Cholangiolocellular Carcinoma Misdiagnosed As Hemangioma: Unique Central Fibrotic Area Devoid of Cancer Cells Detected on a Seven-Year Follow-Up.

Cholangiolocellular carcinoma, a subtype of peripheral-type intrahepatic cholangiocarcinoma, is a relatively rare primary liver tumor. This case report describes a patient with cholangiolocellular carcinoma that was initially misdiagnosed as hemangioma, and ultimately underwent complete tumor resection after a seven-year follow-up period. A 72-year-old female patient with a history of chronic hepatitis C was followed up regularly at the hospital. Computed tomography (CT) performed seven years prior had detected a small tumor (12 mm) with radiographic characteristics suggestive of hemangioma. The tumor increased in size from 12 to 32 mm over the next seven years. On CT, the tumor showed poor central enhancement, indicating reduced blood flow in the central region. Due to the suspicion of malignancy, partial surgical resection was performed. Pathological examination confirmed a diagnosis of cholangiolocellular carcinoma. The carcinoma exhibited vascular invasion and a broad central fibrous area with hyalinization, lacking epithelial cells, and marked by venous obstruction. This case underscores the challenge of distinguishing cholangiolocellular carcinoma from hemangioma, particularly in small tumors with similar radiological features. The findings highlight the importance of employing additional diagnostic modalities such as ultrasound and magnetic resonance imaging, as well as the necessity of biopsy when suspicion arises. This case also describes the unique finding of a central fibrous area without tumor cells, which may have resulted from localized circulatory disturbances potentially caused by tumor embolism in the portal and hepatic veins. This pathological finding provides valuable insights into the nature of this rare tumor.


Paradoxical development of polymyositis-like autoimmunity through augmented expression of autoimmune regulator (AIRE).

Autoimmunity is prevented by the function of the autoimmune regulator [AIRE (Aire in mice)], which promotes the expression of a wide variety of tissue-restricted antigens (TRAs) from medullary thymic epithelial cells (mTECs) and from a subset of peripheral antigen-presenting cells (APCs). We examined the effect of additive expression of human AIRE (huAIRE) in a model of autoimmune diabetes in NOD mice. Unexpectedly, we observed that mice expressing augmented AIRE/Aire developed muscle-specific autoimmunity associated with incomplete maturation of mTECs together with impaired expression of Aire-dependent TRAs. This led to failure of deletion of autoreactive T cells together with dramatically reduced production of regulatory T cells in the thymus. In peripheral APCs, expression of costimulatory molecules was augmented. We suggest that levels of Aire expression need to be tightly controlled for maintenance of immunological tolerance. Our results also highlight the importance of coordinated action between central tolerance and peripheral tolerance under the common control of Aire.


Fibrocyte-like cells mediate acquired resistance to anti-angiogenic therapy with bevacizumab

Bevacizumab exerts anti-angiogenic effects in cancer patients by inhibiting vascular endothelial growth factor (VEGF). However, its use is still limited due to the development of resistance to the treatment. Such resistance can be regulated by various factors, although the underlying mechanisms remain incompletely understood. Here we show that bone marrow-derived fibrocyte-like cells, defined as alpha-1 type I collagen-positive and CXCR4-positive cells, contribute to the acquired resistance to bevacizumab. In mouse models of malignant pleural mesothelioma and lung cancer, fibrocyte-like cells mediate the resistance to bevacizumab as the main producer of fibroblast growth factor 2. In clinical specimens of lung cancer, the number of fibrocyte-like cells is significantly increased in bevacizumab-treated tumours, and correlates with the number of treatment cycles, as well as CD31-positive vessels. Our results identify fibrocyte-like cells as a promising cell biomarker and a potential therapeutic target to overcome resistance to anti-VEGF therapy.


Surfactant protein D attenuates sub-epithelial fibrosis in allergic airways disease through TGF-β.

Surfactant protein D (SP-D) can regulate both innate and adaptive immunity. Recently, SP-D has been shown to contribute to the pathogenesis of airway allergic inflammation and bleomycin-induced pulmonary fibrosis. However, in allergic airways disease, the role of SP-D in airway remodeling remains unknown. The objective of this study was to determine the contribution of functional SP-D in regulating sub-epithelial fibrosis in a mouse chronic house dust mite model of allergic airways disease. C57BL/6 wild-type (WT) and SP-D-/- mice (C57BL/6 background) were chronically challenged with house dust mite antigen (Dermatophagoides pteronyssinus, Dp). Studies with SP-D rescue and neutralization of TGF-β were conducted. Lung histopathology and the concentrations of collagen, growth factors, and cytokines present in the airspace and lung tissue were determined. Cultured eosinophils were stimulated by Dp in presence or absence of SP-D. Dp-challenged SP-D-/- mice demonstrate increased sub-epithelial fibrosis, collagen production, eosinophil infiltration, TGF-β1, and IL-13 production, when compared to Dp-challenged WT mice. By immunohistology, we detected an increase in TGF-β1 and IL-13 positive eosinophils in SP-D-/- mice. Purified eosinophils stimulated with Dp produced TGF-β1 and IL-13, which was prevented by co-incubation with SP-D. Additionally, treatment of Dp challenged SP-D-/- mice with exogenous SP-D was able to rescue the phenotypes observed in SP-D-/- mice and neutralization of TGF-β1 reduced sub-epithelial fibrosis in Dp-challenged SP-D-/- mice. These data support a protective role for SP-D in the pathogenesis of sub-epithelial fibrosis in a mouse model of allergic inflammation through regulation of eosinophil-derived TGF-β.


Exogenous fatty acid binding protein 4 promotes human prostate cancer cell progression.

Epidemiologic studies have found that obesity is associated with malignant grade and mortality in prostate cancer. Several adipokines have been implicated as putative mediating factors between obesity and prostate cancer. Fatty acid binding protein 4 (FABP4), a member of the cytoplasmic fatty acid binding protein multigene family, was recently identified as a novel adipokine. Although FABP4 is released from adipocytes and mean circulating concentrations of FABP4 are linked with obesity, effects of exogenous FABP4 on prostate cancer progression are unclear. In this study, we examined the effects of exogenous FABP4 on human prostate cancer cell progression. FABP4 treatment promoted serum-induced prostate cancer cell invasion in vitro. Furthermore, oleic acid promoted prostate cancer cell invasion only if FABP4 was present in the medium. These promoting effects were reduced by FABP4 inhibitor, which inhibits FABP4 binding to fatty acids. Immunostaining for FABP4 showed that exogenous FABP4 was taken up into DU145 cells in three-dimensional culture. In mice, treatment with FABP4 inhibitor reduced the subcutaneous growth and lung metastasis of prostate cancer cells. Immunohistochemical analysis showed that the number of apoptotic cells, positive for cleaved caspase-3 and cleaved PARP, was increased in subcutaneous tumors of FABP4 inhibitor-treated mice, as compared with control mice. These results suggest that exogenous FABP4 might promote human prostate cancer cell progression by binding with fatty acids. Additionally, exogenous FABP4 activated the PI3K/Akt pathway, independently of binding to fatty acids. Thus, FABP4 might be a key molecule to understand the mechanisms underlying the obesity-prostate cancer progression link.


ブレオマイシン肺線維症モデルにおけるFAKシグナル阻害薬の抗線維化効果

ブレオマイシン肺線維症モデルにおけるFAKシグナル阻害薬の抗線維化効果


Antifibrotic effects of focal adhesion kinase inhibitor in bleomycin-induced pulmonary fibrosis in mice.

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase involved in various biological functions, including cell survival, proliferation, migration, and adhesion. FAK is an essential factor for transforming growth factor to induce myofibroblast differentiation. In the present study, we investigated whether the targeted inhibition of FAK by using a specific inhibitor, TAE226, has the potential to regulate pulmonary fibrosis. TAE226 showed inhibitory activity of autophosphorylation of FAK at tyrosine 397 in lung fibroblasts. The addition of TAE226 inhibited the proliferation of lung fibroblasts in response to various growth factors, including platelet-derived growth factor and insulin-like growth factor I, in vitro. TAE226 strongly suppressed the production of type I collagen by lung fibroblasts. Furthermore, treatment of fibroblasts with TAE226 reduced the expression of -smooth muscle actin induced by transforming growth factor , indicating the inhibition of differentiation of fibroblasts to myofibroblasts. Administration of TAE226 ameliorated the pulmonary fibrosis induced by bleomycin in mice even when used late in the treatment. The number of proliferating mesenchymal cells was reduced in the lungs of TAE226-treated mice. These data suggest that FAK signal plays a significant role in the progression of pulmonary fibrosis and that it can become a promising target for therapeutic approaches to pulmonary fibrosis.

American Journal of Respiratory Cell and Molecular Biology


Suppressive effect of RAS inhibitor manumycin A on aberrant crypt foci formation in the azoxymethane-induced rat colorectal carcinogenesis model.

Manumycin A suppressed ACF formation in the AOM-induced colorectal carcinogenesis model, demonstrating that RAS inhibitors may be very effective for chemoprevention of colorectal cancers.

The mean number of ACF in the 8-week manumycin A group (72.9 ± 20.1) was significantly lower than in the vehicle group (155.6 ± 56.7, P < 0.01), and it was significantly lower even in the 4-week manumycin A group than in the vehicle group (92.2 ± 13.0 vs 222.3 ± 83.3, P < 0.01). The positive rate for phosphorylated ERK in the manumycin A group (13.5 ± 19.2%) was significantly lower than in the vehicle group (50.2 ± 19.8%, P < 0.01). The positive rate for Ki-67 in the manumycin A group (2.2 ± 3.4%) was significantly lower than in the vehicle group (14.7 ± 8.2%, P < 0.01). There were significantly more terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling-positive cells in tissue samples from the manumycin A group versus the vehicle group (8.6 ± 9.7% vs 2.9 ± 2.0%, P < 0.05).

Journal of Gastroenterology and Hepatology


Inhibitory effect of soluble EP2 receptor on ovarian tumor growth in nude mice and utility of TMPRSS4 as a conbinatorial molecular target

We have previously reported that FuEP2/Ex2, a soluble decoy receptor for PGE2, suppresses tumor growth in an orthotopic xenograft model. To examine whether it has further uses, we examined the effect of FuEP2/Ex2 in an intraperitoneal metastasis model of ovarian cancer cells. We established FuEP2/Ex2-expressing ovarian cancer cells (SKOV/ip-FuEP2/Ex2) and injected them intraperitoneally into female nude mice. Mice injected with SKOV/ip-FuEP2/Ex2 had no ascitic fluid and showed smaller tumor lesions compared to mice injected with vector control cells, with decreased microvessel density and M2 macrophages. To identify molecular targets for combination treatment, we conducted cDNA microarray analysis and found three genes encoding enzyme [matrix metalloproteinase-7 (MMP-7), transmembrane protease serin 4 (TMPRSS4) and cytocrome P450 1B1 (CYP1B1)] to be upregulated in SKOV/ip-FuEP2/Ex2-derived tumors. Administration of TMPRSS4 inhibitor further reduced tumor weight and decreased the number of Ki-67-positive cells in SKOV/ip-FuEP2/Ex2-injected mice. These data indicate a possible EP-targeting strategy using FuEP2/Ex2 in the treatment of ovarian cancer and suggest that dual targeting of EP-mediated signaling and TMPRSS4 may enhance therapeutic value.


