A Novel Missense Mutation of DKC1 In Dyskeratosis Congenita With Pulmonary Fibrosis.

Dyskeratosis congenita (DC) is a rare inherited multisystem disorder caused by mutations in seven genes involved in telomere biology, with approximately 20% of cases having pulmonary complications. DKC1 mutations exhibit a severe disease phenotype of DC that develops in early childhood. Here, we report a unique case of DC with pulmonary fibrosis diagnosed at the age of 46. A novel missense mutation(p.Arg65Lys) of DKC1 was detected, and predicted to show a weak mutagenic effect. In spite of the steroid and immunosuppressive treatment, he died of an acute exacerbation seven months after the initial visit. This case suggests that mutation subtypes can cause heterogeneity in DC and pulmonary fibrosis.

Carcinoembryonic antigen-related cell adhesion molecules as surrogate markers for EGFR inhibitor sensitivity in human lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LADCA) patients with epidermal growth factor receptor (EGFR) mutations are in general associated with relatively high clinical response rate to EGFR-tyrosine kinase inhibitors (TKIs) but not all responded to TKI. It has therefore become important to identify the additional surrogate markers regarding EGFR-TKI sensitivity. METHODS: We first examined the effects of EGFR-TKIs, gefitinib and erlotinib, upon cell proliferation of lung adenocarcinoma cell lines. We then evaluated the gene profiles related to EGFR-TKI sensitivity using a microarray analysis. Results of microarray analysis led us to focus on carcinoembryonic antigen-related cell adhesion molecule (CEACAM) family, CEACAM 3, 5, 6, 7, and 19, as potential further surrogate markers of EGFR-TKI sensitivity. We then examined the correlation between the status of CEACAM 3, 5, 6, 7, and 19 immunoreactivity in LADCA and clinicopathological parameters of individual cases. RESULTS: In the cases with EGFR mutations, the status of all CEACAMs examined was significantly higher than that in EGFR wild-type patients, but there were no significant differences in the status of CEACAMs between TKI responder and nonresponder among 22 patients who received gefitinib therapy. However, among 115 EGFR mutation-negative LADCA patients, both CEACAM6 and CEACAM3 were significantly associated with adverse clinical outcome (CEACAM6) and better clinical outcome (CEACAM3). CONCLUSION: CEACAMs examined in this study could be related to the presence of EGFR mutation in adenocarcinoma cells but not represent the effective surrogate marker of EGFR-TKI in LADCA patients. However, immunohistochemical evaluation of CEACAM3/6 in LADCA patients could provide important information on their clinical outcome.

First Report of Guinea Grass Smut Caused by Conidiosporomyces ayresii in Japan.

Guinea grass (Panicum maximum Jacq.) is an important C-4 perennial plant that grows in southern Japan. In March 2010, a smut disease was found in grass that is cultivated in the Ishigaki Islands, Okinawa, in southernmost Japan. Spikelets of susceptible cultivars were swollen and filled with gray spore masses and seed production was substantially reduced. Two single-spore isolates of a smut fungus were obtained from infected spikelets and deposited at the NIAS Genebank, Japan as MAFF511519 and 511520. The 28S rDNA sequences of the isolates were analyzed as described by Boekhout et al. (1). The 28S rDNA sequence (GenBank Accession No. AB647346) of isolate MAFF511519 matched that of Conidiosporomyces ayresii (GenBank Accession No. AY819017) isolated from P. maximum with 99.8% similarity. Spores were pale brown to brown, globose to subglobose, verrucose, and 14 to 16 × 15 to 18 µm in diameter with relatively thick walls of 2 to 3 µm. With scanning electron microscopy, warts appeared dense and short with pointed tips. Spores germinated under wet conditions and produced masses of basidiospores. Basidiospores were aseptate, long, cylindrical, straight to slightly curved, 20 to 37 × 2 to 3 µm, and often germinated into Y-shaped conidia. This description matches previous descriptions (3) of C. ayresii (Berk.) Vánky (Tilletia ayresii Berk.) of the smut pathogen of guinea grass (2). The smut fungus was identified as C. ayresii on the basis of morphology and molecular phylogenetic analysis. To produce inoculum, the isolates were grown on potato dextrose agar at 25°C in the dark for 7 days. Two plants of cv. Ryukyu 5-gou with half-flowering heads were grown in a greenhouse for approximately 1 month and then inoculated by atomizing them with conidial suspensions of each isolate (106 conidia/ml). A plant sprayed with sterilized distilled water served as the control. Inoculated heads were covered with plastic bags for 48 h at 25°C. After 30 days, all inoculated plants were symptomatic with swollen spikelets releasing gray spores. Controls remained asymptomatic. The smut fungus was reisolated from released gray spores, confirming Koch's postulates. To our knowledge, this is the first report of smut caused by C. ayresii on guinea grass in Japan. References: (1) T. Boekhout et al. Stud. Mycol. 38:175, 1995. (2) J. M. Lenné and P. Trutmann. Diseases of Tropical Pasture Plants. CAB International, Wallingford, UK, 1994. (3) K. Vánky and R. Bauer. Mycotaxon 43:427, 1992.

