CREB regulates TNF-α-induced GM-CSF secretion via p38 MAPK in human lung fibroblasts.

BACKGROUND: Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that mediates eosinophilic differentiation, migration and survival, causing respiratory tract inflammation. GM-CSF is also known to be secreted from respiratory tract structural cells. However, the mechanisms of GM-CSF secretion have not been well established. METHODS: Human fetal lung fibroblasts and human primary asthmatic lung fibroblasts were used for the study of tumor necrosis factor alpha (TNF-α)-induced GM-CSF secretion. GM-CSF secretion and mRNA expression were measured by enzyme-linked immunosorbent assay and quantitative real-time reverse transcription polymerase chain reaction, respectively. Knockdown of cAMP response element-binding protein (CREB) in fibroblasts was carried out by using specific small interfering RNAs of CREB. RESULTS: Among respiratory tract structural cells, pulmonary fibroblasts exhibited increased GM-CSF secretion and mRNA expression after stimulation with TNF-α in a concentration-dependent manner. Moreover, a p38 mitogen-activated protein kinase (MAPK) inhibitor controlled TNF-α-induced GM-CSF secretion, and roflumilast and rolipram, inhibitors of phosphodiesterase-4, suppressed TNF-α-induced GM-CSF secretion. Consistent with this, forskolin also completely blocked GM-CSF secretion, and similar results were observed in response to cAMP treatment, suggesting that cAMP signaling suppressed TNF-α-induced GM-CSF secretion in human lung fibroblasts. Furthermore, CREB was phosphorylated through p38 MAPK but not cAMP signaling after TNF-α stimulation, and GM-CSF secretion was inhibited by CREB knockdown. Finally, these effects were also demonstrated in human primary lung fibroblasts in a patient with asthma. CONCLUSIONS: CREB signaled independent of cAMP signaling and was phosphorylated by p38 MAPK following TNF-α stimulation, playing a critical role in GM-CSF secretion in human lung fibroblasts.

Safety and efficacy of high-dose leukocytapheresis in patients with refractory asthma.

OBJECTIVE AND DESIGN: An open-label, non-randomized, single-arm study was performed to investigate the safety and efficacy of high-dose leukocytapheresis (pulse LCAP) for refractory asthma. SUBJECTS: Six patients who fulfilled the ATS workshop criteria for refractory asthma were enrolled and completed this clinical study. TREATMENT: After 4 weeks of observation, pulse LCAP using a large LCAP filter, Cellsorba(®) CS-180S, was performed twice with a 1-week interval at a target dose of 5 L per treatment session. METHODS: The clinical response was assessed by monitoring the peak expiratory flow rate (PEFR) twice a day. The asthma control test (ACT) was used to evaluate the condition of asthma symptoms. The fraction of exhaled nitric oxide (FeNO) as a biomarker for eosinophilic airway inflammation was measured using a chemiluminescence analyzer. RESULTS: PEFR in the morning or the evening and the sum total of the score on the ACT were increased after two consecutive sessions of pulse LCAP. FeNO decreased after pulse LCAP. CONCLUSIONS: The results suggest the efficacy of pulse LCAP for refractory asthma.

Glasgow prognostic score and body mass index predict short-term discontinuation of the antifibrotic agents pirfenidone and nintedanib.

