Yoji Nagashima

Comprehensive mutational analysis of the VHL gene in sporadic renal cell carcinoma: Relationship to clinicopathological parameters

To delineate more precisely the somatic von Hippel‐Lindau disease (VHL) gene alteration as well as to elucidate its etiologic role in renal tumorigenesis, we examined a total of 240 sporadic renal cell carcinomas (RCCs) for somatic VHL gene alterations by DNA‐SSCP followed by sequencing, methylation‐specific PCR assay, microsatellite LOH study, and Southern blot analysis. Intragenic mutation of the VHL gene was found exclusively in clear‐cell or variant‐type RCCs at a frequency of 51% (104/202). Hypermethylation of the VHL promoter region was detected in an additional 11 clear‐cell RCCs. Microsatellite analysis demonstrated that LOH of the VHL locus was found in 140/155 (90%) informative clear‐cell RCCs. The VHL gene therefore seems to be inactivated in a two‐hit manner by intragenic mutation or hypermethylation plus allelic loss in clear‐cell RCC. Genomic rearrangement of the VHL gene detected by Southern analysis was not found (0/216 cases); this is in contrast to germ lines in which Southern aberrations consisted of 7–19% of the mutations. Clinicopathologic data demonstrated that VHL mutation/LOH did not vary according to tumor progression in clear‐cell RCC, including tumor diameter, stage, grading, distant metastasis, and lymph node metastasis. Interestingly, VHL mutation was significantly less frequent in RCCs occurring in younger (≦ 55 years) than that in older (≧ 56 years) patients. These data suggested that the inactivation of the VHL tumor‐suppressor gene is a specific genetic change in clear‐cell RCC, and that it may occur at an early or first step in the clear‐cell tumorigenic pathway rather than as a late event.

Diet-Induced Adipose Tissue Inflammation and Liver Steatosis Are Prevented by DPP-4 Inhibition in Diabetic Mice

OBJECTIVE Diet composition alters the metabolic states of adipocytes and hepatocytes in diabetes. The effects of dipeptidyl peptidase-4 (DPP-4) inhibition on adipose tissue inflammation and fatty liver have been obscure. We investigated the extrapancreatic effects of DPP-4 inhibition on visceral fat and the liver. RESEARCH DESIGN AND METHODS We investigated diet-induced metabolic changes in β-cell–specific glucokinase haploinsufficient (Gck+/−) diabetic mice. We challenged animals with a diet containing a combination of sucrose and oleic acid (SO) or sucrose and linoleic acid (SL). Next, we assessed the effects of a DPP-4 inhibitor, des-fluoro-sitagliptin, on adipose tissue inflammation and hepatic steatosis. RESULTS The epididymal fat weight and serum leptin level were significantly higher in Gck+/− mice fed SL than in mice fed SO, although no significant differences in body weight or adipocyte size were noted. Compared with SO, SL increased the numbers of CD11c+ M1 macrophages and CD8+ T-cells in visceral adipose tissue and the expression of E-selectin, P-selectin, and plasminogen activator inhibitor-1 (PAI-1). DPP-4 inhibition significantly prevented adipose tissue infiltration by CD8+ T-cells and M1 macrophages and decreased the expression of PAI-1. The production of cytokines by activated T-cells was not affected by DPP-4 inhibition. Furthermore, DPP-4 inhibition prevented fatty liver in both wild-type and Gck+/− mice. DPP-4 inhibition also decreased the expressions of sterol regulatory element–binding protein-1c, stearoyl-CoA desaturase-1, and fatty acid synthase, and increased the expression of peroxisome proliferator–activated receptor-α in the liver. CONCLUSIONS Our findings indicated that DPP-4 inhibition has extrapancreatic protective effects against diet-induced adipose tissue inflammation and hepatic steatosis.