Rapid improvement of osseous sarcoidosis after the treatment of pulmonary aspergillosis by itraconazole.

Osseous sarcoidosis is relatively uncommon, and treatment with corticosteroids is not always effective. Moreover, patients with an advanced stage of pulmonary sarcoidosis are sometimes infected with aspergillus in the cavities of the pulmonary lesions, and long-term use of corticosteroids should be prohibited to prevent the development of fatal invasive pulmonary aspergillosis. Here, we described a unique case of osseous sarcoidosis with pulmonary aspergillosis, showing a rapid improvement of the osseous symptoms just after the administration of the antifungal agent, itraconazole. Itraconazole is likely to become a candidate among new therapeutic agents for osseous sarcoidosis.

Mycobacterial hypersensitivity pneumonitis requires TLR9-MyD88 in lung CD11b+ CD11c+ cells.

Mycobacteria are among the most common causes of hypersensitivity pneumonitis (HP), but controversy persists with regard to the involvement of the infectious potency of the organism in mycobacterial HP (hot tub lung). This study aimed to establish a mouse model of hot tub lung to clarify its pathophysiology. Mice were exposed intranasally to formalin-killed Mycobacterium avium from a patient with hot tub lung (HP strain) or chronic pulmonary infection (non-HP strain), and bronchoalveolar lavage fluids and lung tissues were evaluated for allergic inflammation. Dead M. avium HP strain, but not non-HP strain, elicited marked HP-like pulmonary inflammation in wild-type mice. Although the inflammation was induced in mice lacking CD4 or CD8, the induction of HP-like responses was prevented in mice lacking myeloid differentiation factor (MyD)88 or Toll-like receptor (TLR)9. Cultured lung CD11c+ cells responded to M. avium in a TLR9-dependent manner, and reconstitution of TLR9-/- mice with lung CD11c+ cells from wild-type mice restored the inflammatory responses. Further investigation revealed that pulmonary exposure to M. avium HP strain increased the number of lung CD11b+ CD11c+ cells (dendritic cells) through TLR9 signalling. Our results provide evidence that hot tub lung develops via the mycobacterial engagement of TLR9-MyD88 signalling in lung CD11b+ dendritic cells independent of the mycobacterial infectious capacity.

Massive pleural effusion and bronchopleural fistula in Wegener's granulomatosis.

Wegener's granulomatosis (WG) is characterized by systemic granulomatous necrotizing vasculitis, primarily affecting the respiratory tract and kidneys. We describe a rare case in a 28-year-old woman with WG, presenting with a massive lateral pleural effusion, accompanied by an aseptic bronchopleural fistula formed during immunosuppressive treatment. Although any organ can be involved in WG, only left pleuritis and a purpuric lesion on the neck were detected in this case. The pleural effusion and bronchopleural fistula resolved following immunosuppressive treatment for six months. Thus, WG should be considered in the differential diagnosis of a massive pleural effusion, and fistula formation is a possible complication of treatment. Moreover, immunosuppressive treatment was sufficient to resolve the massive pleural effusion and fistula formation without infection (120 words).