BACKGROUND: The antifibrotic agents pirfenidone and nintedanib have been shown to be effective in patients with idiopathic pulmonary fibrosis (IPF). However, discontinuation of antifibrotic drugs is a major clinical concern because of the lack of alternative treatment options. Therefore, we identified factors that may be useful for predicting the termination of antifibrotic agents. METHODS: We retrospectively recruited 280 IPF patients treated with antifibrotic drugs between 2009 and 2018 from seven regional core hospitals in Gunma prefecture, Japan. RESULTS: At four months, the short-term discontinuation group exhibited a significantly worse prognosis in the pirfenidone group and a poorer prognosis in the nintedanib group compared to that in the continuation group. The discontinuation group of pirfenidone at 4 months exhibited lower albumin and higher C-reactive protein (CRP) levels in the sera compared to the group that continued treatment for more than 4 months. In multivariate analysis, the Glasgow prognostic score (GPS), well known as a predictor of cancer prognosis, which comprises serum CRP and albumin levels, predicted early discontinuation and prognosis in the pirfenidone group, whereas the body mass index (BMI) predicted early discontinuation of nintedanib. A high GPS, with both albumin <3.5 g/dL and CRP >1.0 mg/dL, was associated with a poorer prognosis in the pirfenidone group. CONCLUSION: GPS and BMI were significant factors for short-term pirfenidone and nintedanib discontinuation, respectively. Initial evaluation of GPS and BMI prior to antifibrotic therapy may contribute to less interrupted IPF management, thus leading to better prognostic outcomes in patients with IPF.

Lysophosphatidic acid inhibits CC chemokine ligand 5/RANTES production by blocking IRF-1-mediated gene transcription in human bronchial epithelial cells.

Lysophosphatidic acid (LPA) is a phospholipid mediator that exerts a variety of biological responses through specific G-protein-coupled receptors (LPA(1)-LPA(5) and P2Y5). LPA is thought to be involved in airway inflammation by regulating the expression of anti-inflammatory and proinflammatory genes. Chemokines such as CCL5/RANTES are secreted from airway epithelium and play a key role in allergic airway inflammation. CCL5/RANTES is a chemoattractant for eosinophils, T lymphocytes, and monocytes and seems to exacerbate asthma. We stimulated CCL5/RANTES production in a human bronchial epithelial cell line, BEAS-2B, with IFN-γ and TNF-α. When LPA was added, CCL5/RANTES mRNA expression and protein secretion were inhibited, despite the presence of IFN-γ and TNF-α. The LPA effect was attenuated by Ki16425, a LPA(1)/LPA(3) antagonist, but not by dioctylglycerol pyrophosphate 8:0, an LPA(3) antagonist. Pertussis toxin, the inhibitors for PI3K and Akt also attenuated the inhibitory effect of LPA on CCL5/RANTES secretion. We also identify the transcription factor IFN regulatory factor-1 (IRF-1) as being essential for CCL5/RANTES production. Interestingly, LPA inhibited IFN-γ and TNF-α-induced IRF-1 activation by blocking the binding of IRF-1 to its DNA consensus sequence without changing IRF-1 induction and its nuclear translocation. Ki16425, pertussis toxin, and PI3K inhibitors attenuated the inhibitory effect of LPA on IRF-1 activation. Our results suggest that LPA inhibits IFN-γ- and TNF-α-induced CCL5/RANTES production in BEAS-2B cells by blocking the binding of IRF-1 to the CCL5/RANTES promoter. LPA(1) coupled to G(i) and activation of PI3K is required for this unique effect.

Differential Discontinuation Profiles between Pirfenidone and Nintedanib in Patients with Idiopathic Pulmonary Fibrosis.

Antifibrotic agents have been widely used in patients with idiopathic pulmonary fibrosis (IPF). Long-term continuation of antifibrotic therapy is required for IPF treatment to prevent disease progression. However, antifibrotic treatment has considerable adverse events, and the continuation of treatment is uncertain in many cases. Therefore, we examined and compared the continuity of treatment between pirfenidone and nintedanib in patients with IPF. We retrospectively enrolled 261 consecutive IPF patients who received antifibrotic treatment from six core facilities in Gunma Prefecture from 2009 to 2018. Among them, 77 patients were excluded if the antifibrotic agent was switched or if the observation period was less than a year. In this study, 134 patients treated with pirfenidone and 50 treated with nintedanib were analyzed. There was no significant difference in patient background, discontinuation rate of antifibrotic treatment over time, and survival rate between the two groups. However, the discontinuation rate due to adverse events within one year of antifibrotic treatment was significantly higher in the nintedanib group than in the pirfenidone group (76% vs. 37%, p < 0.001). Furthermore, the discontinuation rate due to adverse events in nintedanib was higher than that of pirfenidone treatment throughout the observation period (70.6% vs. 31.2%, p = 0.016). The pirfenidone group tended to be discontinued due to acute exacerbation or transfer to another facility. The results of this study suggest that better management of adverse events with nintedanib leads to more continuous treatment that prevents disease progression and acute exacerbations, thus improving prognosis in patients with IPF.