Induction of retinol-binding protein 4 and placenta-specific 8 expression in human prostate cancer cells remaining in bone following osteolytic tumor growth inhibition by osteoprotegerin

New drugs that inhibit the osteoprotegerin (OPG)/receptor activator of NF-κB ligand (RANKL)/RANK pathway have demonstrated efficacy for the treatment of bone metastasis. Toxicities induced by these drugs, however, including osteonecrosis of the jaw and hypocalcemia, may adversely affect therapy. The aim of this study was to identify additional therapeutic targets that can be combined with OPG/RANKL/RANK pathway inhibition in the treatment of prostate cancer bone metastasis. We established a stable transfectant that produces high levels of OPG mRNA and protein from PC-3 human prostate cancer cells (PC3-OPG). The culture medium of PC3-OPG cells significantly inhibited the differentiation of mouse monocytes into mature osteoclasts. Furthermore, when PC3-OPG cells were injected into the bones of nude mice, bone destruction and tumor-induced osteoclast formation were reduced. Injection into bone of the mixtures containing equal amounts of green fluorescent protein (GFP)-expressing PC-3 cells (PC3-GFP) and PC3-OPG cells also reduced bone destruction, compared to the control mixture. PC3-GFP cells were subsequently isolated from bone tumors and used for microarray analysis to assess changes in gene expression following osteolytic tumor growth inhibition by OPG. We selected the top 10 upregulated genes based on results from microarrays and confirmed mRNA expression of each gene by RT-PCR. The expression patterns of retinol-binding protein 4 (RBP4) and placenta-specific 8 (PLAC8) were consistent with microarray results. Expression of these genes was also increased in the bone tumors of PC3-GFP/PC3-OPG-injected mice. Knockdown of both RBP4 and PLAC8 by siRNA inhibited the growth of PC-3 cells in vitro. Thus, RBP4 and PLAC8 may become new therapeutic targets for prostate cancer bone metastasis, in combination with OPG/RANKL/RANK pathway inhibition.


Antifibrotic effects of CXCR4 antagonist in bleomycin-induced pulmonary fibrosis in mice.

Circulating fibrocytes had been reported to migrate into the injured lungs, and contribute to fibrogenesis via chemokine-chemokine receptor systems including CXCL12-CXCR4 axis. Here we hypothesized that blockade of CXCR4 might inhibit the migration of fibrocytes to the injured lungs and the subsequent pulmonary fibrosis. To explore the antifibrotic effects of blockade of CXCR4, we used a specific antagonist for CXCR4, AMD3100, in bleomycin-induced pulmonary fibrosis model in mice. Administration of AMD3100 significantly improved the loss of body weight of mice treated with bleomycin, and inhibited the fibrotic lesion in subpleural areas of the lungs. The quantitative analysis demonstrated that treatment with AMD3100 reduced the collagen content and fibrotic score (Aschcroft score) in the lungs. Although AMD3100 did not affect cell classification in bronchoalveolar lavage fluid on day 7, the percentage of lymphocytes was reduced by AMD3100 on day 14. AMD3100 directly inhibited the migration of human fibrocytes in response to CXCL12 in vitro, and reduced the trafficking of fibrocytes into the lungs treated with bleocmycin in vivo. These results suggest that the blockade of CXCR4 might be useful strategy for therapy of patients with pulmonary fibrosis via inhibiting the migration of circulating fibrocytes.

The Journal of Medical Investigation : JMI


Molecular mechanisms of apoptosis induction by 2-dodecylcyclobutanone, a radiolytic product of palmitic acid, in human lymphoma U937 cells

The irradiation of fat-containing food forms 2-dodecylcyclobutanone (2-DCB) from palmitic acid (PA). In this study, we investigated whether 2-DCB and PA induce apoptosis in human lymphoma U937 cells. We found that cell viability decreased by 2-DCB and apoptosis was induced by 2-DCB and PA. 2-DCB and PA significantly enhanced the formation of intracellular reactive oxygen species (ROS). Apoptosis induced by 2-DCB and PA was strongly prevented by an antioxidant, N-acetyl-L: -cysteine. The treatment with 2-DCB and PA resulted in the loss of mitochondrial membrane potential, and Fas, caspase-8 and caspase-3 activation. Pretreatment with a pan-caspase inhibitor (z-VAD) significantly inhibited apoptosis induced by 2-DCB and PA. Moreover, 2-DCB and PA also induced Bax up-regulation, the reduction in Bcl-2 expression level, Bid cleavage and the release of cytochrome c from the mitochondria to the cytosol. In addition, an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) was observed after the treatment with 2-DCB and PA. Our results indicated that intracellular ROS generation, the modulation of the Fas-mitochondrion-caspase-dependent pathway and the increase in [Ca(2+)](i) involved in apoptosis are induced by 2-DCB and PA in U937 cells.


Autopsy case of amebic granulomatous meningoencephalitis caused by Balamuthia mandrillaris in Japan

Balamuthia mandrillaris is a free-living ameba that causes amebic encephalitis. Herein, we report an autopsy case of Balamuthia encephalitis proven with polymerase chain reaction (PCR) and immunohistochemistry from paraffin-embedded brain biopsy specimens. A 68-year-old Japanese male presented at a hospital with progressive right hemiparesis approximately 3 months before his death. An open-brain biopsy specimen showed diffuse meningitis with massive coagulative necrosis. The perivascular spaces contained numerous lymphocytes, histiocytes and giant cells, although the etiology was not determined. The patient deteriorated into coma and died from cerebral herniation. Autopsy revealed abundant trophozoites and cysts in the subarachnoid and Virchow-Robin's spaces. Electron-micrographs of the amebic cysts showed a characteristic triple-walled envelope. The amebas were identified as Balamuthia mandrillaris based on immunohistochemical analysis from the autopsy and biopsy specimens. Primer sets designed to amplify approximately 200 bp bands of mitochondrial 16S rRNA gene of Balamuthia by PCR produced positive results from the biopsy specimens but negative results from the autopsy specimens. In summary, PCR to amplify shorter segments of DNA may be of diagnostic value in detecting suspected cases of balamuthiasis in formalin-fixed, paraffin-embedded specimens. Increased awareness and timely diagnosis of Balamuthia encephalitis might lead to earlier initiation of therapy and improved outcome.


Nerve growth factor derived from bronchial epithelium afer chronic mite antigen exposure contributes to airway hyperresponsiveness by inducing hyperinnervaiton, and is inhibited by in vivo siRNA.

Bronchial asthma is a chronic allergic airway inflammatory disease. Neurotrophins, including nerve growth factor (NGF), play an important role in the pathogenesis of asthma. However, the effects of NGF derived from epithelium on airway hyperresponsiveness (AHR) after antigen sensitization/exposure remain uncertain. In this study, we examined the role of NGF on AHR after chronic antigen exposure and the effect of inhibiting NGF by in vivo siRNA on AHR exacerbation. We generated chronic mouse models of bronchial asthma using house-dust mite antigen (Dermatophagoides pteronyssinus; Dp). NGF concentrations in bronchoalveolar lavage fluid (BALF), lung histopathology, hyperresponsiveness, and related neuronal peptides and cytokines in supernatants of lung homogenates were determined. NGF in BALF was increased in a dose- and time-dependent manner, and was expressed primarily in bronchial epithelium. Nerve fibres and substance P-positive fibres were detected in subepithelium of Dp-sensitized and challenged mice over 4 weeks of mite antigen exposure. AHR was positively correlated with NGF concentration and nerve fibre innervation. AHR, modulation of innervation, and increased substance P were inhibited by in vivo administration of siRNA that targeted NGF, although the inhibition of NGF did not affect allergic inflammation and subepithelial fibrosis. These findings suggest that NGF derived from bronchial and alveolar epithelium plays an important role in AHR after chronic exposure to mite antigen. NGF inhibition could potentially manage bronchial asthma, including AHR.


High susceptibility to zymbal gland and intestinal carcinogenesis in diabetic Otsuka Long-Evans Tokushima Fatty rats.

Diabetes mellitus (DM) and obesity are believed to be risk factors for colorectal cancer in humans. In experiment 1, male nondiabetic Long-Evans Tokushima Otsuka (LETO) rats and Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model animal of type 2 DM, were whole-body X-irradiated (4 Gy) at 6 and 8 weeks of age and euthanized at 78 weeks of age (n=15, respectively). The incidences of small intestine adenocarcinoma in LETO and OLETF rats were 0% and 30%, respectively. In experiment 2, male LETO and OLETF rats (n=24, respectively) were given s.c. injections of 15 mg/kg azoxymethane (AOM) once weekly for 3 weeks and euthanized at 36 weeks of age. The incidences of Zymbal gland tumors in LETO and OLETF rats were 0% and 67%, respectively (P<0.001), whereas those of small intestine adenocarcinoma were 0% and 43% (P<0.001) and those of cecum/colon adenocarcinoma were 46% and 79% (P<0.05), respectively. Fatty change of hepatocytes was common in OLETF rats (63%) but not in LETO rats. Serum triglyceride and free fatty acid levels in OLETF rats were significantly higher than in LETO rats at sacrifice, whereas serum insulin levels in OLETF rats were very diverse. These data suggest that hyperlipidemia plays a significant role in high susceptibility to lower intestinal tract carcinogenesis in OLETF rats; this strain is susceptible to AOM-induced Zymbal gland carcinogenesis.


A mutation in the immunoproteasome subunit PSMB8 causes autoinflammation and lipodystrophy in humans.

Proteasomes are multisubunit proteases that play a critical role in maintaining cellular function through the selective degradation of ubiquitinated proteins. When 3 additional subunits, expression of which is induced by IFN-, are substituted for their constitutively expressed counterparts, the structure is converted to an immunoproteasome. However, the underlying roles of immunoproteasomes in human diseases are poorly understood. Using exome analysis, we found a homozygous missense mutation (G197V) in immunoproteasome subunit, type 8 (PSMB8), which encodes one of the subunits induced by IFN- in patients from 2 consanguineous families. Patients bearing this mutation suffered from autoinflammatory responses that included recurrent fever and nodular erythema together with lipodystrophy. This mutation increased assembly intermediates of immunoproteasomes, resulting in decreased proteasome function and ubiquitin-coupled protein accumulation in the patient's tissues. In the patient's skin and B cells, IL-6 was highly expressed, and there was reduced expression of PSMB8. Downregulation of PSMB8 inhibited the differentiation of murine and human adipocytes in vitro, and injection of siRNA against Psmb8 in mouse skin reduced adipocyte tissue volume. These findings identify PSMB8 as an essential component and regulator not only of inflammation, but also of adipocyte differentiation, and indicate that immunoproteasomes have pleiotropic functions in maintaining the homeostasis of a variety of cell types.


