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Growing evidence indicates that hepatocyte growth factor (HGF) possesses potent antifibrotic activity. Furthermore, macrophages migrate to inflamed sites and have been linked to the progression of fibrosis. In this study, we utilized macrophages as vehicles to express and deliver the HGF gene and investigated whether macrophages carrying the HGF expression vector (HGF-M) could suppress peritoneal fibrosis development in mice. We obtained macrophages from the peritoneal cavity of mice stimulated with 3% thioglycollate and used cationized gelatin microspheres (CGMs) to produce HGF expression vector-gelatin complexes. Macrophages phagocytosed these CGMs, and gene transfer into macrophages was confirmed in vitro. Peritoneal fibrosis was induced by intraperitoneal injection of chlorhexidine gluconate (CG) for three weeks; seven days after the first CG injection, HGF-M was administered intravenously. Transplantation of HGF-M significantly suppressed submesothelial thickening and reduced type III collagen expression. Moreover, in the HGF-M-treated group, the number of α-smooth muscle actin- and TGF-β-positive cells were significantly lower in the peritoneum, and ultrafiltration was preserved. Our results indicated that the transplantation of HGF-M prevented the progression of peritoneal fibrosis and indicated that this novel gene therapy using macrophages may have potential for treating peritoneal fibrosis.

Mammalian spermatogenesis is characterized by disproportionate germ cell apoptosis. The high frequency of apoptosis is considered a safety mechanism that serves to avoid unfavorable transmission of paternal aberrant genetic information to the offspring as well as elimination mechanism for removal of overproduced immature or damaged spermatogenic cells. The molecular mechanisms involved in the induction of germ cell apoptosis include both intrinsic mitochondrial Bcl-2/Bax and extrinsic Fas/FasL pathways. However, little is known about the nuclear trigger of those systems. Recent studies indicate that epigenomes are essential in the regulation of gene expression through remodeling of the chromatin structure, and are genome-like transmission materials that reflect the effects of various environmental factors. In spermatogenesis, epigenetic errors can act as the trigger for elimination of germ cells with abnormal chromatin structure, abnormal gene expression and/or morphological defects (disordered differentiation). In this review, we focus on the relationship between global changes in epigenetic parameters and germ cell apoptosis in mice and other mammals.

Prothymosin alpha (ProTα) is a nuclear protein expressed in virtually all mammalian tissues. Previous studies have shown that ProTα exhibits protective effects against ischemia-induced cell death in various cell types. Recently, the 6-residue peptide P6Q (NEVDQE), the modified form of the active 6-residue core (51–56) in ProTα, has also been shown to have protective effects against retinal ischemia. However, it remains to be elucidated whether P6Q is effective against acute kidney injury (AKI). Therefore, we investigated the renoprotective effect of P6Q on cisplatin-induced AKI. Cultured HK-2 cells were treated with cisplatin for 24 h and pretreatment with ProTα or P6Q was carried out 30 min before cisplatin treatment. Cell viability was evaluated using the MTT assay. In an in vivo study, 8-week-old male Wistar rats were divided into control, cisplatin treated, and cisplatin treated with P6Q injection groups. In the last of these, P6Q was injected intravenously before cisplatin treatment. Then, we evaluated the renoprotective effect of P6Q. In the study on cultured cells, pretreatment with ProTα or P6Q prevented cisplatin-induced cell death. In the in vivo study, pretreatment with P6Q significantly attenuated cisplatin-induced increase in serum creatinine and blood urea nitrogen levels, renal tubular cell injury, and apoptosis. Moreover, P6Q attenuated the mitochondrial apoptotic pathway and accelerated Akt phosphorylation after cisplatin-induced renal damage. Taken together, our findings indicate that P6Q can attenuate cisplatin-induced AKI and suppress the mitochondrial apoptotic pathway via Akt phosphorylation. These data suggest that P6Q has potential as a preventative drug for cisplatin-induced AKI.

Peritoneal fibrosis is a serious complication of long-term peritoneal dialysis, attributable to inflammation and mitochondrial dysfunction. Mitochonic acid-5 (MA-5), an indole-3-acetic acid derivative, improves mitochondrial dysfunction and has therapeutic potential against various diseases including kidney diseases. However, whether MA-5 is effective against peritoneal fibrosis remains unclear. Therefore, we investigated the effect of MA-5 using a peritoneal fibrosis mouse model. Peritoneal fibrosis was induced in C57BL/6 mice via intraperitoneal injection of chlorhexidine gluconate (CG) every other day for 3 weeks. MA-5 was administered daily by oral gavage. The mice were divided into control, MA-5, CG, and CG + MA-5 groups. Following treatment, immunohistochemical analyses were performed. Fibrotic thickening of the parietal peritoneum induced by CG was substantially attenuated by MA-5. The number of α-smooth muscle actin-positive myofibroblasts, transforming growth factor β-positive cells, F4/80-positive macrophages, monocyte chemotactic protein 1-positive cells, and 4-hydroxy-2-nonenal-positive cells was considerably decreased. In addition, reduced ATP5a1-positive and uncoupling protein 2-positive cells in the CG group were notably increased by MA-5. MA-5 may ameliorate peritoneal fibrosis by suppressing macrophage infiltration and oxidative stress, thus restoring mitochondrial function. Overall, MA-5 has therapeutic potential against peritoneal fibrosis.