Metformin suppresses intestinal polyp growth in ApcMin/+ mice

Metformin is a biguanide derivative that is widely used in the treatment of diabetes mellitus. One of the pharmacological targets of metformin is adenosine monophosphate‐activated protein kinase (AMPK). We investigated the effect of metformin on the suppression of intestinal polyp formation in ApcMin/+ mice. Administration of metformin (250 mg/kg) did not reduce the total number of intestinal polyp formations, but significantly reduced the number of intestinal polyp formations larger than 2 mm in diameter in ApcMin/+ mice. To examine the indirect effect of metformin, the index of insulin resistance and serum lipid levels in ApcMin/+ mice were assessed. These factors were not significantly attenuated by the treatment with metformin, indicating that the suppression of polyp growth is not due to the indirect drug action. The levels of tumor cell proliferation as determined by 5‐bromodeoxyuridine and proliferating cell nuclear antigen immunohistochemical staining, and apoptosis, via transferase deoxytidyl uridine end labeling staining, in the polyps of metformin‐treated mice were not significantly different in comparison to those of control mice. Gene expression of cyclin D1 and c‐myc in intestinal polyps were also not significantly different between those two groups. In contrast, metformin activated AMPK in the intestinal polyps, resulting in the inhibition of the activation of mammalian target of rapamycin, which play important roles in the protein synthesis machinery. Metformin suppressed the polyp growth in ApcMin/+ mice, suggesting that it may be a novel candidate as a chemopreventive agent for colorectal cancer. (Cancer Sci 2008; 99: 2136–2141)

Expression of gelatinase A, tissue inhibitor of metalloproteinases-2, matrilysin, and trypsin(ogen) in lung neoplasms: An immunohistochemical study

Lung cancer is a heterogeneous tumor in terms of clinical and biological behavior, and its aggressiveness depends on its invasive and metastatic properties. Matrix metalloproteinases and serine proteinases are believed to play a crucial role in invasion and metastasis of malignant tumor cells. In the present study, the authors evaluated immunohistochemically the expression of gelatinase A; tissue inhibitor of metalloproteinases-2 (TIMP-2), an inhibitor of gelatinase A; matrilysin; and trypsin(ogen) in 67 lung tumors from a variety of histological types including 17 squamous cell carcinomas, 16 adenocarcinomas, 15 small cell carcinomas, and 12 carcinoids. Interestingly, normal bronchial, bronchiolar, and alveolar epithelial cells expressed gelatinase A, TIMP-2, matrilysin, and trypsin(ogen) at varying frequencies and intensities. Bronchial smooth muscle cells and cartilage cells expressed gelatinase A alone, whereas endothelial cells, fibroblasts, and macrophages expressed gelatinase A and TIMP-2. Gelatinase A was expressed at high levels in most lung tumors examined (47% to 80%). TIMP-2 was also expressed at high levels except in the small cell carcinomas, which showed TIMP-2 expression at a lower frequency (60%) compared with other types of lung tumors (80% to 100%). Although matrilysin was expressed by tumor cells of all the histological types at various frequencies (13% to 63%), its expression was most common in adenocarcinomas. Expression of trypsin(ogen) was observed almost exclusively in adenocarcinomas (56%); other types of lung tumors expressed trypsin(ogen) far less frequently (0% to 12%). The present results, taken together with those of previous studies, suggest that gelatinase A is associated with malignant behavior of all the types of lung tumors, whereas its activity may be controlled by the endogenous inhibitor TIMP-2. The aggressive clinical behavior of small cell carcinoma may be attributable, at least in part, to a loss of the inhibitory effect of TIMP-2, as a significant proportion of these tumors showed negative or low levels of TIMP-2 expression. Matrilysin and trypsin(ogen) expressions are unlikely to be correlated with the aggressiveness of lung tumors. The expression of trypsin (ogen) may rather reflect the differentiation of adenocarcinoma cells toward normal airway epithelial cells.