Inhibitory effect of soluble PDGFRbeta on intraosseous growth of breast cancer cells in nude mice

Bone metastasis is a frequent complication of advanced breast cancer. On the basis of functional and molecular evidence, signaling mediated by the binding of platelet-derived growth factor (PDGF)-BB and -DD to PDGF receptor β (PDGFRβ) is critical for the survival and growth of metastatic breast cancer cells within the bone microenvironment. In this study, we propose a new approach to blocking PDGFRβ signaling using soluble PDGFRβ (sPDGFRβ) as a decoy receptor for PDGF-BB and -DD secreted from tumor cells and bone marrow stromal cells. A bone-seeking TNBCT/Bo cell line was established by in vivo selection from TNBCT human breast cancer cells, which are negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 protein expression. The TNBCT/Bo cells were transfected with a mammalian expression vector encoding the extracellular domain of PDGFRβ. A stable transfectant (TNBCT/Bo-sPDGFRβ) grew at a similar rate to that of control cells under normal culture conditions, although growth stimulation of human fibroblasts with PDGF-BB was neutralized by the culture medium from TNBCT/Bo-sPDGFRβ cells. Intratibial injection of TNBCT/Bo-sPDGFRβ cells into athymic nude mice resulted in a significant decrease in tumor incidence compared with control mice (P < 0.01). This attenuated growth correlated with decreased cancer cell proliferation, angiogenesis, and recruitment of stromal cells, and with an increase in the number of apoptotic cells. These findings suggest that sPDGFRβ is useful for the treatment of breast cancer bone metastasis.


Expression of p53, Ki-67, E-cadherin, N-cadherin and TOP2A in triple-negative breast cancer.

Elucidation of the biological features of triple negative breast cancer (TNBC) is important for deciding treatment strategies. The expression of a number of biomarkers in TNBC was analyzed to elucidate those features. The subjects were 134 TNBC patients. Immunohistochemical staining was employed to analyze for eight biomarkers: cytokeratin 5/6 (CK5/6), epidermal growth factor receptor (EGFR), p53, Ki-67 antigen (Ki-67), E-cadherin, N-cadherin, topoisomerase 2 alpha (TOP2A) and B-cell lymphoma 2 (BCL-2), which were then correlated with the nuclear grade (NG), tumor diameter, and the presence/absence of lymph node metastasis, distant recurrence and lymphatic infiltration. Significantly more high than low NG TNBC exhibited positive p53, Ki-67, E-cadherin and TOP2A. High N-cadherin and TOP2A expression was shown significantly in TNBC with lymphatic infiltration, and N-cadherin was also significantly positively expressed in node metastasis-positive cases. EGFR and CK5/6 were positively expressed in high NG TNBC, but not significantly. Analysis for expression of p53, Ki-67, E-cadherin, N-cadherin and TOP2A is meaningful for deciding treatment strategies for TNBC.


Among triple-negative breast cancers, HER2(0) breast cancer shows a strong tendency to be basal-like compared with HER2(1+) breast cancer: preliminary results.

Diagnosis of triple-negative breast cancer (ER-negative, PgR-negative, HER2-negative; TNBC) is performed by means of immunohistological staining. HER2-negative includes HER2(0) and HER2(1+), based on differences in the staining intensity, but there have been no reports on comparison of these two types in TNBC. Accordingly, this study was designed to investigate the possible differences in the biological characteristics of HER2(0) breast cancer and HER2(1+) breast cancer in TNBC. Tissue specimens from 89 TNBC patients were immunohistochemically stained for CK5/6, EGFR, p53, Ki67, E-cadherin, TOP2A and Bcl-2. The expressions of these markers and the clinicopathological findings were compared between the HER2(0) patient group and the HER2(1+) patient group. When either CK5/6 or EGFR was positive, the specimen was judged to be the basal-like phenotype of breast cancer. The percentages of CK5/6- and/or EGFR-positive specimens in the HER2(0) and HER2(1+) groups were 44.9 and 16.8%, respectively, showing that there was a significantly greater number of basal-like phenotype patients in the HER2(0) group (p < 0.01). The percentage of E-cadherin-positive specimens in the HER2(0) group was 66.6%, which was significantly greater than the 40.0% recorded in the HER2(1+) group (p < 0.05). The respective percentages of TOP2A-positive specimens in the HER2(0) and HER2(1+) groups were 55.0 and 30.0%, and the difference was statistically significant (p < 0.05). In TNBC, HER2(0) breast cancer showed a strong tendency to include more of the basal-like phenotype compared with HER2(1+) breast cancer. The staining results indicated the possibility that HER2(0) breast cancer and HER2(1+) breast cancer have different characteristics.


The solubla EP2 receptor FuEP2/Ex2 suppresses endometrial cancer cell growth in an orthotopic xenograft model in nude mice

Endometrial cancer is one of the most common gynecologic malignancies and many factors influence in its growth and development. As in many other types of cancer, prostaglandin E(2) (PGE(2)) is thought to be an accelerator of cell proliferation and endometrial cancer progression. In this study, we examined the effect of FuEP2/Ex2, a soluble decoy receptor for PGE(2) on growth of endometrial cancer cells. A stable transfectant expressing FuEP2/Ex2 was established from human endometrial cancer Ishikawa cells (Ish-FuEP2/Ex2). Ish-FuEP2/Ex2 cells expressed FuEP2/Ex2 mRNA and protein. Expression levels of E-prostanoid receptor 1 (EP1), EP2, EP3, EP4, and F-prostanoid receptor (FP) were almost the same in Ish-FuEP2/Ex2 and vector control cells. Growth rates of Ish-FuEP2/Ex2 under normal culture conditions were also similar to vector control cells, although PGE(2)-induced growth stimulation was completely inhibited in Ish-FuEP2/Ex2 or by Ish-FuEP2/Ex2 culture medium. Moreover, phosphorylation of extracellular signal-regulated kinase (ERK) and induction of cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF), cyclin D1, and c-fos mRNA by PGE(2) were not observed in Ish-FuEP2/Ex2 and Ish-FuEP2/Ex2 culture medium-treated vector control cells, although they were found when treated with prostaglandin F(2α). An orthotopic xenograft model in athymic nude mice revealed that Ish-FuEP2/Ex2-injected mice had significantly decreased mean tumor area. The proportion of Ki-67-positive cells in the tumor lesion was also significantly lower in Ish-FuEP2/Ex2-injected mice. These findings suggest that an EP-targeting strategy using FuEP2/Ex2 may be of use in the treatment of endometrial cancer.


Blockade of Th1 chemokine receptors ameliorates pulmonary granulomatosis in mice.

Sarcoidosis is a granulomatous disease of unknown aetiology. We identified immunological targets for the treatment of pulmonary granulomatosis using a murine model generated with Propionibacterium acnes. Sensitisation and challenge using heat-killed P. acnes and dendritic cells (DCs) were performed to produce pulmonary granulomatosis in C57BL/6 mice. Immunological analyses using ELISA as well as cDNA microarray analysis were used to search for cytokines or chemokines associated with the formation of granulomas in the lungs. Co-administration of P. acnes and DCs reproducibly induced the formation of pulmonary granulomas, which resembled sarcoid granulomas. The cDNA microarray assay demonstrated that the gene expression of CXCL9 and CXCL10, ligands for CXCR3, and of CCL4, a ligand for CCR5, was strongly upregulated during granulomatosis. ELISA confirmed that levels of CXCL9 and CXCL10 as well as T-helper (Th)1 cytokines and chemokines including tumour necrosis factor-α and interferon-γ were elevated in bronchoalveolar lavage fluid (BALF). The blockade of Th1 chemokine receptors using TAK-779, a dual blocker for CXCR3 and CCR5, led to reduced numbers of CXCR3+CD4+ and CCR5+CD4+ T-cells in BALF. Furthermore, administration of TAK-779 ameliorated the granulomatosis. The targeted inhibition of Th1 chemokines might be useful for inhibiting Th1-biased granulomatous diseases, including sarcoidosis.


Enhancement of osteoclastogenic activity in osteolytic prostate cancer cells by physical contact with osteoblasts

The interaction between prostate cancer cells and osteoblasts is critical for the development of bone metastasis. Metastatic cancer cells may physically contact osteoblasts in the bone microenvironment; however, the biological significance of this interaction is not fully understood. Human prostate cancer cells (the osteolytic cell line PC-3 and the osteoblastic cell line MDA-PCa 2b) and human osteoblasts (hFOB1.19) were cocultured under two different conditions (bilayer and contact conditions). Differential gene expression profiles of prostate cancer cells were then investigated using microarray analysis. Differentially expressed genes were analysed using RT-PCR and western blotting, and the effect of anti-cadherin neutralising antibodies on their expression was assayed. The osteoclastogenic activity of cells grown under these different conditions was also investigated using an in vitro assay. When PC-3 or MDA-PCa 2b cells were cocultured with hFOB1.19 cells under contact conditions, the expression of eight genes was upregulated and that of one gene was downregulated in PC-3 cells compared with gene expression in bilayer culture. No differentially expressed genes were detected in MDA-PCa 2b cells. Four of the eight upregulated genes (interleukin-1β (IL-1β), cyclooxygenase-2 (COX-2), IL-6 and the third component of complement (C3)) have already been reported to participate in osteoclastogenesis. Indeed, a cell lysate of PC-3 cells grown under contact coculture conditions significantly enhanced osteoclastogenesis in vitro (P<0.005). neutralisation of cadherin-11 with a specific antibody inhibited upregulation of COX-2 and C3 mRNA in PC-3 cells. In contrast, neutralisation of N-cadherin induced upregulation of COX-2 mRNA. Physical contact between osteolytic prostate cancer cells and osteoblasts may upregulate osteoclastogenesis-related gene expression in prostate cancer cells and enhance osteoclastogenesis. Additionally, cadherin-11 and N-cadherin are involved in this process. These data provide evidence supporting new therapies of prostate cancer bone metastasis that target direct cancer-cell-osteoblast cell-cell contact.


A retrospective study on TS mRNA expression and prediction of the effects of adjuvant oral 5-fluorouracil in breast cancer

Nucleic acid-metabolizing enzymes, such as thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), thymidine phosphorylase (TP) and orotate phosphoribosyl transferase (OPRT), have attracted attention as candidates for response determinants of 5-fluorouracil (5-FU). Whether the expression levels of these enzymes can be adopted as valuable parameters for 5-FU sensitivity in breast cancer has yet to be elucidated. In the present study, intratumoral mRNA expression of TS, DPD, TP and OPRT were determined in formalin-fixed paraffin-embedded surgical specimens collected from 217 breast cancer patients, using the Danenberg Tumor Profile method, which combines microdissection and real-time-polymerase chain reaction. The significance of these enzymes as prognostic and 5-FU efficacy-predicting factors was evaluated. Our data showed that a low DPD expression is related to a high nuclear grade and other factors including hormone receptor-negativity. Low expression levels of TP were found in hormone receptor-negative tumors. TS and OPRT expression were not related to various clinicopathological factors, but patients with a high TS mRNA expression showed a significantly poorer prognosis in cases where 5-FU was not administered. The efficacy of 5-FU was more significant when administered for more than 6 months in the group with a high TS mRNA expression. These data suggest that TS mRNA expression in breast cancer tissue is an ideal predictor of outcomes for patients with no administration of 5-FU, and of the efficacy of 5-FU.


