Novel non‐alcoholic steatohepatitis model with histopathological and insulin‐resistant features

Although several non‐alcoholic steatohepatitis (NASH) models have been reported to date, few of these models fully reflect the histopathology and pathophysiology of human NASH. The aim of this study was to establish a novel NASH model by feeding a high‐fat (HF) diet and administering both carbon tetrachloride (CCl₄) and the Liver X receptor agonist T0901317. Male C57BL/6J mice were divided into four groups (each n = 5): HF, HF + CCl₄, HF + T0901317, and the novel NASH model (HF + CCl₄ + T0901317). CCl₄ (0.1 mL/kg) and T0901317 (2.5 mg/kg) were intraperitoneally administered four times and five times, respectively. The livers of the novel NASH model group presented a whitish colour. The serum levels of TNF‐α and IL‐6 were significantly increased in the novel NASH model group, and mice in this group exhibited histopathological features and insulin resistance reflective of NASH, i.e., macrovesicular hepatic steatosis, ballooning hepatocytes, Mallory‐Denk bodies, lobular inflammation and fibrosis. The novel NASH model group presented significantly upregulated expression levels of mRNAs related to lipogenesis, oxidative stress, fibrosis and steatosis and significantly downregulated expression levels of mRNAs related to triglyceride export. We successfully established a novel experimental NASH model that exhibits similar histopathology and pathophysiology to human NASH.     

Induction of β-Defensin Expression by Porphyromonas gingivalis-Infected Human Gingival Graft Transplanted in nu/nu Mouse Subdermis

It is important to understand the onset of periodontal disease in terms of bacterial infection and host factors. Host-bacteria interactions can be elicited in human cultured cells and animal models, but these models provide only limited biological information about human host reactions against bacterial attacks. Development of an in vivo model using human gingival tissue is needed. We established an in vivo model using nu/nu mice and evaluated host defense following bacterial infection in human gingiva. Human gingival samples were collected from periodontitis patients and transplanted in nu/nu mouse subdermis. After 2 weeks, human characteristics were confirmed by positive immunohistochemical reactions for human-specific markers. We used this model to investigate human β-defensin-2 (hBD-2), an antimicrobial peptide that contributes to initial defense against bacterial invasion. Using real-time polymerase chain reaction, in situ hybridization, and immunohistochemistry, we investigated whether hBD-2 expression was induced in human gingiva as a response to Porphyromonas gingivalis as a periodontal pathogen. Two hours after infection with bacteria, we detected increased expression of hBD-2 mRNA, which was localized in the epithelium of human gingiva. Using our in vivo model, we concluded that increased hBD-2 may play an important role in early defense from bacterial infection in human gingival epithelium.     

Cytolytic Recombinant Vesicular Stomatitis Viruses Expressing STLV-1 Receptor Specifically Eliminate STLV-1 Env-Expressing Cells in an HTLV-1 Surrogate Model In Vitro

Human T-cell leukemia virus type 1 (HTLV-1) causes serious and intractable diseases in some carriers after infection. The elimination of infected cells is considered important to prevent this onset, but there are currently no means by which to accomplish this. We previously developed “virotherapy”, a therapeutic method that targets and kills HTLV-1-infected cells using a cytolytic recombinant vesicular stomatitis virus (rVSV). Infection with rVSV expressing an HTLV-1 primary receptor elicits therapeutic effects on HTLV-1-infected envelope protein (Env)-expressing cells in vitro and in vivo. Simian T-cell leukemia virus type 1 (STLV-1) is closely related genetically to HTLV-1, and STLV-1-infected Japanese macaques (JMs) are considered a useful HTLV-1 surrogate, non-human primate model in vivo. Here, we performed an in vitro drug evaluation of rVSVs against STLV-1 as a preclinical study. We generated novel rVSVs encoding the STLV-1 primary receptor, simian glucose transporter 1 (JM GLUT1), with or without an AcGFP reporter gene. Our data demonstrate that these rVSVs specifically and efficiently infected/eliminated the STLV-1 Env-expressing cells in vitro. These results indicate that rVSVs carrying the STLV-1 receptor could be an excellent candidate for unique anti-STLV-1 virotherapy; therefore, such antivirals can now be applied to STLV-1-infected JMs to determine their therapeutic usefulness in vivo.     

Molecular diversity of TEX101, a marker glycoprotein for germ cells monitored with monoclonal antibodies: variety of the molecular characteristics according to subcellular localization within the mouse testis

TEX101 was characterized as a unique germ cell marker molecule using the specific monoclonal antibody (mAb), TES101. Although this mAb has strong affinity/specificity for TEX101, TES101 mAb loses its reactivity under reducing conditions. In this study, we have generated new mAbs against TEX101 to compensate for the shortcomings of the TES101 mAb using different approaches. First, we immunized mice with the antigen on a baculovirus expression system and isolated new anti-TEX101 mAbs, 6002 and 6035. Second, we raised the mAb Ts4 from spleen cells of an immunologically naive old mouse. Western blot analysis revealed that the new mAbs possess immunoreactivity under reducing/non-reducing conditions. Immunopositive staining of the mAbs against Bouin-fixed sections was observed in spermatocytes, spermatids and testicular spermatozoa, but not in other cells, similar to paraformaldehyde (PFA)-fixed frozen sections stained with TES101 as previously reported. However, whereas the mAbs 6002/6035 mainly showed immunoreactivity only in spermatocytes in PFA-fixed frozen sections, the reactivity of the mAbs to spermatids and testicular spermatozoa was clearly recovered when the PFA-fixed sections were autoclaved or treated with SDS. Peptide mapping and deglycosylation analysis indicated that the epitopes for TES101, 6002 and 6035 are located within TEX101(25-94), whereas Ts4 recognized N-linked carbohydrate moieties on TEX101 in Triton X-100-soluble mouse testicular extracts but not in the extracellular or water-soluble fractions. These results suggest strongly that the molecular association or structure of N-linked carbohydrate moieties of TEX101 varies according to its subcellular localization within the seminiferous tubules. These new mAbs will be valuable tools for further analysis of TEX101, including its function(s).     