PI3K p110beta positively regulates lipopolysaccharide-induced IL-12 production in human macrophages and dendritic cells and JNK1 plays a novel role.

The PI3K family is thought to participate in TLR signaling, and it has been reported to be a negative regulator of TLR-mediated production of IL-12, a key inducer of Th1 responses. However, the role of individual PI3K subtypes in IL-12 production remains obscure. We defined the distinct regulation of LPS-mediated IL-12 production by p110alpha and p110beta catalytic subunits of PI3K in human APCs. We observed that knockdown of PI3K p110beta by small interfering RNA (siRNA) suppressed both LPS-induced IL-12 protein production and mRNA expression in monocyte-derived macrophages and dendritic cells (DCs). Knockdown of PI3K p110alpha by siRNA reduced LPS-induced IL-12 protein production in both cell types. Conversely, knockdown of PI3K p110alpha suppressed LPS-induced IL-12 mRNA expression in monocyte-derived macrophages but minimally affected monocyte-derived DCs. PI3K p110beta siRNA inhibited JNK activation, but not p38 MAPK or ERK activation, stimulated by LPS, while PI3K p110alpha siRNA did not affect LPS-induced JNK, p38 MAPK, or ERK activation in both cell types. Transfection of siRNA against JNK1, JNK2, and both decreased LPS-induced IL-12 production. Furthermore, PI3K p110beta siRNA attenuated LPS-induced JNK1 phosphorylation, while not affecting JNK2 phosphorylation. Our findings indicate that PI3K p110beta positively controls LPS-induced IL-12 production through the JNK1-dependent pathway in human macrophages and DCs.

Protective effect of resolvin E1 on the development of asthmatic airway inflammation.

Resolvin E1 (RvE1) is an anti-inflammatory lipid mediator derived from the omega-3 fatty acid eicosapentaenoic acid (EPA), and strongly acts in the resolution of inflammation. We previously reported that RvE1 dampens airway inflammation and hyperresponsiveness in a murine model of asthma. In the present study, to elucidate the effects of RvE1 on the development of asthmatic airway inflammation, we investigated whether RvE1 acts on different phases of an OVA-sensitized and -challenged mouse model of asthma. RvE1 treatments at the time of either OVA sensitization or at the time of OVA challenge were investigated and compared with RvE1 treatments at the time of both OVA sensitization and challenge. After RvE1 was administered to mice intraperitoneally at the time of both OVA sensitization and challenge, there were decreases in airway eosinophil and lymphocyte recruitment, as well as a reduction in Th2 cytokine and airway hyperresponsiveness. RvE1 treatment at the time of either OVA sensitization or challenge also improved AHR and airway inflammation. Our results suggest that RvE1 acts on several phases of asthmatic inflammation and may have anti-inflammatory effects on various cell types.

JNK1 and JNK2 differently regulate IL-12 production in THP-1 macrophage cells.

Macrophages play a key role in initiating the innate responses to infection by secreting cytokines such as interleukin-12 (IL-12). This study defined the distinct regulation of lipopolysaccharide (LPS)-mediated IL-12 production by c-jun NH(2)-terminal kinase (JNK)1 and JNK2 isoforms in human macrophages. Knockdown of JNK1 and JNK2 by small interference RNA (siRNA) reduced and enhanced LPS-induced IL-12 p40 production in THP-1 macrophage cells, respectively. The simultaneous knockdown of JNK1 and JNK2 augmented LPS-induced IL-12 production as well as a specific JNK inhibitor. In addition, transfection of siRNA against phosphoinositide 3-kinase (PI3K) p110beta attenuated LPS-induced IL-12 production and JNK1 phosphorylation, while not affecting JNK2 phosphorylation. These findings indicate that JNK1- and JNK2-mediated signaling plays a positive and a negative role, respectively, in LPS-induced IL-12 production and PI3K p110beta controls LPS-induced JNK1 activation, not JNK2 activation, resulting in the positive regulation of IL-12 production in THP-1 macrophage cells.