IκB kinase β inhibitor IMD-0354 suppresses airway remodelling in a Dermatophagoides pteronyssinus-sensitized mouse model of chronic asthma

Nuclear factor (NF)-κB is a transcription factor that regulates cytokine and chemokine production in various inflammatory diseases, including bronchial asthma. IκB kinase (IKK) β is important for NF-κB activation in inflammatory conditions, and is possibly related to airway remodelling. Thus, inhibition of the IKKβ-NF-κB pathway may be an ideal strategy for the management of airway remodelling. We examined the effects of a newly synthesized IKKβ inhibitor, IMD-0354, in a chronic allergen exposure model of bronchial asthma in mice. A chronic mouse model was generated by challenge with house dust mite antigen (Dermatophagoides pteronyssinus). IMD-0354 was administrated intraperitoneally in therapeutic groups. Lung histopathology, hyperresponsiveness and the concentrations of mediators and molecules in supernatants of lung homogenates were determined. NF-κB activation was inhibited by prolonged periods of IMD-0354 administration. IMD-0354 reduced the numbers of bronchial eosinophils. IMD-0354 also inhibited the pathological features of airway remodelling, including goblet cell hyperplasia, subepithelial fibrosis, collagen deposition and smooth muscle hypertrophy. Inhibition of these structural changes by IMD-0354 was the result of the suppressing the production and activation of remodelling-related mediators, such as TGF-β, via inhibition of IKKβ. IMD-0354 inhibited IL-13 and IL-1β production, and it restored the production of IFN-γ. It also ameliorated airway hyperresponsiveness. IKKβ plays crucial roles in airway inflammation and remodelling in a chronic mouse model of asthma. A specific IKKβ inhibitor, IMD-0354, may be therapeutically beneficial for treating airway inflammation and remodelling in chronic asthma.


Low susceptibility to N-ethyl-N-nitrosourea-induced transplacental carcinogenesis in Long-Evans Cinnamon (LEC) rats

The Long-Evans Cinnamon (LEC) rat, an animal model of Wilson's disease, is resistant to a variety of chemical carcinogenesis except liver and colon. In the present study, N-ethyl-N-nitrosourea (ENU)-induced transplacental carcinogenesis was examined in male and female LEC, Long-Evans Agouti (LEA), a sibling line of the LEC rat, and F344 rats (n=21). ENU was administered to pregnant rats as a single s.c. injection at a dose of 60 mg/kg body weight on the 17th day after conception. Cerebral/spinal gliomas and trigeminal/spinal nerve schwannomas developed in both LEA and F344 rats at 30 weeks of age, but no nervous system tumors developed in LEC rats, the difference being statistically significant. Lung adenomas also developed in LEA and F344 rats, but not in LEC rats. Semiquantitative RT-PCR demonstrated that metallothionein (MT)1a, MT2 and O(6)-methylguanine-DNA methyltransferase (MGMT) mRNA levels in the liver of LEC rats were higher than those in F344 and LEA rats. In addition, Western blot analysis showed that MT (MT1 plus MT2) in the liver of LEC rats was also higher than that in other strains. Present results suggest that high levels of MT and/or MGMT contribute to the resistance to nitrosamine-induced carcinogenesis in LEC rats.

The Journal of Medical Investigation : JMI


Soluble EP2 neutralizes prostaglandin E2-induced cell signaling and inhibits osteolytic tumor growth

Prostaglandin E2 (PGE2) plays a key role in osteolytic bone metastasis as well as roles in inflammation, cell growth, and tumor development. PGE2 exerts its effects by binding and activating E-prostanoid receptor (EP). In this study, we propose a new approach for blocking EP-mediated cell signaling using a soluble chimeric EP2 fragment. Mammalian expression vectors encoding several human EP2 cDNAs were introduced into 293 cells and the culture medium was tested for their function as a decoy receptor for PGE2. PGE2 binding assays revealed that culture medium containing the second extracellular region of EP2 (FuEP2/Ex2) had binding activity. FuEP2/Ex2 neutralized PGE2-induced cyclic AMP production, cyclic AMP-responsive element binding protein phosphorylation, and subsequent induction of cyclooxygenase-2, interleukin (IL)-1beta, and IL-6 mRNAs. In human osteoblasts, this culture medium neutralized the induction of receptor activator of nuclear factor-kappaB ligand mRNA. A stable transfectant expressing FuEP2/Ex2 was established from human prostate cancer PC-3 cells (PC3-FuEP2/Ex2). PC3-FuEP2/Ex2 cells grew at similar rates to vector control cells under normal culture conditions, although PGE2-induced growth stimulation was suppressed. Intraosseous injection of PC3-FuEP2/Ex2 cells into the tibia of athymic nude mice revealed that the degrees of tumor growth and osteolysis were decreased compared with control cell-injected mice, with decreased osteoclasts and increased apoptotic cells. Furthermore, the cyclooxygenase-2, IL-1beta, and IL-6 mRNA levels were reduced in the tumor lesions. These data suggest that FuEP2/Ex2 is useful for treating osteolytic bone metastasis and cancers that depend on EP signaling for their growth and development.


Development of Inflammatory Bowel Disease in Long-Evans Cinnamon Rats Based on CD4+CD25+Foxp3+ Regulatory T Cell Dysfunction

A mutant strain with defective thymic selection of the Long-Evans Cinnamon (LEC) rat was found to spontaneously develop inflammatory bowel disease (IBD)-like colitis. The secretion of Th1-type cytokines including IFN-gamma and IL-2 from T cells of mesenteric lymph node cells (MLNs) and lamina propria mononuclear cells, but not spleen cells, in LEC rats was significantly increased more than that of the control Long-Evans Agouti rats through up-regulated expression of T-bet and phosphorylation of STAT-1 leading to NF-kappaB activation. In addition, the number of CD4(+)CD25(+)Foxp3(+) regulatory T (Treg) cells of the thymus, MLNs, and lamina propria mononuclear cells from LEC rats was significantly reduced, comparing with that of the control rats. Moreover, bone marrow cell transfer from LEC rats into irradiated control rats revealed significantly reduced CD25(+)Foxp3(+) Treg cells in thymus, spleen, and MLNs compared with those from control rats. Indeed, adoptive transfer with T cells of MLNs, not spleen cells, from LEC rats into SCID mice resulted in the development of inflammatory lesions resembling the IBD-like lesions observed in LEC rats. These results indicate that the dysfunction of the regulatory system controlled by Treg cells may play a crucial role in the development of IBD-like lesions through up-regulated T-bet, STAT-1, and NF-kappaB activation of peripheral T cells in LEC rats.


Epistasis between hyperglycemic QTLs revealed in a double congenic of the OLETF rat

Glucose homeostasis is believed to be regulated by multiple genetic components, in addition to numerous external factors. It is therefore crucial to dissect and understand what roles each causative gene plays in maintaining proper glucose metabolism. In OLETF (Otsuka Long-Evans Tokushima Fatty) rat, a model of polygenic type 2 diabetes, at least 14 quantitative trait loci (QTLs) influencing plasma glucose levels were identified. In congenic strains some of the OLETF allelic variants were shown to increase glucose levels. In this study the focus was on two of the hyperglycemic loci, Nidd1/of and Nidd2/of. Congenic rats possessing OLETF genome fragment at either locus showed similar levels of mild hyperglycemia. A newly established double congenic rat showed a further aggravation of hyperglycemia. The Nidd1/of locus was also shown to function in the reduction of plasma leptin levels and fat weights, while the Nidd2/of locus led to increased plasma insulin and fat weights. Interestingly, both plasma leptin and fat weights reverted to the control levels in the double congenic rat. These results indicate that there is an epistatic interaction between the two loci. However, it is unlikely that the abnormal level of enhanced glucose homeostasis is mediated, at least not directly, by leptin or fat mass.


Possible application of human c-Ha-ras proto-oncogene transgenic rats in a medium-term bioassay model for carcinogens.

With the aim of developing a medium-term assay for screening of environmental carcinogens, we exposed mammary carcinogen sensitive human c-Ha-ras proto-oncogene transgenic (Hras128) rats to various carcinogens, including compounds that do not normally induce mammary tumors. Seven-week-old Hras128 rats and wild-type littermates received administrations of 3-methylcholanthrene (3-MC), benzo[a]pyrene (B[a]P), anthracene, pyrene, 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 4-(methyl-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK), dimethylarsinic acid (DMA), diethylnitrosamine (DEN) or azoxymethane (AOM) and were sacrificed at week 12 (females) (at week 10 for the 3-MC group) or week 20 (males). Female Hras128 rats receiving NNK, DEN, or DMA showed a significant increase in mammary tumor incidence and/or multiplicity compared to the respective values with olive oil or deionized distilled water (DDW) vehicles. In male Hras128 rats, a significant increase in mammary tumors was also observed in groups administered 3-MC, B[a]P, anthracene, IQ, and NNK. Mutations of transgenes were observed in codons 12 and/or 61 in the induced tumors by PCR-RFLP except in the DEN group in female and in the MeIQx group in male Hras128 rats. Thus various carcinogens, not necessarily limited to those normally targeting the breast, were found to induce mammary carcinomas in Hras128 rats, especially in females, pointing to potential use for medium-term screening.


Alteration of intra-pancreatic target-organ specificity by abrogation of Aire in NOD mice

Factors that determine the spectrum of target organs involved in autoimmune destruction are poorly understood. Although loss of function of autoimmune regulator (AIRE) in thymic epithelial cells is responsible for autoimmunity, the pathogenic roles of AIRE in regulating target-organ specificity remain elusive. In order to gain insight into this issue, we have established NOD mice, an animal model of type 1 diabetes caused by autoimmune attack against beta cell islets, in which Aire has been abrogated. Remarkably, acinar cells rather than beta cell islets were the major targets of autoimmune destruction in Aire-deficient NOD mice, and this alteration of intra-pancreatic target-organ specificity was associated with production of autoantibody against pancreas-specific protein disulfide isomerase (PDIp), an antigen expressed predominantly by acinar cells. Consistent with this pathological change, the animals were resistant to the development of diabetes. The results suggest that Aire not only is critical for the control of self-tolerance but is also a strong modifier of target-organ specificity through regulation of T cell repertoire diversification. We also demonstrated that transcriptional expression of PDIp was retained in the Aire-deficient NOD thymus, further supporting the concept that Aire may regulate the survival of autoreactive T cells beyond transcriptional control of self-protein expression in the thymus.


Essential role of IkappaB kinase alpha in thymic organogenesis required for the establishment of self-tolerance.