Gene expression analysis of renal carcinoma: adipose differentiation-related protein as a potential diagnostic and prognostic biomarker for clear-cell renal carcinoma

The gene expression profiles of 33 renal cell carcinomas (RCCs) and nine normal kidney samples were examined using high‐density oligonucleotide microarrays in an attempt to identify biomolecular markers for the diagnosis of tumour subtypes and also for prediction of prognosis. Hierarchical clustering demonstrated that clear‐cell RCC, chromophobe RCC, and normal kidney tissue showed distinctive gene expression profiles. The mean expression levels of 149 of 12 500 genes were more than three times higher in clear‐cell RCC than in chromophobe RCC and normal kidney tissue. Among the genes whose expression was upregulated in clear‐cell RCC, adipose differentiation‐related protein (ADFP) and nicotinamide N‐methyltransferase (NNMT) were selected for further analysis. Consistent with the results of the microarray, increased levels of ADFP and NNMT mRNA were found more frequently in clear‐cell RCCs than in other non‐clear‐cell tumour subtypes using real‐time quantitative PCR. Immunohistochemistry for ADFP showed strong and unique tumour cell staining patterns in the majority of clear‐cell RCCs. More importantly, patients bearing tumours with higher AFDP mRNA levels showed significantly better survival in both univariate and multivariate analyses. ADFP is a lipid storage droplet‐associated protein and its transcription is considered to be regulated by the von Hippel–Lindau/hypoxia‐inducible factor pathway. It is known that clear‐cell RCC contains abundant lipids and cholesterols. Thus it is likely that sustained upregulation of ADFP following VHL inactivation is involved in the morphological appearance of clear‐cell RCC. Moreover ADFP expression status may provide useful prognostic information as a biomolecular marker in patients with clear‐cell RCC. Copyright

Wide Distribution of Laminin-5 γ2 Chain in Basement Membranes of Various Human Tissues

Laminin 5 (LN5), a heterotrimer of laminin α3, β3, and γ2 chains, is a laminin isoform which strongly promotes adhesion, migration, and scattering of cells through binding to integrins α3β1, α6β1 and α6β4. To get an insight into the physiological functions of LN5, we prepared a mouse monoclonal antibody to human laminin γ2 chain and used it for immunohistochemical analysis of laminin γ2 chain in normal human tissues. The basement membranes of various epithelial tissues, such as the skin, lung, small intestine, stomach, kidney and prostate, were immunostained with the anti-laminin γ2 chain monoclonal antibody. In addition, the basement membrane of the surface germinal epithelium in the ovary was also positive for laminin γ2 chain. These results suggest general roles of LN5 in the anchorage of various types of epithelial cells to the underlying basement membrane and in the expression of their cellular functions. Moreover, deposition of laminin γ2 chain around small arteries and veins was observed in the thymus and spleen. This lymphatic organ-specific expression of vascular LN5 might provide a novel function of LN5 in immune responses.

Renal angiomyolipoma: a radiological classification and update on recent developments in diagnosis and management

Angiomyolipoma is the most common benign solid renal neoplasm observed in clinical practice. Once thought to be a hamartoma and almost always diagnosed by the imaged-based detection of fat, angiomyolipomas are now known to consist of a heterogeneous group of neoplasms. Although all are considered perivascular epithelioid cell tumors, many display different pathology, imaging features, and clinical behavior. The importance of understanding this group of neoplasms is emphasized by the fact that many types of angiomyolipoma contain little to no fat, and despite being benign, sometimes escape a pre-operative diagnosis. These types of angiomyolipomas can all be considered when encountering a renal mass that is both hyperattenuating relative to renal parenchyma on unenhanced CT and T2-hypointense, features that reflect their predominant smooth muscle component. We review recent developments and provide a radiological classification of angiomyolipomas that helps physicians understand the various types and learn how to both diagnose and manage them.