Brianodins A-D, briarane-type diterpenoids from soft coral Pachyclavularia sp

Four new briarane-type diterpenoids, brianodins A-D ( 1- 4), were isolated from a soft coral, Pachyclavularia sp., and the structures and relative stereochemistry of 1- 4 were elucidated on the basis of spectroscopic data. The absolute configurations of 3 and 4 were assigned by the MTPA method. Brianodin A ( 1) showed a modest cytotoxicity.     

Fractalkine expression and the recruitment of CX3CR1+ cells in the prolonged mesangial proliferative glomerulonephritis

BACKGROUND: We established the reversible and the prolonged models of mesangial proliferative glomerulonephritis (GN) with anti-Thy 1 antibody 1-22-3. However, the essential factors leading to the prolonged glomerular alterations have not been identified. METHODS: The expressions of several chemokines and cytokines were compared in the reversible and the prolonged models. Expression of fractalkine and the number of the fractalkine receptor CX3CR1-positive cells in the glomeruli in the prolonged model were significantly higher than those in the reversible model. Then, the localization of fractalkine and the characteristics of CX3CR1+ cells were analyzed in glomeruli. To elucidate the significance of the fractalkine expression, we analyzed the expression in the model treated with angiotensin II receptor antagonist, candesartan. RESULTS: Immunostaining of fractalkine was detected on endothelial cells on the fifth day, and fractalkine staining also was detected in the mesangial area on day 14. Major parts of the CX3CR1+ cells in the glomeruli were macrophages, especially ED3+ cells. Candesartan treatment ameliorated the glomerular morphological findings at six weeks after disease induction. Although the treatment did not ameliorate the morphological finding at two weeks, decreased expression of fractalkine and CX3CR1+ were already detected at two weeks in rats treated with candesartan. CONCLUSIONS: Fractalkine expression and the recruitment of CX3CR1+ cells in glomeruli might play an important role in the development of the prolonged disease. These expressions could be predictors of the prolonged disease of the mesangial proliferative glomerulonephritis.     

Late recurrence after resection of mass-forming intrahepatic cholangiocarcinoma: report of a case

The outcome after surgical resection for intrahepatic cholangiocarcinoma has not been satisfactorily evaluated due to its malignant behavior. Surgical resection, however, has the potential to improve the prognosis and may allow surgeons to experience rare cases with long survival. This report presents the case of a patient who developed recurrence 9?years after resection of intrahepatic cholangiocarcinoma. A 76-year-old female was diagnosed to have intrahepatic cholangiocarcinoma and underwent an extended right posterior subsegmentectomy. The gross appearance showed a mass-forming type tumor. The histopathological examination revealed well to moderately differentiated adenocarcinoma associated with portal vein invasion. Subcutaneous metastasis in the head as the first sign of relapse was diagnosed 9?years after hepatectomy. The histopathological findings of the subcutaneous tumor were similar to those of the intrahepatic cholangiocarcinoma, thus suggesting metastasis from intrahepatic cholangiocarcinoma. Positron emission tomography with 2-[fluorine-18]-fluoro-2-deoxy-d-glucose was useful for detecting multiple metastases. Long-term follow-up for more than 5?years is recommended because the present case shows that late recurrence of intrahepatic cholangiocarcinoma occurs even 5?years after resection.     

Correlation between mechanical stress by finite element analysis and 18F‐fluoride PET uptake in hip osteoarthritis patients

¹⁸F‐fluoride positron emission tomography (¹⁸F‐fluoride PET) is a functional imaging modality used primarily to detect increased bone metabolism. Increased ¹⁸F‐fluoride PET uptake suggests an association between increased bone metabolism and load stress at the subchondral level. This study therefore examined the relationship between equivalent stress distribution calculated by finite element analysis and ¹⁸F‐fluoride PET uptake in patients with hip osteoarthritis. The study examined 34 hips of 17 patients who presented to our clinic with hip pain, and were diagnosed with osteoarthritis or pre‐osteoarthritis. The hips with trauma, infection, or bone metastasis of cancer were excluded. Three‐dimensional models of each hip were created from computed tomography data to calculate the maximum equivalent stress by finite element analysis, which was compared with the maximum standardized uptake value (SUV_max) examined by ¹⁸F‐fluoride PET. The SUV_max and equivalent stress were correlated (Spearman's rank correlation coefficient ρ = 0.752), and higher equivalent stress values were noted in higher SUV_max patients. The correlation between SUV_max and maximum equivalent stress in osteoarthritic hips suggests the possibility that ¹⁸F‐fluoride PET detect increased bone metabolism at sites of stress concentration. This study demonstrates the correlation between mechanical stress and bone remodeling acceleration in hip osteoarthritis. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:78–83, 2015.