IkappaB kinase (IKK) alpha exhibits diverse biological activities through protein kinase-dependent and -independent functions, the former mediated predominantly through a noncanonical NF-kappaB activation pathway. The in vivo function of IKKalpha, however, still remains elusive. Because a natural strain of mice with mutant NF-kappaB-inducing kinase (NIK) manifests autoimmunity as a result of disorganized thymic structure with abnormal expression of Rel proteins in the thymic stroma, we speculated that the NIK-IKKalpha axis might constitute an essential step in the thymic organogenesis that is required for the establishment of self-tolerance. An autoimmune disease phenotype was induced in athymic nude mice by grafting embryonic thymus from IKKalpha-deficient mice. The thymic microenvironment that caused autoimmunity in an IKKalpha-dependent manner was associated with defective processing of NF-kappaB2, resulting in the impaired development of thymic epithelial cells. Thus, our results demonstrate a novel function for IKKalpha in thymic organogenesis for the establishment of central tolerance that depends on its protein kinase activity in cooperation with NIK.


Prevention of lethal hepatic injury in Long-Evans Cinnamon(LEC) rats by D-galactosamine hydrochloride

Repeated injections of D-galactosamine hydrochloride (GalN) increase the survival rate of Long-Evans Cinnamon (LEC) rats, an animal model of Wilson's disease. The aim of the present study was to investigate the mechanism of GalN for prevention of spontaneous lethal hepatic injury in LEC rats. Male LEC rats were given a single subcutaneous injection of 300 mg/kg of GalN or vehicle (0.9% NaCl) at 14 weeks, and killed at 28 weeks of age. Next, 6-week-old male LEC rats were given weekly subcutaneous injections of 300 mg/kg of GalN or vehicle for 3 or 12 weeks, and their hepatic 8-hydroxydeoxy-2'-guanosine (8-OHdG), glutathione peroxidase (GPX), and catalase activities were measured. None of GalN-treated rats died of hepatic injury (0/12), whereas the mortality rate of control rats given 0.9% NaCl was 17% (2/12). GalN administration for 12 weeks decreased the hepatic 8-OHdG, and GalN administration for either 3 or 12 weeks increased the glutathione peroxidase activity. GalN administration increased the serum level of alanine aminotransferase, and accelerated megalocytic degeneration of the hepatocytes. GalN treatment is effective in preventing lethal hepatitis in LEC rats and decrease of oxidative DNA damage by GalN plays an important role in increase of the survival rate.


Laminin 5 expression protects against anoikis at aerogenous spread and lepidic growth of human lung adenocarcinoma

Adenocarcinoma of the lung is characterized by frequent aerogenous spread (AE) and advancement along the alveolar wall (BAC growth). To elucidate the mechanism of AE metastasis and BAC growth in human lung adenocarcinoma, we established an in vivo orthotopic animal model and an in vitro culture. Investigation of expression levels of integrins, laminins and Type IV collagens, which are the major regulating molecules for cell attachment and anoikis was carried out and a clear correlation between the expression level of laminin 5 (LN5) and the BAC growth was observed using an orthotopic animal model. Introduction of LN5 cDNA to A549 cells increased anoikis resistance in an expression dependent manner. Cells with LN5 overexpression resisted with anoikis after treatment with PI3K-Akt and ERK inhibitors. The amount of phosphorylated focal adhesion kinase (FAK) was also higher in LN5 overexpressing cells. Major tyrosine residues of the EGF receptor at 1068, 1086 and 1173, except at 1148, remained phosphorylated only in the LN5 overexpressing cells even without EGF stimulation, that indicates the ligand independent activation of EGF receptor. BAC growth ratio and AE was confirmed to be significantly correlated with LN5 expression in surgically resected human lung adenocarcinomas by immunohistochemistry. Our results indicate that the activation of the EGF receptor by overexpressing LN5-integrin-FAK signaling pathway may play a crucial role in BAC growth and AE metastasis in human lung adenocarcinoma.


Imatinib as a Novel Antifibrotic Agent in Bleomycin-induced Pulmonary Fibrosis in Mice

Imatinib mesylate is a potent and specific tyrosine kinase inhibitor against c-ABL, BCR-ABL, and c-KIT, and has been demonstrated to be highly active in chronic myeloid leukemia and gastrointestinal stromal tumors. We examined the antifibrotic effects of imatinib using a bleomycin-induced lung fibrosis model in mice because imatinib also inhibits tyrosine kinase of platelet-derived growth factor receptors (PDGFRs). Imatinib inhibited the growth of primary murine lung fibroblasts and the autophosphorylation of PDGFR-beta induced by PDGF. Administration of imatinib significantly prevented bleomycin-induced pulmonary fibrosis in mice, partly by reducing the number of mesenchymal cells incorporating bromodeoxyuridine. Analysis of bronchoalveolar lavage cells demonstrated that imatinib did not suppress early inflammation on Days 7 and 14 caused by bleomycin. These results suggest that imatinib has the potential to prevent pulmonary fibrosis by inhibiting the proliferation of mesenchymal cells, and that imatinib might be useful for the treatment of pulmonary fibrosis in humans.

American Journal of Respiratory and Critical Care Medicine


Development of autoimmunity against transcriptionally unrepressed target antigen in the thymus of Aire-deficient mice

Autoimmune regulator (AIRE) gene mutation is responsible for the development of organ-specific autoimmune disease with monogenic autosomal recessive inheritance. Although Aire has been considered to regulate the elimination of autoreactive T cells through transcriptional control of tissue-specific Ags in thymic epithelial cells, other mechanisms of AIRE-dependent tolerance remain to be investigated. We have established Aire-deficient mice and examined the mechanisms underlying the breakdown of self-tolerance. The production and/or function of immunoregulatory T cells were retained in the Aire-deficient mice. The mice developed Sjogren's syndrome-like pathologic changes in the exocrine organs, and this was associated with autoimmunity against a ubiquitous protein, alpha-fodrin. Remarkably, transcriptional expression of alpha-fodrin was retained in the Aire-deficient thymus. These results suggest that Aire regulates the survival of autoreactive T cells beyond transcriptional control of self-protein expression in the thymus, at least against this ubiquitous protein. Rather, Aire may regulate the processing and/or presentation of self-proteins so that the maturing T cells can recognize the self-Ags in a form capable of efficiently triggering autoreactive T cells. With the use of inbred Aire-deficient mouse strains, we also demonstrate the presence of some additional factor(s) that determine the target-organ specificity of the autoimmune disease caused by Aire deficiency.


Inhibitory effects of Centella asiatica on azoxymethane-induced aberrant crypt focus formation and carcinogenesis in the intestines of F344 rats

Effects of the water extract of Centella asiatica Linn. on formation of azoxymethane (AOM)-induced aberrant crypt foci (ACF) and intestinal tumorigenesis in male F344 rats were investigated. Treatment with the extract significantly decreased the number of larger ACF (with four or more crypts per focus) in the large intestine in the early stage, while the number of methylated DNA adducts was not decreased compared with that in the AOM-treated group. In the post-initiation stage, the extract significantly decreased the total number of ACF and the number of larger ACF, accompanied by a decrease in the 5-bromo-2'-deoxyuridine-labeling index and an increase in the induction of apoptotic cells in the colonic mucosa. The incidences of neoplasms, the numbers of adenocarcinomas in the small intestines and entire intestines, and sizes of neoplasms in the entire intestines in rats fed C. asiatica extract at a dose of 10 mg/kg were smaller than those in rats given AOM alone (p < 0.05). The extract at a dose of 100 mg/kg significantly reduced the multiplicity of neoplasms in the small intestine (p < 0.05). These results suggest that inhibition of the formation of AOM-induced ACF by C. asiatica extract is associated with modification of cell proliferation and induction of apoptosis in colonic crypts and that the extract has a chemopreventive effect on colon tumorigenesis.


Platelet-derived growth factor stimulates glucose transport in skeletal muscles of transgenic mice specifically expressing PDGF receptor in the muscle, but does not affect blood glucose levels

Insulin stimulates the disposal of blood glucose into skeletal muscle and adipose tissues by the translocation of GLUT4 from intracellular pools to the plasma membrane, and consequently the concentration of blood glucose levels decreases rapidly in vivo. Phosphatidylinositol (PI) 3-kinase and Akt play a pivotal role in the stimulation of glucose transport by insulin, but detailed mechanisms are unknown. We and others reported that not only insulin but also platelet-derived growth factor (PDGF) and epidermal growth factor facilitate glucose uptake through GLUT4 translocation by activation of PI 3-kinase and Akt in cultured cells. However, opposite results were also reported. We generated transgenic mice that specifically express the PDGF receptor in skeletal muscle. In these mice, PDGF stimulated glucose transport into skeletal muscle in vitro and in vivo. Thus, PDGF apparently shares with insulin some of the signaling molecules needed for the stimulation of glucose transport. The degree of glucose uptake in vivo reached approximately 60% of that by insulin injection in skeletal muscle, but blood glucose levels were not decreased by PDGF in these mice. Therefore, PDGF-induced disposal of blood glucose into skeletal muscle is insufficient for rapid decrease of blood glucose levels.


Endothelin-1(1 31) levels are increased in atherosclerotic lesions of the thoracic aorta of hypercholesterolemic hamsters

The novel vaso-constricting 31-amino acid-length endothelin-1 [ET-1(1-31)] is selectively produced by human mast cell chymase via its action on big ET-1. However, the pathological role of ET-1(1-31) in atherosclerosis remains unclear. The aim of this study was to clarify vasoconstrictive response and expression of ET-1(1-31) in atherosclerotic aorta. Syrian golden hamster, was used for preparing the atherosclerotic models by the administration of a high cholesterol diet (HC), treatment with the nitric oxide synthase inhibitor (Nomega-nitro-L-arginine methylester, L-NAME) alone, or both (HC and L-NAME) for 40 weeks. Early atherosclerosis was observed in the case of HC or L-NAME alone treatments respectively and severe atherosclerosis was observed in the case of combined HC and L-NAME treatment. Vasoconstriction induced by ET-1(1-31) was not altered by the atherosclerotic changes, but the expression pattern of ET-1(1-31) was different at each stage of the atherosclerotic aorta. ET-1(1-31) was observed rarely in normal aortas or in early atherosclerotic lesions, but ET-1(1-31) expression was dramatically increased in aortic neointima and adventitia in a state of atherosclerosis with severe inflammation. ET-1(1-31) might play in a role of promoting atherosclerosis, and especially be involved in inflammatory mediation during the progression of atherosclerosis.