Transfer of Heme Oxygenase 1 cDNA by a Replication-Deficient Adenovirus Enhances Interleukin 10 Production from Alveolar Macrophages That Attenuates Lipopolysaccharide-Induced Acute Lung Injury in Mice

By using a direct, intratracheal inoculation of an adenovirus encoding heme oxygenase 1 (Ad.HO-1), model gene therapy for acute lung injury induced by inhaled pathogen was performed. Data demonstrated that Ad.HO-1 administration is as effective as the pharmacologic upregulation of the endogenous HO-1 gene expression by hemin to attenuate neutrophilic inflammations of the lung after aerosolized lipopolysaccharide (LPS) exposure. Interestingly, immunohistochemical analysis revealed that the HO-1 gene was transferred not only to the airway epithelium, but to the alveolar macrophages (AMs). Moreover, overexpression of exogenous HO-1 in the macrophages provided a high level of endogenous interleukin 10 (IL-10) production from the macrophages, and additional experiments using IL-10 knockout mice demonstrated that the increase in IL-10 in the macrophages was critical for the resolution of neutrophilic migration in the lung after LPS exposure. These results suggest that AMs not only are barriers for efficient gene transfer to the respiratory epithelium, but also represent logical targets for Ad-mediated, direct, in vivo gene therapy strategies for inflammatory disorders in humans.

Interstitial pneumonia in Hermansky-Pudlak syndrome: significance of florid foamy swelling/degeneration (giant lamellar body degeneration) of type-2 pneumocytes

Abstract Although usual interstitial pneumonia (UIP)-like IP has been known as the most serious complication of Hermansky-Pudlak syndrome (HPS), its pathologic features and pathogenesis are poorly understood. We investigated biopsied and autopsied lung tissues from five patients who died of UIP-like IP associated with HPS (HPSIP). The salient histopathologic features of HPSIP observed were: (1) alveolar septa displaying florid proliferation of type-2 pneumocytes (2PCs) with characteristic foamy swelling/degeneration; (2) patchy fibrosis with lymphocytic and histiocytic infiltration centered around respiratory bronchioles, occasionally showing constrictive bronchiolitis; and (3) honeycomb change without predilection for the lower lobes or subpleural area. Those peculiar 2PCs were histochemically characterized by the over accumulation of phospholipid, immunohistochemically by a weak positivity for surfactant protein, and ultrastructurally by the presence of numerous giant lamellar bodies that compressed the nucleus with occasional cytoplasmic disruption, together suggesting a form of cellular degeneration with an over accumulation of surfactant (giant lamellar body degeneration). The present study strongly indicates that there is a basic defect in the formation/secretion process of surfactant by the 2PCs in HPS, which may well be the triggering factor for the HPSIP development. Other factors, such as macrophage dysfunction, may be working synergistically for further acceleration of the inflammatory process.

Collecting Duct (Bellini Duct) Renal Cell Carcinoma: A Nationwide Survey in Japan

PURPOSE Collecting duct carcinoma, a rare type of renal cell carcinoma, remains poorly understood. To analyze the nature of collecting duct carcinoma a retrospective survey was performed in Japan. MATERIALS AND METHODS This survey was done from August 2001 to April 2003. A total of 281 institutions throughout Japan were requested to document all cases of collecting duct carcinoma. All pertinent clinical information was compiled, including patient age, sex, mode of presentation, evaluation modality, preoperative laboratory data, surgery type, macroscopic and microscopic findings, and survival data. Two urological pathologists reviewed microscopic slides of all tumor specimens to confirm collecting duct carcinoma. RESULTS Two pathologists confirmed collecting duct carcinoma in 81 of the 120 cases documented as collecting duct carcinoma. Mean patient age was 58.2 years and males comprised 71.6% of all patients. The mode of presentation was classified as symptomatic in 65.4% of cases, incidental in 24.7% and not available in 9.9%. Regional lymph node metastasis was histologically detected in 44.2% of patients who underwent lymph node dissection, while 32.1% of the population had distant metastasis at presentation. Although postoperative adjuvant therapy against metastasis or recurrence was performed in 25 patients, no obvious responses were identified except in 1 with lung metastases, who showed a partial response to combined gemcitabine and carboplatin therapy. At a median followup of 15 months 1, 3, 5 and 10-year disease specific survival was 69.0%, 45.3%, 34.3% and 13.7%, respectively. CONCLUSIONS We report what is to our knowledge the largest known series of collecting duct carcinoma. Since advanced or recurrent collecting duct carcinoma is resistant to standard treatment modalities, new treatment strategies are needed for advanced collecting duct carcinoma.