[Effective gemcitabine plus S-1 treatment in elderly non-small cell lung cancer patients with multiple bone metastases].

An 81-year-old man presented with right shoulder pain 10 months after surgery for non-small cell lung cancer (p- T3N0M0, Stage II b). FDG-PET revealed an increased uptake in the multiple bone and mediastinal lymph nodes, suggestive of recurrence of lung cancer. He was given combined chemotherapy of gemcitabine plus S-1 as first-line treatment. This regimen was performed as a phase I trial for an elderly patient with advanced non-small cell lung cancer, and the treatment proved effective. No toxic events were observed due to this therapy, so he was treated as an outpatient for one year. He was considered to have good quality of life.

[A case of cryptogenic organizing pneumonia with eosinophilic infiltration which was difficult to distinguish from nonspecific interstitial pneumonia].

A 57-year-old man presented with shortness of breath of four months duration which had recently become worse. A chest X-ray and computed tomography (CT) showed diffuse ground-glass opacities of the bilateral lower lungs suggesting interstitial pneumonia. The number of eosinophils was increased in the bronchoalveolar lavage fluid (BALF) (52%) and peripheral blood. A histological examination of the specimen obtained by TBLB revealed organized pneumonia with slight infiltration of inflammatory cell. Because the images were not typical of chronic eosinophilic pneumonia, video-assisted thoracic surgery biopsy was performed. The histological findings of the resected specimen showed organizing pneumonia with infiltration of eosinophils in the alveolar walls. He had not taken any medication prior to coming to the hospital and he was negative for medicine-related pneumonia. The oral administration of prednisolone (0.5 mg/kg) improved his symptoms and also CT findings.

[Antigen presenting cells in allergy].

Allergic response is the result of a complex immunologic cascade leading to the dysregulated production of Th2 cytokines. Antigen presenting cells (APCs), mainly dendritic cells (DCs), provide T cells with antigen and costimulatory signals as well as with polarizing signals, such as signals of cytokines, chemokines, and costimulatory molecules. In addition, DCs are also important for the induction of immunological tolerance. In this review, the author discusses the contribution of APCs to allergic responses and new strategies for the treatment of allergic diseases using DC subsets that either deliver signals to modify polarization of T cells or induce tolerance.

Resolvin E1 dampens airway inflammation and hyperresponsiveness in a murine model of asthma.

Resolvin E1 (RvE1; 5S, 12R, 18R-trihydroxyeicosapentaenoic acid) is an anti-inflammatory lipid mediator derived from the omega-3 fatty acid eicosapentaenoic acid (EPA). It has been recently shown that RvE1 is involved in the resolution of inflammation. However, it is not known whether RvE1 is involved in the resolution of asthmatic inflammation. To investigate the anti-inflammatory effect of RvE1 in asthma, a murine model of asthma was studied. After RvE1 was administered to mice intraperitoneally, there were decreases in: airway eosinophil and lymphocyte recruitment, specific Th2 cytokine, IL-13, ovalbumin-specific IgE, and airway hyperresponsiveness (AHR) to inhaled methacholine. Moreover, RvE1-treated mice had significantly lower mucus scores compared to vehicle-treated mice based on the number of goblet cells stained with periodic acid-schiff (PAS). These findings provide evidence that RvE1 is a pivotal counterregulatory signal in allergic inflammation and offer novel multi-pronged therapeutic approaches for human asthma.

Five-antituberculosis Drug-resistance Genes Detection Using Array System.

