Seiichi Miwa

Enhanced Mdm2 activity inhibits pRB function via ubiquitin-dependent degradation

Retinoblastoma gene product (pRB) plays critical roles in regulation of the cell cycle and tumor suppression. It is known that downregulation of pRB can stimulate carcinogenesis via abrogation of the pRB pathway, although the mechanism has not been elucidated. In this study, we found that Mdm2, a ubiquitin ligase for p53, promoted ubiquitin‐dependent degradation of pRB. pRB was efficiently ubiquitinated by wild‐type Mdm2 in vivo as well as in vitro, but other RB family proteins were not. Mutant Mdm2 with a substitution in the RING finger domain showed dominant‐negative stabilization of pRB. Both knockout and knockdown of Mdm2 caused accumulation of pRB. Moreover, Mdm2 inhibited pRB‐mediated flat formation of Saos‐2 cells. Downregulation of pRB expression was correlated with a high level of expression of Mdm2 in human lung cancers. These results suggest that Mdm2 regulates function of pRB via ubiquitin‐dependent degradation of pRB.

High expression of Cks1 in human non-small cell lung carcinomas

Enhanced degradation of cyclin-dependent kinase (CDK) inhibitor p27(Kip1) is known to be a powerful prognostic marker in many types of human cancers. Human CDK subunit 1 (Cks1) and S-phase kinase associated protein 2 (Skp2) are components of the SCF(Skp2) complex, which acts as a ubiquitin ligase for p27(Kip1). There are no reports about the involvement of Cks1 in the pathogenesis of human cancer. Here we show high expression of Cks1 in non-small cell lung cancers (NSCLCs) using Western blotting and quantitative real-time RT-PCR. The Skp2 mRNA expression level was high in squamous cell carcinomas and was inversely related with the p27(Kip1) protein level in individual clinical samples. In contrast, Cks1 mRNA expression had no such relationship with p27(Kip1), although Cks1 mRNA was significantly elevated in adenocarcinomas. These results suggest that high expression of Skp2 and Cks1 may be involved in the pathogenesis of NSCLCs via different mechanisms.

Intravenous Immunoglobulin Therapy for Refractory Interstitial Lung Disease Associated with Polymyositis/Dermatomyositis

Interstitial lung disease (ILD) associated with polymyositis/dermatomyositis (ILD-PM/DM), including amyopathic dermatomyositis (ADM), is recognized as an important condition because it frequently causes death, despite intensive therapy with high-dose corticosteroid and immunosuppressive agents, such as cyclosporine A and cyclophosphamide. Intravenous immunoglobulin therapy (IVIG) has shown efficacy for myopathy associated with PM/DM, but its usefulness for ILD-PM/DM is unclear. This study was designed to investigate the efficacy of IVIG for refractory ILD-PM/DM. A review was made of medical charts of five patients (2 men and 3 women) who were treated with IVIG for refractory ILD-PM/DM resistant to high-dose corticosteroid and cyclosporine A and/or cyclophosphamide. One patient had acute ILD-PM and four patients had acute ILD-ADM. Of the five patients, one patient with ILD-PM and one patient with ILD-ADM survived. No adverse reactions were seen due to IVIG treatment. There were no critical differences in the clinical parameters and clinical courses between survivors and nonsurvivors. IVIG treatment is safe and could be an effective salvage therapy for refractory ILD-PM/DM in certain cases, suggesting that further controlled trials are worthwhile.

Increased expression of YKL-40, a chitinase-like protein, in serum and lung of patients with idiopathic pulmonary fibrosis

BACKGROUND YKL-40, a mammalian member of chitinase-like proteins, has been shown to play a role in pathological conditions leading to tissue remodeling and fibrosis. Recently, YKL-40 was found to be increased in severe asthma, suggesting that YKL-40 contributes to airway remodeling; however, no data are available about YKL-40 expression in idiopathic pulmonary fibrosis (IPF). The present study was conducted to investigate YKL-40 expression in the serum and lung of IPF patients, and to determine its clinical significance. METHODS Using an enzyme-linked immunosorbent assay, we measured YKL-40 levels in the serum of 63 IPF patients and in bronchoalveolar lavage fluid (BALF) of 18 IPF patients. YKL-40 levels were also assessed in the serum and BALF of healthy subjects. We further investigated the relationship between serum YKL-40 levels and clinical parameters. Additionally, immunohistochemical staining for YKL-40 was performed in lung specimens of IPF patients and control subjects. RESULTS Serum and BALF YKL-40 levels were significantly higher in IPF than in controls (serum: 245.8+/-180.2ng/ml vs. 116.0+/-58.3ng/ml; BALF: 17.8+/-19.1ng/ml vs. 0.3+/-0.9ng/ml, respectively). Serum YKL-40 levels significantly correlated positively with serum KL-6 levels and AaDO(2), and negatively with DLco and PaO(2). Immunohistochemical study revealed enhanced YKL-40 expression in alveolar macrophages and bronchiolar epithelia adjacent to fibrotic lesions in IPF, but not in controls. CONCLUSIONS These data suggest that YKL-40 is increased in the circulation and lungs of IPF patients, suggesting that this glycoprotein is associated with the pathophysiology of IPF.