Direct inhibition of EGF receptor activation in vascular endothelial cells by gefitinib ('Iressa', ZD1839)

The development of gefitinib ('Iressa', ZD1839) by targeting the EGFR tyrosine kinase is a recent therapeutic highlight. We have reported that gefitinib is antiangiogenic in vitro, as well as in vivo. In this study, we asked if the anti-angiogenic action of gefitinib is due to a direct effect on activation of vascular endothelial cells by EGF. EGF, as well as VEGF, caused pronounced angiogenesis in an avascular area of the mouse cornea, and i.p. administration of gefitinib almost completely blocked the response to EGF, but not to VEGF. Immunohistochemical analysis demonstrated phosphorylation of EGFR by EGF in the neovasculature, and gefitinib markedly reduced this effect. Gefitinib also inhibited downstream activation of ERK 1/2 via EGFR in cultured microvascular endothelial (HMVE) cells. These findings suggest that the anti-angiogenic effect of gefitinib in the vascular endothelial cells of neo-vasculature is partly attributable to direct inhibition of EGFR activation, and that endothelial cells in malignant tumors play a critical role in the cancer therapeutic efficacy of gefitinib.


KATP Channel Knockout Mice Crossbred with Transgenic Mice Expressing a Dominant-Negative Form of Human Insulin Receptor have Glucose Intolerance but not Diabetes

Impaired insulin secretion and insulin resistance are thought to be two major causes of type 2 diabetes mellitus. There are two kinds of diabetic model mice: one is a K(ATP) channel knockout (Kir6.2KO) mouse which is defective in glucose-induced insulin secretion, and the other is a transgenic mouse expressing the tyrosine kinase-deficient (dominant-negative form of) human insulin receptor (hIR(KM)TG), and which has insulin resistance in muscle and fat. However, all of these mice have no evidence of overt diabetes. To determine if the double mutant Kir6.2KO/hIR(KM)TG mice would have diabetes, we generated mutant mice by crossbreeding, which would show both impaired glucose-induced insulin secretion and insulin resistance in muscle and fat. We report here that: 1) blood glucose levels of randomly fed and 6 h fasted double mutant (Kir6.2KO/hIR(KM)TG) mice were comparable with those of wild type mice; 2) in intraperitoneal glucose tolerance test (ipGTT), Kir6.2KO/hIR(KM)TG mice had an impaired glucose tolerance; and 3) during ipGTT, insulin secretion was not induced in either Kir6.2KO/hIR(KM)TG or Kir6.2KO mice, while the hIR(KM)TG mice showed a more prolonged insulin secretion than did wild type mice; 4) hyperinsulinemic euglycemic clamp test revealed that Kir6.2KO, Kir6.2KO/hIR(KM)TG and hIR(KM)TG mice, showed decreased whole-body glucose disposal compared with wild type mice; 5) Kir6.2KO, but not Kir6.2KO/hIR(KM)TG mice had some obesity and hyperleptinemia compared with wild type mice. Thus, the defects in glucose-induced insulin secretion (Kir6.2KO) and an insulin resistance in muscle and fat (hIR(KM)TG) were not sufficient to lead to overt diabetes.


NF-κB-inducing kinase establishes self-tolerance in a thymic stroma-dependent manner

Physical contact between thymocytes and the thymic stroma is essential for T cell maturation and shapes the T cell repertoire in the periphery. Stromal elements that control these processes still remain elusive. We used a mouse strain with mutant NF-kappaB-inducing kinase (NIK) to examine the mechanisms underlying the breakdown of self-tolerance. This NIK-mutant strain manifests autoimmunity and disorganized thymic structure with abnormal expression of Rel proteins in the stroma. Production of immunoregulatory T cells that control autoreactive T cells was impaired in NIK-mutant mice. The autoimmune disease seen in NIK-mutant mice was reproduced in athymic nude mice by grafting embryonic thymus from NIK-mutant mice, and this was rescued by supply of exogenous immunoregulatory T cells. Impaired production of immunoregulatory T cells by thymic stroma without normal NIK was associated with altered expression of peripheral tissue-restricted Ags, suggesting an essential role of NIK in the thymic microenvironment in the establishment of central tolerance.


Localization of the 31-amino-acid endothelin-1 in hamster tissue

Endothelin (ET)-1(1-31) is a novel vasoconstrictor peptide produced by human mast cell chymase, which selectively cleaves big ET-1 at the Try(31)-Gly(32) bond. We investigated the localization of ET-1(1-31) in various hamster tissues by immunohistochemistry and compared it to the distribution of ET-1(1-21). We found that the localization and amount of ET-1(1-31) were different from those of ET-1(1-21) in each tissue. ET-1(1-31)-like immunoreactivities (IR) in the heart, lung, and adrenal gland were observed in the same areas as ET-1(1-21) but were significantly weaker, suggesting that ET-1(1-31) might play a role only in mast cell/chymase-related pathological conditions in these tissues. In the liver, ET-1(1-31)-like IR was strongly detected in Kupffer cells where ET-1(1-21)-like IR was seen more weakly. In the kidney, ET-1(1-31)-like IR was slightly higher than ET-1(1-21). These results suggest that ET-1(1-31) might have physiological roles distinct from those of ET-1(1-21) in some hamster tissues.


Enhancement of hepatocarcinogenesis by kojic acid in rat two-stage models after initiation with N-bis(2-hydroxypropyl)nitrosamine or N-diethylnitrosamine


Increased Insulin Sensitivity and Hypoinsulinemia in APS Knockout Mice

A tyrosine kinase adaptor protein containing pleckstrin homology and SH2 domains (APS) is rapidly and strongly tyrosine phosphorylated by insulin receptor kinase upon insulin stimulation. The function of APS in insulin signaling has heretofore remained unknown. APS-deficient (APS(-/-)) mice were used to investigate its function in vivo. The blood glucose-lowering effect of insulin, as assessed by the intraperitoneal insulin tolerance test, was increased in APS(-/-) mice. Plasma insulin levels during fasting and in the intraperitoneal glucose tolerance test were lower in APS(-/-) mice. APS(-/-) mice showed an increase in the whole-body glucose infusion rate as assessed by the hyperinsulinemic-euglycemic clamp test. These findings indicated that APS(-/-) mice exhibited increased sensitivity to insulin. However, overexpression of wild-type or dominant-negative APS in 3T3L1 adipocytes did not affect insulin receptor numbers, phosphorylations of insulin receptor, insulin receptor substrate-1, or Akt and mitogen-activated protein kinase. The glucose uptake and GLUT4 translocation were not affected by insulin stimulation in these cells. Nevertheless, the insulin-stimulated glucose transport in isolated adipocytes of APS(-/-) mice was increased over that of APS(+/+) mice. APS(-/-) mice also showed increased serum levels of leptin and adiponectin, which might explain the increased insulin sensitivity of adipocytes.


The role of mast cells in acute tubulo-interstitial nephritis with uveitis

We describe the clinicopathological characteristics of two patients with acute tubulo-interstitial nephritis with uveitis (TINU) with mast cells infiltrating the interstitium. The pathogenesis of TINU remains unknown, but a T-cell-mediated immune response was suggested to be involved. Recent studies have shown that infiltrating mast cells are closely associated with the development of renal interstitial fibrosis in glomerulonephritis. To address the role of mast cells in the renal interstitial injury in TINU, immunohistochemical studies were performed in renal biopsy sections using anti-human mast cell tryptase antibody specific for mast cells. In addition, we tried to detect CD68-positive macrophages to compare with the localisation of mast cells within the renal interstitium. Mast cells and macrophages could be detected in renal interstitial lesions of both patients. Massive infiltration of macrophages into interstitial lesions was observed, whereas mast cells were detected in a sporadic rather than a clustered manner, and associated with fibrotic lesions. Repeat renal biopsy findings suggested the involvement of these cells in the renal interstitial injury because the number of infiltrating mast cells and macrophages in the interstitium decreased with the improvements in clinical symptoms and pathological lesions. CONCLUSION: The present study showed that mast cells might play an important role in the development of renal interstitial injury in tubulo-interstitial nephritis with uveitis.


A case of acute tubulointerstitial nephritis and uveitis syndrome with mast cells

肥満細胞浸潤を認めたぶどう膜炎を伴う間質性腎炎症候群の1女児例

We describe the clinicopathological characteristics of a patient with acute tubulointerstitial nephritis and uveitis syndrome (TINU) with mast cells infiltrating in the interstitium. Repeated biopsies were performed before and after treatment of prednisolone. The pathogenesis of TINU remains unknown, but the T cell-mediated immune response or autoantibodies is considered to be involved. Recently, mast cells have been postulated to play a role in renal interstitial fibrosis. We investigated the localization of mast cells in the biopsy sections by immunohistochemistry. First renal biopsy showed that the mast cells infiltrated into the interstitial fibrotic lesion. Second biopsies showed the number of mast cells is decreased with the improvement of clinical symptoms and pathological lesions. These suggested that mast cells play an important role in the development of renal inerstitial injury in TINU.

ステロイド治療により臨床症状と検査所見の改善がみられたぶどう膜炎を伴う間質性腎炎症候群 (TINU) の1女児例を経験した．本症例の生検腎組織について免疫染色法で肥満細胞を同定し，間質病変におけるその役割を検討した．肥満細胞は，腎間質線維化部位に浸潤し，治療後の組織所見の改善とともに浸潤肥満細胞数は減少した．この結果からTINUの間質障害に肥満細胞が関与する可能性が示唆された．

Japanese Journal of Pediatric Nephrology


Cytological analysis of glycogen-rich carcinoma of the breast: report of two cases

Glycogen-rich carcinoma is a rare special histologic subtype of breast cancer and its incidence is estimated to be 1.4% in breast malignancies. However, its precise characteristics in cytological specimens have not yet been fully clarified. Fifty-nine-year-old and 53-year-old women underwent fine-needle aspiration biopsy cytology (FNABC) of a breast tumor, confirming malignancy. A mastectomy with axillary dissection was performed. Cytologically, a moderate amount of eosinophilic, finely granular cytoplasm was seen in the majority of the tumor cells, however, foamy and vacuolated cytoplasm was noted in some tumor cells. Histologically, the tumor cells of both cases had clear and granular cytoplasm, which showed a positive reaction with periodic acid-Schiff, eliminated by diastase. While clear cytoplasm in the tumor cells in the FNABC seemed to be a pivotal cytological characteristic of glycogen-rich carcinoma, it may not be a major component of cytological specimens. Routine periodic acid-Schiff staining may be required to diagnose glycogen-rich carcinoma in cytological methods.

The Journal of Medical Investigation : JMI


An immunohistochemical evaluation of biomarkers and apoptotic cells in ductal carcinoma in situ of breast


Cases Report of Malignant Mucocele-like Lesions

Mucocele-like lesions (MLL) of the breast have been reported as extremely rare as well as benign, but now it is believed they can be both malignant and benign. This paper describes two cases of malignant MLL, both subjected to immunohistochemical staining. Case 1: A 42-year-old woman with multiple malignant MLLs without evidence of a mass at presentation to our hospital after biopsy, but whose ultrasonogram showed small multiple hypoechoic lesions. Case 2: A 70-year-old woman, whose left breast cancer was found on routine mammography after modified radical mastectomy for right breast cancer. Pathologically, MLL with intraductal carcinoma was diagnosed. Case 1 underwent two lumpectomies in 3 years, but even now new lesions have developed. However, the patient refused to have another operation. C-erbB2 was negative in both cases. When malignant MLL is histologically of low grade, excisional biopsy is sufficient for a single MLL with intraductal carcinoma, while it may be necessary to perform a subcutaneous mastectomy for multiple lesions.