Detection of resistance to drugs for Mycobacterium tuberculosis takes about two months from the sample collection using culture-based methods. To test a rapid method for detection of resistance for five antituberculosis drugs using DNA microarray and to examine its potential for clinical use, we employed a DNA microarray for detection of seven mutations genes related to resistance of five kinds of antituberculous drugs using Mycobacterium tuberculosis DNA isolated from sputum. The results of microarray analysis were compared with the results of a standard culture method of Lowenstein-jensen drug sensitivity testing system. DNA microarray analysis showed a high sensitivity (>90%) for all five drugs. Specificity of rifampicin and ethambutol were nearly 90%, however specificity of isoniazid (60%) and kanamycin (67%) were not enough. The amount of Mycobacterium tuberculosis DNA required for microarray analysis corresponded to at least 1-9 Acid-Fast Bacilli per 10 fields by carbolfuchsin staining. DNA microarray analysis appears to be useful for estimation of drug resistances, nevertheless its limitations. To minimize misunderstanding, it is necessary to confirm the number of bacilli in the sputum, and culture method is needed for comparison when use the PCR-based array system.

[A case of elderly non-small cell lung cancer effectively treated by combined chemotherapy of gemcitabine and S-1].

The patient was an 86-year-old female who suffered from advanced non-small cell-lung cancer (cT4N3M1, Stage IV). She was treated with combined chemotherapy of gemcitabine and S-1 as first-line therapy. This regimen was performed as a phase I trial for elderly patients with advanced non-small cell lung cancer. The treatment assessment was evaluated as partial response. No toxic events were observed due to this therapy, so she was treated as an outpatient. She was considered to have good quality of life.

[A case of large cell neuroendocrine carcinoma in a patient with sarcoidosis].

A 60-year-old female smoker presented with bloody sputum and back pain. A computed tomographic (CT) scan of the chest revealed a mass lesion in S(1+2) of the left lung and hilarmediastinal lymphadenopathy. TBLB revealed small cell carcinoma. At first, we thought that the patient's clinical staging was c-T4N2M0 IIIB disease. However, it was pointed out she had had hilar-mediastinal lymphadenopathy 7 years previously. Though FDG-PET revealed an increased uptake in the hilar-mediastinal lymph node, she was determined to have surgery. The resected specimen revealed the swollen lymph nodes to be sarcoidosis, accompanying a large cell neuroendocrine carcinoma (LCNEL). She was treated with a combination chemotherapy consisting of cisplatin and VP-16 and radiotherapy concurrently. Chest CT revealed a partial response of the primary site after 3 courses of chemoradiotherapy. Information on concomitant malignancies accompanying sarcoidosis is limited. Although the main types of concomitant malignancy are lung cancer, and lymphoma, there has been no description of LCNEC as a concomitant malignancy. In sarcoidosis patients, clinicians should be alert to the possibility of concomitant malignancy.

Acute pancreatitis in a patient treated with micafungin.

CASE SUMMARY: A 73-year-old man (height, 158.2 cm; weight, 49.8 kg) presented with upper abdominal tenderness after 3 weeks of treatment with 150 mg/d of micafungin (3 mg/kg . d) (Mycamine, Astellas Pharma US Inc., Deerfield, Illinois) intravenously for pulmonary aspergillosis accompanied by [DOSAGE ERROR CORRECTED] pulmonary Mycobacterium avium complex (MAC) infection. Pulmonary aspergillosis was noninvasively diagnosed by a fungus lump in a cavity in the right upper lung field with a high value of 1,3-beta-D-glucan and a positive result for aspergillosis antigen. The patient had a medical history of gastrectomy due to gastric cancer and idiopathic thrombocytopenic purpura (ITP). He had been prescribed 800 mg/d of clarithromycin, 400 mg/dL of rifampicin, and 750 mg/d of ethambutol hydrochloride for pulmonary MAC infection for 2 years and 5 mg/d of prednisolone for ITP for 7 years. No traditional or homeopathic medicine had been received/administered. Laboratory tests at the onset of abdominal pain revealed a white blood cell count of 4300/microL with 51% neutrophils. There was no eosinophilia. Platelet count was 15,100/muL, with normal coagulation. Immunoglobulin G and immunoglobulin M were 1720 and 154 mg/dL, respectively. The patient had no history of allergy, biliary tract disease, hyperlipidemia, or hypercalcemia. He did not report alcohol use. The laboratory findings, magnetic resonance imaging, and upper abdominal tenderness were consistent with acute pancreatitis. After cessation of all drugs, his symptoms improved with bowel rest and parenteral nutrition. His laboratory measurements normalized thereafter. All drugs, except micafungin, were readministered for pulmonary MAC infection and ITP, and itraconazole was administered for pulmonary aspergillosis after the recovery from pancreatitis. During 16 months of follow-up, the pancreatitis did not recur. DISCUSSION: We performed a literature search of all available English-language articles published on MEDLINE between January 1966 and January 2007 using the key terms micafungin (text and indexed terms) and pancreatitis (text and indexed terms). Based on the search of MEDLINE, there have been no reports of acute pancreatitis associated with micafungin. The Naranjo adverse drug reaction (ADR) probability scale was used to assess the probability of micafungin-associated acute pancreatitis. A score of 6 was obtained, indicating a probable ADR from micafungin treatment. CONCLUSION: We report a case of acute pancreatitis probably associated with micafungin use in an elderly patient.