Serum Indoleamine 2,3-Dioxygenase Activity Predicts Prognosis of Pulmonary Tuberculosis

ABSTRACT Tuberculosis (TB) continues to be a major health problem, and there are few biomarkers for predicting prognosis. Indoleamine 2,3-dioxygenase (IDO), a potent immunoregulatory molecule, catalyzes the rate-limiting step of tryptophan (Trp) degradation in the kynurenine (Kyn) pathway. An increase in IDO activity determined by the serum Trp/Kyn ratio has been shown to be associated with poor prognosis in cancers and bacteremia. In TB, however, there are no studies measuring serum IDO activity to determine its clinical significance. We evaluated serum IDO activity with 174 pulmonary TB (PTB) patients and 85 controls, using liquid chromatography/electrospray ionization tandem mass spectrometry. IDO activity was estimated by calculating the serum Kyn-to-Trp ratio. PTB patients had significantly higher Kyn concentrations and IDO activity and significantly lower Trp concentrations (P < 0.0001, P < 0.0001, and P < 0.0001, respectively) than the controls. Of 174 PTB patients, 39 (22.4%) died. The patients who died had significantly higher concentrations of Kyn and significantly lower Trp concentrations, resulting in significantly higher IDO activity (P < 0.0001, P < 0.0001, and P < 0.0001, respectively). In a receiver operating characteristic (ROC) analysis, serum IDO activity had the highest area under the curve (0.850), and this activity was an independent prognostic factor in multivariate analysis. These results suggest that serum IDO activity can be used as a novel prognostic marker in PTB.

Efficacy of Clarithromycin and Ethambutol for Mycobacterium avium Complex Pulmonary Disease. A Preliminary Study

RATIONALE Patients with Mycobacterium avium complex pulmonary disease are frequently administered a combination of clarithromycin, ethambutol, and rifampicin. However, rifampicin is known to reduce the serum levels of clarithromycin. It remains unclear whether a reduction in clarithromycin serum levels influences the clinical outcome of the Mycobacterium avium complex pulmonary disease treatment regimen. OBJECTIVES To compare a three-drug regimen (clarithromycin, ethambutol, and rifampicin) to a two-drug regimen (clarithromycin and ethambutol) for the treatment of Mycobacterium avium lung disease. METHODS In a preliminary open-label study, we randomly assigned newly diagnosed, but as-yet untreated, patients with disease caused by Mycobacterium avium complex without HIV infection to either the three-drug or the two-drug regimen for 12 months. The primary endpoint was the conversion of sputum cultures to negative after 12 months of treatment. Patient data were analyzed using the intention-to-treat method. MEASUREMENTS AND MAIN RESULTS Of 119 eligible patients, 59 were assigned to the three-drug regimen and 60 to the two-drug regimen. The rate of sputum culture conversion was 40.6% with the three-drug regimen and 55.0% with the two-drug regimen (difference, -14.4% [95% confidence interval, -32.1 to 3.4]). The incidence of adverse events leading to the discontinuation of treatment was 37.2 and 26.6% for the three-drug and the two-drug regimens, respectively. CONCLUSIONS This preliminary study suggests that treatment with clarithromycin and ethambutol is not inferior to treatment with clarithromycin, ethambutol, and rifampicin for Mycobacterium avium complex lung disease. Our findings justify a larger clinical trial to compare long-term clinical outcomes for the two treatment regimens. Clinical trial registered with http://www.umin.ac.jp/english/ (UMIN000002819).