Pirfenidone in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease without proven effective therapy. A multicentre, double-blind, placebo-controlled, randomised phase III clinical trial was conducted in Japanese patients with well-defined IPF to determine the efficacy and safety of pirfenidone, a novel antifibrotic oral agent, over 52 weeks. Of 275 patients randomised (high-dose, 1,800 mg x day(-1); low-dose, 1,200 mg x day(-1); or placebo groups in the ratio 2:1:2), 267 patients were evaluated for the efficacy of pirfenidone. Prior to unblinding, the primary end-point was revised; the change in vital capacity (VC) was assessed at week 52. Secondary end-points included the progression-free survival (PFS) time. Significant differences were observed in VC decline (primary end-point) between the placebo group (-0.16 L) and the high-dose group (-0.09 L) (p = 0.0416); differences between the two groups (p = 0.0280) were also observed in the PFS (the secondary end-point). Although photosensitivity, a well-established side-effect of pirfenidone, was the major adverse event in this study, it was mild in severity in most of the patients. Pirfenidone was relatively well tolerated in patients with IPF. Treatment with pirfenidone may decrease the rate of decline in VC and may increase the PFS time over 52 weeks. Additional studies are needed to confirm these findings.

Association between mycobacterial genotypes and disease progression in Mycobacterium avium pulmonary infection.

BACKGROUND: Non-tuberculous mycobacterial lung disease, most commonly caused by Mycobacterium avium infection, tends to show variable disease progression, and significant disease predictors have not been adequately established. METHODS: Variable numbers of tandem repeats (VNTR) were evaluated in 16 mycobacterial interspersed repetitive unit (MIRU) loci from M avium isolates cultured from respiratory specimens obtained from 2005 to 2007. Specifically, the association between VNTR profiles and disease progression was assessed. RESULTS: Among the 37 subjects who provided positive respiratory cultures for M avium during the 2005-6 period, 15 subjects were treated within 10 months following a microbiological diagnosis of progressive M avium lung disease. Nine subjects underwent long-term follow-up (>24 months) without treatment for stable M avium lung disease. Based on a neighbour-joining cluster analysis used to classify M avium-positive subjects according to the VNTR profile, subjects with progressive versus stable lung disease were found to be grouped together in distinct clusters. Further analysis using logistic regression modelling showed that disease progression was significantly associated with the genetic distance of the M avium isolate from an appropriately selected reference (age-adjusted odds ratio 1.95; 95% confidence interval 1.16 to 3.30; p = 0.01 for the most significant model). A best-fit model could be used to predict the progression of M avium lung disease when subjects from the 2005-6 period were combined with those from 2007 (p = 0.003). CONCLUSION: Progressive lung disease due to M avium infection is associated with specific VNTR genotypes of M avium.

Acute stress reduces intraparenchymal lung natural killer cells via beta-adrenergic stimulation.

There are lines of evidence that natural killer (NK) cells are sensitive to physical and psychological stress. Alterations in the immune system including NK cells are known to differ among tissues and organs. The effect of stress on the lung immune system, however, has not been well documented in spite of the fact that the lungs always confront viral or bacterial attacks as well as tumour cell metastasis. In this study, we intended to investigate the effect of restraint stress on lung lymphocytes including NK cells. C57BL/6 mice were exposed to 2 h restraint stress. The concentration of plasma epinephrine significantly rose immediately after the release from restraint as compared to home-cage control mice. Flow cytometric analysis revealed that the numbers of most lymphocyte subsets including NK cells were decreased in the lungs and blood but not in the spleen, immediately after restraint stress. Immunohistochemical examination revealed that the number of NK cells was decreased in the intraparenchymal region of the lungs, while the number of alveolar macrophages did not change. The decrease in the number of NK cells in the lungs and blood was reversed by the administration of propranolol, a nonselective beta adrenergic antagonist. Taken together, our findings suggest that acute stress reduces the number of intraparenchymal lung NK cells via activation of beta adrenergic receptors.

Interstitial lung disease associated with drug therapy.

