumonitis, complete A-V block and pleural effusion during interferon-alpha therapy].

A 76-year-old man with postoperative renal cell carcinoma accompanied by multiple lung metastasis was being treated with recombinant interferon-alpha. After administration of 3 MU/day on 3 days/week for 1 month, he complained of headache and tinnitus. During continuous treatment for 3 months, he complained of appetite loss, low-grade fever and dyspnea. He was then referred to our Department of Internal Medicine. Electrocardiography indicated a complete A-V block, and chest radiography (CXR) showed a reticular shadow in both lower lung fields and bilateral pleural effusion. Chest computed tomography (CT) indicated subpleural emphysematous changes, multiple nodules, consolidation shadow with ground glass opacity in both lower lobes, and bilateral pleural effusion. The findings in the bronchoalveolar lavage (BAL) fluid included increases in the numbers of lymphocytes and eosinophils. We reached a diagnosis of interferon-alpha-induced pneumonitis on the basis of the patient's clinical course, and the CXR, chest CT and BAL fluid findings. Treatment with methylprednisolone pulse therapy for 3 days and then administration of prednisolone for 1 month resulted in marked improvement in the complete A-V block and interstitial pneumonitis. At day 7 after discontinuation of prednisolone, the serum level of C-reactive protein increased, and CXR showed bilateral pleural effusion. We therefore believe that the pleural effusion was probably also induced by interferon-alpha. Interferon is an effective drug for chronic hepatitis C and malignant diseases. Many complications have been reported during interferon therapy. However, although these complications, such as interstitial pneumonitis, complete A-V block and pleural effusion, have rarely been reported, careful attention is required during interferon therapy in case any appear.

Double cancer (lung and colon cancer) that showed complete remission with irinotecan and cisplatin combined chemotherapy.

We report a rare case of double (colon and lung) cancer which showed complete remission with chemotherapy with irinotecan (CPT-11) and cisplatin (CDDP). The patient was a 67-year-old man who was diagnosed as having double cancer (stage IIIb pulmonary adenocarcinoma and stage 0 [or 1] well-differentiated adenocarcinoma of the ascending colon). Two courses of chemotherapy (CPT-11, 60 mg/m2, days 1 and 8; CDDP, 30 mg/m2, days 1 and 8) were performed. The combination therapy of CPT-11 and CDDP was very effective. In Japan, there have been few published reports describing the use of CPT-11 for the treatment of gastrointestinal cancer. We think that the use of CPT-11 in gastrointestinal cancer is promising.

[Hypersensitivity pneumonitis caused by Lyophyllum aggregatum in two sisters].

We encountered two sisters with hypersensitivity pneumonitis caused by Lyophyllum aggregatum. In Case 1, the patient was a 47-year-old woman who consulted Iiyama Red Cross Hospital because of a non-productive cough. She had worked with Lyophyllum aggregatum cultures for 7 years. In 1996, she complained of a dry cough, and had had a severe cough in November 1998. She was admitted to Iiyama Red Cross Hospital because of dyspnea on effort. Laboratory data revealed a high level of C-reactive protein. Chest radiography and CT scanning showed ground glass opacities in both lower lobes, and centrilobular nodules. Bronchoalveolar lavage fluid revealed a decrease in the CD4/CD8 ratio, and the lymphocyte fraction was very high. Transbronchial lung biopsy revealed lymphocytic granuloma and interstitial thickening, and the lymphocyte stimulation test was positive for Lyophyllum aggregatum. Case 2 occurred in the 51-year-old sister of Patient 1. She had worked with Lyophyllum aggregatum cultures for 10 years. In 1994, she had experienced dyspnea and a dry cough while working. In January 1999, she was admitted to Iiyama Red Cross Hospital because of a dry cough, sputum and chest oppression. Chest radiography and CT scanning revealed ground glass opacities in both lower lobes and centrilobular nodules. The CD4/CD8 ratio in the bronchoalveolar lavage fluid was low, and the lymphocyte fraction conspicuously high. The transbronchial lung biopsy revealed lymphocytic granuloma and interstitial thickening. The lymphocyte stimulation test was negative for Lyophyllum aggregatum. She was discharged and returned to work, but the dry cough and low-grade fever recurred. However, the occupational provocation test is not regarded as positive because chest radiography, laboratory examinations, and a pulmonary function test were not performed. The symptoms of both cases were eliminated only on isolation of the antigen. There has not been any previous report of hypersensitivity pneumonitis caused by Lyophyllum aggregatum.