Drug Lymphocyte Stimulation Test in the Diagnosis of Adverse Reactions to Antituberculosis Drugs

BACKGROUND Tuberculosis (TB) is a worldwide infectious disease. Recently, standard therapy has become very effective for treating patients with TB; however, as a result of this powerful regimen, serious side effects have become an important problem. The aim of this prospective study was to evaluate the usefulness of the drug lymphocyte stimulation test (DLST) to determine anti-TB drugs causing side effects. METHOD Four hundred thirty-six patients with TB were admitted to our hospital for treatment between January 2002 and August 2007. DLST was performed in patients who had certain adverse drug reactions during TB treatment. The causative drug was identified by the drug provocation test (DPT). The tested drugs were mainly isoniazid (INH), rifampin (RIF), ethambutol (EMB) and pyrazinamide (PZA). RESULTS Of 436 patients, 69 patients (15.8%) had certain adverse drug reactions to anti-TB drugs. Of the 261 agents that underwent the DLST and DPT, 28 agents (10.7%) in 20 patients (28.9%) were positive by DLST, and 67 agents (25.7%) in 46 patients (66.6%) were identified as causative drugs by DPT. The sensitivity of DLST was only 14.9% for all drugs (INH, 14.3%; RIF, 13.6%; EMB, 14.3%; PZA, 0%). CONCLUSIONS DLST offers little contribution to the detection of causative agents in patients with adverse anti-TB drug reactions.

Usefulness of serum procalcitonin levels in pulmonary tuberculosis

There are very few data on serum procalcitonin (PCT) levels in pulmonary tuberculosis (PTB) patients who are negative for HIV. We assessed serum PCT in consecutive patients diagnosed with pulmonary tuberculosis or community-acquired pneumonia (CAP) on admission to discriminate between PTB and CAP, and examined the value of prognostic factors in PTB. 102 PTB patients, 62 CAP patients, and 34 healthy volunteers were enrolled. Serum PCT in PTB patients was significantly lower than in CAP patients (mean±sd 0.21±0.49 versus 4.10±8.68 ng·mL−1; p<0.0001). By receiver-operating characteristic curve analysis, serum PCT was an appropriate discrimination marker for PTB and CAP (area under the curve 0.866). PTB patients with ≥0.5 ng·mL−1 (normal cut-off) had significantly shorter survival than those with <0.5 ng·mL−1 (p<0.0001). Serum PCT is not habitually elevated in HIV-negative PTB patients and is a useful biomarker for discriminating between PTB and CAP; however, when serum PCT is outside the normal range, it is a poor prognostic marker.

Effects of MdmX on Mdm2‐mediated downregulation of pRB

Mdm2, a RING‐finger type ubiquitin ligase, is overexpressed in a variety of human cancers. It promotes ubiquitination of the tumor suppressor p53 and can function as an oncogene by largely downregulating p53. Recently, we reported that Mdm2 degrades retinoblastoma tumor suppressor protein (pRB) via the ubiquitin–proteasome system. In the present study, we assessed the effects of MdmX, a structural homolog of Mdm2, on the Mdm2‐mediated ubiquitination of pRB. MdmX is known to negatively regulate p53 function by enhancing the Mdm2‐mediated ubiquitination and degradation of p53. Interestingly, MdmX inhibited the Mdm2‐mediated pRB ubiquitination. Furthermore, an MdmX siRNA decreased the endogenous pRB level, while MdmX overexpression stimulated pRB functions in cultured cells. Therefore, MdmX may have different roles in the regulation of Mdm2 activity for ubiquitination of pRB and p53.

Indoleamine 2,3-dioxygenase in the pathogenesis of tuberculous pleurisy.

BACKGROUND Pleural fluid is a frequent manifestation in pulmonary diseases, such as lung cancer and infectious diseases, including pulmonary tuberculosis (TB). The enzyme indoleamine 2,3-dioxygenase (IDO) catalyses tryptophan through the kynurenine pathway, and is considered a crucial immunoregulatory molecule mediating immune tolerance. Recent studies have shown IDO activity to be a novel prognostic factor not only in cancer patients but also in those with infectious diseases, including pneumonia and pulmonary TB. However, no studies have measured and determined the clinical significance of IDO activity in pleural fluid. METHODS We enrolled 92 patients, including 34 with tuberculous pleurisy (TBP), 36 with malignant pleuritis and 15 with parapneumonic effusions. IDO activity was evaluated using liquid chromatography/electrospray ionisation tandem mass spectrometry, and was estimated by calculating kynurenine-to-tryptophan ratio. RESULTS Pleural fluid from patients with TBP had significantly higher kynurenine concentrations and significantly lower tryptophan concentrations, resulting in significantly higher IDO activity compared with pleural effusion or serum from non-tuberculous pleuritis (all P < 0.001). Pleural tissue from TBP showed enhanced IDO expression in epithelioid granuloma regions by immunohistochemistry. CONCLUSIONS These results suggest that IDO is strongly involved in the pathogenesis of TBP.