Biliary atresia (BA) is a disease of the newborn that is characterized by progressive, inflammatory and sclerosing cholangiopathy. Innate immune responses to viral components are thought to be involved in the pathogenesis of BA. It is also reported that some chemokines, such as CCL5, are possibly involved in the pathogenesis of experimental animal model of BA. We treated human biliary epithelial HuCCT1 cells with polyinosinic-polycytidylic acid (poly IC), an authentic double-stranded RNA (dsRNA) which mimics viral RNA, and analyzed the CCL5 expression by quantitative reverse transcription-PCR (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). To examine the regulation mechanisms of CCL5, we subjected the cells to RNA interference (siRNA) against Toll-like receptor 3 (TLR3), interferon (IFN)-β, NF-κB p65 and IFN regulatory factor (IRF) 3. Immunohistochemical staining for CCL5 was also performed in tissues from patients with BA. Poly IC induced CCL5 expression in HuCCT1 cells. CCL5 expression induced by poly IC was inhibited by the knockdown of TLR3, p65 or IRF3, but it was not affected by knockdown of IFN-β. Immunohistochemical staining showed that CCL5 was strongly expressed in biliary epithelial cells of patients with BA. The current study suggests that TLR3 signaling induces CCL5 expression via NF-κB and IRF3 in bile duct cells, and this pathway may be involved in the pathogenesis of BA.
The study was designed to investigate calcification in developing rat mandibular bone using whole mount staining, micro-computed tomography (micro-CT) and scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX). Wistar rats at embryonic days 16, 18, and 20 and postnatal weeks 1 and 6 were used. Rats were fixed with 4% paraformaldehyde and heads were resected, frozen and sectioned for histology, then analysed with SEM-EDX. Some of the specimens were observed with micro-CT. Other rats were fixed and stained with alcian blue and alizarin red for whole mount staining. Histology and whole mount staining showed that osteoid was deposited around Meckel’s cartilage at day 15 and developed into bone at day 16. Accumulation of Ca and P was identified in the bone matrix with SEM-EDX. The area of bone expanded until week 6. The Ca/P ratio increased, whereas the C/Ca and C/P ratios decreased during development. Micro-CT demonstrated an increase in radio-opacity with bone development. The results suggest that rat mandibular bone formation is initiated around Meckel’s cartilage at day 15. Deposition and maturation of the calcium phosphate mineral increase gradually with decrease in the organic component as the rat mandible develops.
Thiamine pyrophosphatase (TPPase) cytochemistry is an established method for specific labeling of the trans-Golgi cisterns in tissue sections. Herein, we combined this enzyme cytochemical method with array tomography using scanning electron microscopy (SEM), a new imaging technique based on collection of backscattered electron (BSE) images of consecutive resin-embedded sections on glass slides, to detect the entire three-dimensional (3D) organization of the Golgi apparatus with sufficient spatial resolution. As the signal intensity of BSE depends on the atomic number of the materials, lead precipitates confined to the trans-Golgi cisterns after TPPase cytochemistry were clearly observed by BSE-mode SEM. The mild fixative used for TPPase cytochemistry also enabled accurate identification of target gonadotropes in the composite pituitary tissue by immunocytochemical staining. By 3D reconstruction of the entire trans-Golgi cisterns based on serial ultrathin section images of tissues after TPPase cytochemistry, we detected ultrastructural differences in the 3D configuration of the Golgi apparatus between cerebellar Purkinje cells and pituitary gonadotropes. The appropriate combination of enzyme cytochemistry and/or immunostaining with array tomography will further clarify the relationship between the organization and functional states of the Golgi apparatus.
Long-term treatment with antiepileptic drugs (AED) is associated with an elevated risk of bone fracture due to decreased bone mineral density (BMD). Phenytoin has been shown to affect bone metabolism adversely, whereas newly developed AEDs have not been studied. This study evaluated the effects of topiramate and lamotrigine on bone metabolism in rats. Five-week-old male Sprague-Dawley rats were treated orally with phenytoin (20 mg/kg), topiramate (5 or 20 mg/kg), or lamotrigine (2 or 10 mg/kg) daily for 12 weeks. Phenytoin reduced bone strength, measured by maximum load to failure of the femoral mid-diaphysis, along with reduced femur total BMD. Serum tartrate-resistant acid phosphatase-5b levels significantly increased after phenytoin treatment, while serum osteocalcin levels decreased after topiramate 20 mg/kg treatment. Furthermore, osteoblast surface and bone mineralizing surface were significantly lowered by topiramate. Lamotrigine treatment did not affect bone strength, BMD, or bone turnover. We demonstrated that phenytoin treatment significantly increased bone resorption and lowered BMD and bone strength. Since lamotrigine did not affect bone metabolism, it can be concluded that lamotrigine is safety medicine for bone health. Topiramate was associated with decreased bone formation, which may affect bone strength and BMD with chronic use. Thus, patients taking topiramate should be monitored for changes in BMD to avoid risk of fracture.
Although the morbidity rate of prostate cancer has increased with 2.3 times in these 10 years in Japan, little is known about the changes in the prevalence of latent prostate cancer. To understand changes in the prevalence of latent prostate cancer, a retrospective analysis was performed. Forensic autopsy findings from Tochigi Prefecture between September 2012 and February 2014 were collected. Two cross sections, from the base and apex of the prostate, were examined histopathologically. The prevalence of latent prostate cancer was compared with findings from forensic autopsies performed between August 2002 and July 2005 in the same region. The prevalence of latent prostate cancer in both groups was similar, showing an overall prevalence of 13.6% and 12.2% and a Gleason score >6 of 6.2% and 7.1%, respectively. When prevalence was compared by cause of death, the values were similar for both groups. The prevalence of latent prostate cancer in this Japanese population did not show any significant change over the past 10 years. The dramatic increase in morbidity rate for prostate cancer could be from the increase in prostate-specific antigen screening and subsequent referral to urologists.
Cordycepin is an active component extracted from Traditional Chinese medical herb Cordyceps militaris. Many reports demonstrated that cordycepin harbors antitumor activity in a broad spectrum of cancer types. In this study the actions and the underneath molecular mechanisms of cordycepin were investigated in renal cell carcinoma Caki-1 cell line. Results showed that cordycepin induced apoptotic cell death and inhibited cell migration in Caki-1 cells. Quantitative real-time PCR results and western blot analyses indicated cordycepin dose-dependently decreased microRNA-21 expression and Akt phosphorylation levels in Caki-1 cells, but increased PTEN phosphatase levels. Block of cordycepin-induced microRNA-21 decrease or PTEN increase in Caki-1 cells by transfection of microRNA-21 mimic or PTEN siRNA significantly attenuated cordycepin-induced cell death and inhibition of cell migration. Taken together, findings in present study suggested that cordycepin induced apoptotic cell death in renal cell carcinoma through regulation of microRNA-21 and PTEN phosphatase. Furthermore, present study also firstly illustrated that cordycepin inhibited cell migration of renal cell carcinoma, which also involved microRNA-21 and PTEN phosphatase.