Evaluation of gefitinib efficacy according to body mass index, body surface area, and body weight in patients with EGFR-mutated advanced non-small cell lung cancer.

PURPOSE: In patients with epidermal growth factor receptor (EGFR)-mutated, advanced, non-small cell lung cancer (NSCLC), common gefitinib-sensitive EGFR mutations that predict a greater response to therapy include the exon 19 deletion and L858R point mutation. The objective of this study was to evaluate whether body surface area (BSA), body weight (BW), and body mass index (BMI) affect gefitinib efficacy in such patients. METHODS: The medical charts of 138 consecutive patients with advanced NSCLC harboring sensitive EGFR mutations, who underwent gefitinib treatment, were reviewed. The median BSA and BW were used as cutoff values to evaluate their impact on gefitinib efficacy. BMI was categorized as underweight (<18.5 kg/m2), normal (18.5-25 kg/m2), and overweight (≥25 kg/m2). RESULTS: The median BSA and BW were 1.48 m2 and 53 kg, respectively. The overall response rate, progression-free survival (PFS), and overall survival (OS) were 65.2%, 12.2, and 24.2 months, respectively. There were no significant differences in clinical outcomes according to BSA, BW, or BMI alone. Subgroup analysis based on the mutation type and BSA revealed no significant differences in PFS between the groups; however, the median OS in those with exon 19 deletion combined with low BSA was significantly favorable compared with the other groups. CONCLUSIONS: Gefitinib efficacy in patients with NSCLC harboring sensitive EGFR mutations did not differ according to BSA, BW, and BMI. However, OS was superior in patients with both the exon 19 deletion and low BSA.

DNA microarray genotyping of N-acetyltransferase 2 polymorphism using carbodiimide as the linker for assessment of isoniazid hepatotoxicity.

The antituberculous drug isoniazid (INH) is acetylated by N-acetyltransferase 2 (NAT2), and the frequency of INH-induced hepatotoxicity is determined by the NAT2 genotype. NAT2 genotyping is not done routinely in hospital laboratories because of its difficulty. Use of microarrays for research is becoming common and its expectations of clinical application are increasing. In this study, we attempted to develop an easier method of NAT2 genotyping for clinical use. We devised a novel oligonucleotide-based DNA microarray for NAT2 genotyping using a recently developed technique for attaching oligonucleotide probes to poly carbodiimide-coated glass slides, which achieves a stronger hybridization signal and better specificity than the more widely used aminosilane-coated slides. To assess the validity of this microarray, four clones with NAT2 mutations and DNA from 42 tuberculosis patients were investigated by the microarray method and by restriction fragment length polymorphism analysis. The results of genotyping by these two methods were in agreement. Analysis of the relationship between the NAT2 phenotype determined by the DNA microarray and the risk of INH-induced hepatotoxicity revealed that slow acetylators had a significantly higher risk. These findings suggest that our microarray may be clinically useful for predicting drug reactions to INH.