Huang, L., Ogushi, F., Tani, K., Ogawa, H., Kawano, T., Endo, T., Izumi, K., Ueno, J., Nishitani, H. and Sone, S. Thrombin Promotes Fibroblast Proliferation during the Early Stages of Experimental Radiation Pneumonitis. Radiat. Res. 156, 45-52 (2001). To clarify the role of thrombin in the pathogenesis of radiation-induced pneumonitis, we measured the thrombin activity and fibroblast growth-inducing activity in bronchoalveolar lavage fluid obtained from the irradiated lungs of rats at 1, 2, 4, 8 and 18 weeks after irradiation. Thrombin activity was not detected in the bronchoalveolar lavage fluid from unirradiated rats, but the bronchoalveolar lavage fluid from irradiated rats showed significantly increased thrombin activity which reached a maximum at 4 weeks after treatment. Higher fibroblast growth-inducing activity was detected in the bronchoalveolar lavage fluid from irradiated rats at 4 and 18 weeks than in fluid from unirradiated rats. Bronchoalveolar lavage fluid from irradiated rats that were pretreated with the thrombin inhibitors antithrombin III and argatroban showed significantly inhibited fibroblast growth-inducing activity and thrombin activity at 4 weeks. However, these thrombin inhibitors did not inhibit fibroblast growth-inducing activity in bronchoalveolar lavage fluid from irradiated rats at 18 weeks. Purified rat thrombin similarly induced proliferation of fibroblasts derived from irradiated and unirradiated rats. These findings suggest that thrombin may play an important role as a fibroblast growth-inducing factor during the early stages of radiation pneumonitis.


So-called mesothelial/monocytic incidental cardiac excrescences obtained during valve replacement surgery: report of three cases and literature review.

We present three cases of so-called mesothelial/monocytic incidental cardiac excrescences (MICE) of the heart and a brief review of related literature. Case 1 was a 51-year-old woman who underwent mitral- and aortic-valve replacement. A tissue sample was submitted as a thrombus attached to the left atrial endocardium. Case 2 was a 69-year-old woman who underwent mitral-valve replacement. The sample was incidentally obtained as whitish clot-like fragments, but its exact origin was not known. Case 3 was a 68-year-old woman who underwent mitral-valve replacement for suspected infective endocarditis. The sample adherent to the pericardium was removed after valvular surgery. Histologically, these lesions were composed of a mixture of plump histiocytoid cells, a papillary arrangement of cuboidal cells, various sized vacuoles, and fibrin. The nests of cuboidal cells resembled cancer cells but showed features of mesothelial cells and no proliferative activity, immunohistochemically or ultrastructurally. In all cases, a suction tube placed in the left atrium was occasionally used to remove overflowing intrapericardial fluid during the surgery. The tip of the suction tube was covered with spiral wire, which is likely to transfer the stripped pericardial mesothelial cells to the left atrium. The significance of MICE is their possibility of being misdiagnosed as metastatic carcinoma by pathologists and a risk of arterial embolization by mesothelial debris clinically.


Identification of a locus for susceptibility to renal cell carcinoma in the Long-Evans Cinnamon rat.

The Long-Evans Cinnamon (LEC) mutant rat shows higher incidence of renal cell carcinomas induced by a treatment with the chemical carcinogen N-diethylnitrosamine, as compared to the normal control rat. We performed the first genome-wide scan for genes responsible for susceptibility to chemically induced renal cell carcinoma in an F2 intercross obtained by mating the LEC and Fischer-344 (F344) rats. The genotype of 71 (F344 x LEC) F2 progenies was determined with the use of 338 simple sequence length polymorphisms (SSLPs) spread over the genome. The F2 rats which carried renal cell carcinoma were shown to possess the incidence of homozygosity of the LEC allele which is higher than that of the other genotypes at SSLP markers on chromosome 5 (chi2 = 17.5 for D5Rat21). Our linkage analysis has led to the revelation of a novel gene that influences susceptibility to renal cell carcinoma on rat chromosome 5.

The Journal of Veterinary Medical Science


Effects of D-galactosamine hydrochloride and partial hepatectomy on spontaneous hepatic injury and hepatocarcinogenesis in Long-Evans Cinnamon rats


Role of Copper Accumulation in Spontaneous Renal Carcinogenesis in Long-Evans Cinamon Rats

Spontaneous renal cell tumors in totals of 223 male and female Long-Evans Cinnamon (LEC) rats of 51-120 weeks old, 157 male F344 rats of 51-120 weeks old, and 14 male Long-Evans Agouti (LEA) rats of 51-70 weeks old were examined histologically. The incidences of renal cell tumors increased with age in male and female LEC rats, but no tumors developed in F344 or LEA rats. Dilated atypical tubules of the kidneys were observed at high incidence in aged LEC rats. Copper staining of LEC rat kidneys showed a positive reaction in proximal tubules of the cortex and the outer stripe of the medulla. The renal copper concentration of LEC rats reached a peak in the period of necrotizing hepatitis with renal tubular necrosis, and was higher than that in F344 rats for up to 106 weeks. In contrast, the renal iron concentration of LEC rats was lower than that in F344 rats except in the period of necrotizing hepatitis. Long-term treatment of LEC rats with D-penicillamine, a copper-chelating agent, inhibited accumulation of copper, but not iron, in the kidneys, and inhibited the development of karyomegaly of proximal tubules and dilated atypical tubules. These results suggest that persistent copper accumulation after toxic necrosis of tubules is the major cause of spontaneous renal carcinogenesis in LEC rats.


Modifying effects of a mixture of acetaminophen, aspirin, dipyrone and ethenzamide on a multiorgan initiation model and its carcinogenicity in male F344 rats


Non-carcinogenicity of Capsaicinoids in B6C3F1 Mice


Comparative tumorigenicity of 1- and 3-nitrobenzo[a]pyrenes, and 3,6- and 1,6-dinitrobenzo[a]pyrenes in F344/DuCrj rats

Our earlier study revealed that 1- and 3-nitrobenzo[a]pyrene (NBP), 1,6- and 3,6-dinitrobenzo[a]pyrene (DNBP), nitrated derivatives of benzo[a]BP (BP), are present in the environment. These derivatives are potent mutagens for Salmonella tester strains and we have preliminarily reported them to be carcinogenic in F344/DuCrj rats. In this study, the tumorigenic action of 1- and 3-NBP, 1.6- and 3,6-DNBP, and BP induced by subcutaneous injection into rats was found to differ according to the NO2-substitution in the BP structure. The chemicals were suspended in equal volumes of beeswax and tricaprylin, and rats were subcutaneously injected with single doses of 500, 1000, and 2000 microg for 1- and 3-NBP, and of 8, 40, 200, and 1000 microg for 3,6- and 1,6-DNBP, and BP as a positive control. 3,6-DNBP and BP induced tumors in a dose-dependent manner at the injection site. Rats given 1000 microg of 3,6-DNBP (2924 nmol) and BP (3968 nmol) developed subcutaneous tumors at the rate of 70 and 80%, respectively, and those given a minimum dose of 23 nmol for 3.6-DNBP and 32 nmol for BP per rat developed tumors at a rate of 4.8 and 18.2%, respectively. However, rats given 500 and 1000 microg of 1- and 3-NBP did not develop any tumors while those given a high dose, 2000 microg, of each chemical developed tumors at only one of ten animals used. It was concluded, therefore, that these chemicals are weak carcinogens. Histologically, most of the tumors were malignant fibrous histiocytomas. Rats given various doses of 1,6-DNBP did not develop any tumors at the injection site. The failure of 1,6-DNBP to induce tumors may involve its metabolites because of the lower mutagenicity of its reduction products, 1-nitroso-6-NBP and 1-amino-6-NBP. It is suggested, therefore, that tumorigenicities of NBPs and DNBPs differ according to the NO2-substitution on the chemical structure, which may be due to the possible nitroreduction of the chemicals.


A case with Small Cell Carcinoma of the Esophagus:Measurement of Tumor Markers,Including Pro-gastrin-Releasing Peptide

Small cell carcinoma of the esophagus is a rare clinical entity and the accumulation of information is necessary to clarify its clinical behavior. We report a 69-year-old Japanese man with this rare disease with systemic metastases, including liver, bone and lymphnodes. The patient was treated with systemic chemotherapy consisting of 300 mg/m2 of carboplatin on day 1, and 80 mg/m2 of etoposide on days 1, 2 and 3. Although transient relief of subjective symptoms was obtained, the disease showed systemic progress, and the patient died on day 25 of chemotherapy. During the clinical course of the disease, serum pro-gastrin-releasing peptide (proGRP) decreased upon systemic chemotherapy from elevated level (54.6 pg/ml) to normal range (19.2 pg/ml). Further study is warranted to examine whether measurement of serum proGRP may yield valuable information on the diagnosis and monitoring activities of esophageal small cell carcinoma.


Assessment of the biological malignancy of hepatocellular carcinoma: relationship to clinicopathological factors and prognosis

It is difficult to determine the prognosis of patients with hepatocellular carcinoma (HCC). Assessment of the clinicopathological and biological malignancy of HCC may help in determining treatment strategies and predicting outcome. The tumor DNA content, p53 protein expression, proliferating cell nuclear antigen labeling index, and argyrophilic proteins of nuclear organizer regions were used as markers of biological malignancy. A correlation between these biological parameters and clinicopathological factors was sought. DNA aneuploidy was observed in 31 of 80 tumors (38.8%). Aneuploidy increased as differentiation decreased. The overall survival rate of patients with aneuploid tumors was significantly poorer than that of patients with diploid tumors. p53 overexpression was observed in 18 of 80 tumors (22.5%). The incidence of p53 positivity increased significantly with increasing tumor size and poorer differentiation. The overall survival rate of p53-positive patients was significantly worse than that of p53-negative patients. The proliferating cell nuclear antigen labeling index and the mean number of argyrophilic proteins of nuclear organizer regions were higher in more poorly differentiated lesions. We conclude that DNA ploidy and p53 expression are useful prognostic indicators in HCC. Cell proliferation increases as HCC progresses. With progression, tumors tend to become more poorly differentiated.


Effects of tacrolimus (FK506) on induction of glutathione S-transferase P-positive foci and liver cell regeneration after partial hepatectomy in F344 rats


Hyperinsulinemia but no diabetes in transgenic mice homozygously expressing the tyrosine kinase-deficient human insulin receptor.

We generated transgenic mice homozygous for the tyrosine kinase-deficient human insulin receptor (hIRK1030M(+/+)) under control of the insulin receptor promoter. Similar growth patterns and results of glucose tolerance tests were observed among normal, heterozygous, and homozygous mice. Insulin tolerance test indicated no significant difference in the hypoglycemic response to insulin among the three genotypes. However, the serum insulin levels of the homozygous mice before and after glucose loading (201.42 +/- 58.15 pg/ml to 578.57 +/- 49.03 pg/ml) were significantly higher than in the control mice (100.92 +/- 19.55 pg/ml to 356.36 +/- 55.08 pg/ml; p < 0.01 and p < 0.01, respectively) and heterozygous mice (74.46 +/- 18.55 pg/ml to 352.33 +/- 52.43 pg/ml; p < 0.005 and p < 0.01, respectively). Immunohistological evidence of pancreatic islets showed no significant difference among the three genotypes. Taken together, these results suggest that the tyrosine kinase-deficient insulin receptor causes hyperinsulinemia but not diabetes in these homozygous transgenic mice.