Drug-associated interstitial lung disease (ILD) is not uncommon, with diverse patterns ranging from benign infiltrates to the potentially fatal acute respiratory distress syndrome. As acute respiratory failure due to drug-associated ILD has an unpredictable onset and rapid time course, establishing a diagnosis is often difficult. An accurate diagnosis is based on clinical, radiological (including high-resolution computed tomography) and histological manifestations, although is often only possible by exclusion. Cancer chemotherapy is commonly associated with acute disease that, on pathology, is often diffuse alveolar damage. Furthermore, a combination of drugs with or without radiotherapy can increase the risk of ILD. This article reviews treatments for non-small-cell lung cancer (NSCLC) that are associated with the development of ILD and how systematic evaluation of the possible role of these drugs in ILD is warranted. A difference between Japan and the rest of the world in reporting rates of ILD when gefitinib ('Iressa') has been used in advanced NSCLC is also discussed. However, the difference remains unexplained, leaving important epidemiological and mechanistic questions.

Expression of androgen receptor and 5alpha-reductase types 1 and 2 in early gestation fetal lung: a possible correlation with branching morphogenesis.

The bioactive and potent androgen 5alpha-dihydrotestosterone (DHT) has been postulated to be involved in the development of branching morphogenesis in the human fetal lung, but its expression has not been examined. We therefore examined the expression of the androgen receptor (AR) and 5alpha-reductases (type 1 and type 2), which catalyse the conversion of testosterone into DHT, in the human fetal lung using immunohistochemistry and reverse transcription-PCR (RT-PCR). Immunoreactive AR was detected predominantly in the nuclei of epithelial cells of the budding component of the early gestational fetal lung. 5alpha-Reductase type 1 immunoreactivity was detected in the cytoplasm of epithelial cells, whereas immunoreactivity for 5alpha-reductase type 2 was not detected in the samples of human fetal lung examined. RT-PCR also confirmed the presence of AR and 5alpha-reductase in all fetal lung and epithelial cell lines. The results of our present study suggest that DHT may play an important role in epithelial cells, which might include precursor cells, in which both AR and 5alpha-reductases are expressed during branching morphogenesis of the human fetal lung.

Correlation between the airway branching patterns and the heterogeneous distribution of the lesions in bleomycin-injured mouse lung: a 3-D morphometric study.

The possible roles of airway branching patterns on the pathogenesis of lung diseases, especially on the heterogeneous distribution of the lesions, were examined through three-dimensional (3-D) morphometric analysis of a mouse lung injury, induced by bleomycin. On serial sections of a mouse lung damaged by subcutaneous injection of bleomycin, we performed a computer-assisted reconstruction of the lung for visualizing the relation of airways with the distribution of the lesions, and defined the features of bronchial routes to each lesion by four parameters: LTB; the distance from the hilum, Ng; generation numbers, RD(min); irregularity of airway branching, and thetaTB; the grade of airway recurrence. Among these four parameters, only thetaTB was found to correlate with the severity of lesions (p < 0.05 by an ANOVA test), which was proved by a posthoc test (p < 0.0001). These results showing the acini supplied by recurrent branches are more prone to be damaged than those by non-recurrent branches suggest that branching patterns may underlie the heterogeneous distribution of lesions in diffuse interstitial lung diseases.

Irregularity of airway branching in a mouse bronchial tree: a 3-D morphometric study.

Limited information on the degree of irregularity of branching patterns of a bronchial tree may obscure the cause of heterogeneous distribution of the lesions in a variety of lung diseases. We reconstructed three dimensional (3-D) images from hilum to terminal bronchioles of a mouse lung, and defined the irregularity of airway branching by diameter-based morphometric analysis. The relative diameter ratios of a daughter to the parent branch (D1/D0) and those of a minor to major daughter branches (D1/D2) were calculated, and irregular dichotomies were found to be distributed in 48% of bifurcations. D1/D0 is well correlated with D1/D2, and is proved to indicate regular and irregular branching, as well as D1/D2. Irregular branches with D1/D0 smaller than 0.4 correspond to typical lateral branches, taking off from major bronchi. Our novel 3-D morphometric analysis showed the first portrayal of the 3-D structures of mouse bronchial airways, which provides a quantitative description of branching patterns leading to the correlation with distribution of lesions in the diseased lung.

In situ detection of unexpected patterns of mutant p53 gene expression in non-small cell lung cancers.

Many solid tumors, including non-small cell lung cancers (NSCLCs), are characterized by heterogenous expression of p53 protein in the neoplastic cells. To analyse the molecular implications of this finding, we examined topographic distribution of p53 mutations using in situ polymerase chain reaction (PCR) in primary NSCLCs, showing distinct patterns of variable p53 overexpression by immunohistochemistry. Unique sets of primers for each mutation were designed, and optimal PCR conditions were determined by standard PCR using DNA from cloned mutants or cell lines established from these tumors. All tumor cell nuclei, regardless of the status of p53 overexpression, demonstrated homogeneous distribution of mutant p53 with specific primers, indicating that only subgroups of the mutated cells overexpressed p53 protein. In situ reverse transcription (RT)-PCR was applied to detect mutant mRNA in the individual tumor cells using specific primers. We found that in each case the distribution of mutant p53 mRNA coincided with that of immunohistochemical overexpression of p53 protein. Our results suggest that the regulation of mutant p53 expression, but not the genotype, is heterogeneous in the neoplastic cells. The topographic genomapping of p53 in NSCLC using in situ PCR provides a novel approach to view molecular mechanisms of lung carcinogenesis.

Papillary serous carcinoma of the peritoneum: analysis of clonality of peritoneal tumors.

Papillary serous carcinoma of the peritoneum (PSCP) is a primary neoplasm of peritoneal origin, and is histologically difficult to differentiate from papillary serous carcinoma of the ovary (PSCO). PSCP is frequently accompanied by many peritoneal tumors, and has been managed as a disseminated disease. In previous reports, however, the clonality of the tumors has not been fully discussed. Recently, the significant roles of the p53 and BRCA1 genes in PSCP have been reported. In this study, we investigated immunohistochemical staining for p53 proteins, and investigated p53 gene mutations, using DNA sequencing analysis, to clarify the clonality of PSCP tumors. Immunohistochemically, all the tumor samples demonstrated nuclear overexpression of p53 proteins, and the DNA sequencing analysis of the p53 gene showed diverse point mutations at codons 167 and 192 in two of four anatomically different tumors. In conclusion, the possibility of polyclonality of PSCP tumors is suggested.

Transition from squamous cell carcinoma to adenocarcinoma in adenosquamous carcinoma of the lung.

The heterogeneity of tumor cells is frequently observed in lung cancer, but the clonality of these cells has not yet been established. The distinct components of 12 lung adenosquamous carcinomas were compared by genetic alterations of p53 and K-ras, chromosomal abnormalities at 9p21 and 9q31-32, and immunohistochemical reactions. The immunoreactivity of p53 was consistent in both adenocarcinomatous and squamous cell carcinomatous components as well as in the transitional areas, retaining the morphological characteristics of the distinct components. The same p53 mutation was found in both components of each tumor with p53 overexpression. No K-ras mutations were detected in any of the tumors examined. Three of the four tumors with chromosomal abnormalities detected, one at 9p21 and two at 9q31-32, had coincident abnormalities between the distinct components, whereas one tumor deleted homozygously at 9p21 (D9S259) in the adenocarcinomatous component with loss of heterozygosity in the other component. The expression of squamous cell carcinoma-related antigen in adenocarcinomatous components was significantly higher than that of lung adenocarcinomas (57 +/- 5.8% vs. 1.0 +/- 0.5%, P < 0.0001), whereas Mucin 1 expression is less in these components (9.0 +/- 4.9% vs. 55 +/- 8.2%, P = 0.003). These results suggest monoclonal transition from squamous cell carcinoma to adenocarcinoma in lung adenosquamous carcinoma.

Bilateral pneumothoraces with multiple bullae in a patient with asymptomatic bronchiolitis obliterans 10 years after bone marrow transplantation.

A 16-year-old boy developed bronchiolitis obliterans (BO) 10 years after BMT for myelodysplastic syndrome. Although the patient complained of almost no dyspnea on exertion, he had mild hypercapnea with a markedly reduced forced expiratory volume of 0.32 l. Chest X-rays showed occasional bilateral minimal pneumothoraces, which is in accordance with the existence of multiple small bullae found on the pleural surface at video-assisted thoracic surgery. Histologic examination of the biopsied lung revealed BO. This case indicates that BO in adolescence following BMT and possible chronic GVHD may be masked because of lung immaturity at BMT, and BO after BMT may be associated with multiple pleural bullae.