Biochemical and Biophysical Research Communications


p53 Gene Mutation in N-(4-hydroxybutyl) nitrosamine-induced Urinary Bladder Tumors and N-methyl-N-nitrosourea-induced Colon Tumors of Rats.


The Tokushima Journal of Experimental Medicine


Distribution of the kexin family proteases in pancreatic islets. --- PACE4C is specifically expressed in B cells of pancreatic islets

Kexin Family Proteasesはペプチドホルモン等のプロセシング酵素として同定され, 血糖調節ホルモンのインスリン等のプロセシングにも関与することが示唆されていたが, 本研究では我々が新規にクローニングしたPACE4CをはじめとするKexin Family Proteasesとインスリン等との膵島細胞での局在, 共存関係を初めて明らかにし, その成果は雑誌表紙にも掲載された.


Loss of heterozygosity on chromosome arm 17p in small cell lung carcinomas, but not in neurofibromas, in a patient with von Recklinghausen neurofibromatosis.

The formation of SCLC may result from several genetic alterations, including inactivation of tumor-suppressor gene on chromosome 17p, most likely P53, although it still is unknown whether or not a mutation of the NF1 gene on 17q was involved in the development of SCLC in this patient.


Hepatic Cytomegalovirus Involvement in Autopsy Cases

Hepatic involvement of cytomegalovirus (CMV) was studied in 15 autopsy cases with disseminated or solitary CMV infection. Formalin-fixed, paraffin-embedded tissue sections were examined by immunohistochemistry (IHC) using a monoclonal antibody against early and late CMV antigens, and by in situ hybridization (ISH) using biotinylated CMV DNA probes. Three cases showed cytomegalic cells in liver sections by conventional staining, five showed hybridization with CMV DNA probes and seven showed reaction with the monoclonal antibody in the liver. CMV infection was detected not only in cytomegalic cells but also in many non-cytomegalic cells by IHC and ISH, proving these techniques to be superior to routine histological examination. The inflammatory reaction in the liver was not prominent. The reason for the weak inflammatory response in the liver of our present cases, and the possible availability of IHC and ISH for analysis of liver biopsy and bronchoalveolar lavage specimens from immunocompromised hosts were discussed.


Tumor promoting potential in male F344 rats and mutagenicity in Salmonella typhimurium of dipyrone

For assessment of the carcinogenic potential and the mutagenicity of dipyrone, an antipyretic anodyne, -[(2,3-dihydro-1,5-dimethyl-3-oxo-2-phenyl-1H-pyrazol-4-yl) methylamino]-methanesulfonic acid sodium salt monohydrate, three experiments were conducted using dipyrone A produced in Japan and/or dipyrone B obtained from the Federal Republic of Germany. (i) Carcinogenic potential of dipyrone A for rat liver: 8 week old male F344 rats were pretreated with 0.01% diethylnitrosamine (DEN) in drinking water for 2 weeks and, after 1 week of resting, administered 0.4% dipyrone in drinking water, 5 days a week, for 72 weeks. After an 8 week recovery period, all surviving rats were killed at 83 weeks. Hepatocellular carcinomas developed at a higher incidence in the DEN + dipyrone group (18 of 29 rats, 62%) than in the DEN alone group (9 of 29 rats, 31%), the difference being statistically significant (P less than 0.05). No carcinogenic activity of dipyrone was demonstrated in the groups given 0.4% dipyrone for 72 weeks or 0.4% dipyrone for 25 weeks, followed by 0.05% phenobarbital (PB) for 50 weeks. However, glutathione S-transferase P positive (GST-P+) preneoplastic hepatic foci in these groups were observed at a higher incidence than in the untreated control group (P less than 0.01). (ii) Effect of dipyrone A and dipyrone B on induction of DEN-initiated GST-P+ hepatic foci in a medium-term bioassay system: 0.4% dipyrone A in drinking water and 0.57% dipyrone A or dipyrone B in powdered diet after DEN initiation had similar enhancing effects on the development of GST-P+ foci (P less than 0.001). (iii) The Ames mutation test in Salmonella: both dipyrone A and dipyrone B proved weakly mutagenic for strain TA100 in the presence or absence of S9 fraction.


Histopathological and Immunohistochemical Studies on Nickel Sulfide-induced Tumors in F344 Rats

Twenty-five tumors induced in F344 male rats were examined histologically and immunohistochemically using antibodies against myoglobin, myosin, desmin and cathepsin B. Eight were from rats which had been given intramuscular (im) injection and 17 were from rats which had been given subcutaneous (sc) injection of 5 mg of Ni3S2. Among 10 rhabdomyosarcomas, myoglobin was detectable in 3, myosin in 8, and desmin in all, but cathepsin B was present in none. Out of 8 malignant fibrous histiocytomas, cathepsin B was detectable in all, but the other antigens were absent. In a leiomyosarcoma, only desmin was detected. In two fibrosarcomas, none of the markers were detected. In four undetermined tumors, one reacted only with anti-desmin antibody, two with only anti-cathepsin B antibody, and one with none of the antibodies. Of the three myogenic markers utilized in this study, anti-desmin antibody appeared to be the most sensitive. Cathepsin B was found mainly in the histiocytic cells of malignant fibrous histiocytoma. Thus, desmin appears to be particularly valuable in distinguishing immature myogenic tumors from other primitive tumors, while cathepsin B is useful in distinguishing malignant fibrous histiocytoma from other pleomorphic mesenchymal tumors.


Lack of carcinogenicity of phenytoin in (C57BL/6 x C3H)F1 mice

Groups of 50 B6C3F1 mice of each sex were given 0.012% or 0.006% phenytoin in their powdered diet for 78 wk and were then fed a basal diet for 8 wk. Control groups of 50 mice of each sex were fed powdered basal diet for 86 wk. Mean total intakes of phenytoin per mouse were 301 and 150 mg in males, and 292 and 154 mg in females, respectively. The survival rates of each group at week 86 were 72-86% in males, and 86-94% in females. Liver-cell tumors, alveolar tumors, and Harderian-gland adenomas in male mice, malignant lymphomas and/or leukemias in female mice, and a few tumors in other organs of both sexes were found. The total number of hepatocellular tumors in mice treated with the high dose of phenytoin was significantly smaller than that of control mice in males (p less than 0.05). However, hepatocellular carcinomas developed 15 to 3 wk earlier in a few mice of phenytoin-treated males than in the controls. In other organs, no significant increase of any particular tumor type was observed in the treated groups of both sexes. Thus, phenytoin was not carcinogenic in B6C3F1 mice in this study.

Journal of Toxicology and Environmental Health


Induction of subcutaneous tumors in rats by 3,7- and 3,9- dinitrofluoranthene

Two dinitrofluoranthenes (DNFs) derived from 3-nitrofluoranthene were purified to over 99%, and tested for tumorigenicity in F344/DuCrj male rats. Rats were inoculated s.c. with 0.05 mg 3,7- or 3,9-DNF twice a week for 10 weeks. All 21 rats given 3,7-DNF and 10 of 11 rats (91%) given 3,9-DNF developed tumors at the injection site by 48 weeks after the first injection. Twenty of the 21 tumors induced by 3,7-DNF and seven of the 10 tumors induced by 3,9-DNF were classified as malignant fibrous histiocytoma, but one tumor in the 3,7-DNF-treated group and three tumors induced by 3,9-DNF showed typical features of rhabdomyosarcoma. The first tumors in 3,9-DNF-treated rats appeared on day 88 (on day average 117), 10 weeks earlier than in 3,7-DNF-treated ones (on day average 186).


Tumorigenicity Test of 1,3- and 1,8-Dinitropyrene in BALB/c Mice

1,3-Dinitropyrene (DNP) and 1,8-DNP (CAS: 42397-65-9) are very potent mutagens and induce a frameshift-type mutation in the Salmonella test system. Each compound was tested for tumorigenicity in BALB/c mice by sc inoculation of 0.05 mg of the compound once a week for 20 weeks. Tumors developed at the site of injection of 1,8-DNP in 6 of 15 mice up to 60 weeks after the first injection. The incidence of tumors was statistically significant at a P-value of less than .05 but not of less than .01. Therefore, the carcinogenicity of 1,8-DNP in BALB/c mice was concluded to be weaker than that of benzo[a]pyrene [(BP) CAS: 50-32-8], which induced a 100% tumor incidence when it was injected at the same dose as that of 1,8-DNP. No tumors occurred at the injection site in mice given 1,3-DNP. Most of the tumors induced by 1,8-DNP and BP showed histologic features characteristic of malignant fibrous histiocytoma.

Journal of the National Cancer Institute


Dinitro derivatives of pyrene and fluoranthene in diesel emission particulates and their tumorigenicity in mice and rats

A number of mutagens/carcinogens in diesel emission particulates are produced and distributed into the atmosphere. On the basis of the results of chemical analysis, it was found that most of the mutagenicity in diesel particulate extracts is due to super-mutagens such as 1,3-, 1,6- and 1,8-DNP, and 3,7- and 3,9-DNF. 3,7- and 3,9-DNF are new mutagens which were isolated in this study and they induced a frameshift type mutation in the Salmonella microsome test. Each derivative of DNP and DNF was detected at a concentration of 0.01 to 0.03 ppm in the particulates while the diesel engine was used under the condition of idling. 1,6- and 1,8-DNP were tumorigenic at the injection site in BALB/c male mice when a total of 2 and 1 mg, respectively, of the compounds was given subcutaneously. The incidences for tumors by 60 weeks were at a ratio of 50 and 30%, respectively, for 1,6- and 1,8-DNP-treated mice. 4-NQO and BaP, as positive control, induced tumors at the injection site in BALB/c mice when a total of 2 and 1 mg, respectively, of the compound was given. The incidences of tumors were observed at a high ratio of 90 and 93.4%, respectively, for 4-NQO- and BaP-treated mice. Histologically the tumors were diagnosed as malignant fibrous histiocytomas in all the tumors which developed. No tumors occurred at the injection site in mice given injections of 1,3-DNP. Tumorigenicity tests of 3,7- and 3,9-DNF are now being attempted using F344/DuCrj male rats. This animal experiment is now in progress. In the 3,9-DNF-treated rats (a total of 1 mg per rat), subcutaneous tumors developed in 8 of 11 rats up to 150 days after injection, and a part of a tumor resected from a rat showed the typical features of rhabdomyosarcoma.

Developments in Toxicology and Environmental Science


後腹膜悪性線維性組織球腫の1例

後腹膜悪性線維性組織球腫の1例