Back ground: Krüppel-like transcription factor 5 (KLF5) is a transcription factor regulating cell proliferation, angiogenesis and differentiation. It has been recently reported that Am80, a synthetic retinoic acid receptor α-specific agonist, inhibits the expression of KLF5. In the present study, we have examined the expression of KLF5 in fibrotic peritoneum induced by chlorhexidine gluconate (CG) in mouse and evaluated that Am80, as an inhibitor of KLF5, can reduce peritoneal fibrosis. Methods: Peritoneal fibrosis was induced by intraperitoneal injection of CG into peritoneal cavity of ICR mice. Am80 was administered orally for every day from the start of CG injection. Control mice received only a vehicle (0.5% carboxymethylcellulose solution). After 3 weeks of treatment, peritoneal equilibration test (PET) was performed and peritoneal tissues were examined by immunohistochemistry. Results: The expression of KLF5 was less found in the peritoneal tissue of control mice, while KLF5 was expressed in the thickened submesothelial area of CG-injected mice receiving the vehicle. Am80 treatment reduced KLF5 expression and remarkably attenuated peritoneal thickening, accompanied with the reduction of type III collagen expression. The numbers of transforming growth factor β-positive cells, α-smooth muscle actin-positive cells and infiltrating macrophages were significantly decreased in Am80-treated group. PET revealed the increased peritoneal permeability in CG mice, whereas Am80 administration significantly improved the peritoneal high permeability state. Conclusions: These results indicate the involvement of KLF5 in the progression of experimental peritoneal fibrosis and suggest that Am80 may be potentially useful for the prevention of peritoneal fibrosis through inhibition of KLF5 expression.

γ-Glutamyl carboxylase (GGCX) is a vitamin K (VK)-dependent enzyme that catalyzes the γ-carboxylation of glutamic acid residues in VK-dependent proteins. The anticoagulant warfarin is known to reduce GGCX activity by inhibiting the VK cycle and was recently shown to disrupt spermatogenesis. To explore GGCX function in the testis, here, we generated Sertoli cell-specific Ggcx conditional knockout (Ggcx scKO) mice and investigated their testicular phenotype. Ggcx scKO mice exhibited late-onset male infertility. They possessed morphologically abnormal seminiferous tubules containing multinucleated and apoptotic germ cells, and their sperm concentration and motility were substantially reduced. The localization of connexin 43 (Cx43), a gap junction protein abundantly expressed in Sertoli cells and required for spermatogenesis, was distorted in Ggcx scKO testes, and Cx43 overexpression in Sertoli cells rescued the infertility of Ggcx scKO mice. These results highlight GGCX activity within Sertoli cells, which promotes spermatogenesis by regulating the intercellular connection between Sertoli cells and germ cells.

Background and Aims Anti-glomerular basement membrane (GBM) glomerulonephritis (GN), characterized by glomerular crescent formation, requires early treatment because of poor prognosis. Hydroxychloroquine (HCQ) is a well-known antimalarial drug. In addition, it has immunomodulatory, anti-inflammatory, and autophagy inhibitory effects and its recognized in the treatment of autoimmune disease such as SLE. However, its effect for anti-GBM GN is unknown. In this study, we investigated the effect of HCQ against anti-GBM GN in rats. Method 7 week old male, WKY rats were induced by the administration of anti-GBM serum (50μg/rat). We administered either HCQ (50mg/kg) or vehicle (Phosphate-buffered saline) from day 0 to day 7 after the induction of nephritis. Renal function was assessed by measuring serum creatinine, proteinuria, hematuria. Urine was collected for 24 hours on day 1, 3, 5, and 7. Rats were sacrificed on day 7 after induction of anti-GBM GN. Renal histological changes were assessed by PAS staining, and Masson trichrome stain, and macrophage was assessed by ED-1 stain. Mitogen-Activated Protein Kinase (MAPK) was evaluated by western blotting (WB) and inflammatory cytokines were evaluated by ELISA using urine. Results HCQ treatment suppressed renal function decline. Histologically, extracellular and intracellular cells were increased from day 1, fibrinoid necrosis and ED-1 positive cells were observed from day 3. Rats with anti-GBM GN had high levels of interferon-α, interleukin-6, monocyte chemotactic protein-1, and tumor necrosis factor-α. These changes were significantly suppressed by HCQ. In addition, HCQ suppressed phosphorylation of JNK/p38 MAPK. Conclusion Our study showed that HCQ could attenuate anti-GBM GN and have an anti-inflammatory effect by inhibiting JNK/p38 MAPK activation. HCQ may have therapeutic potential in anti-GBM GN.

Background and Aims Peritoneal fibrosis is one of important complications induced by long-term peritoneal dialysis. Mitochondrial dysfunction causes an increase of oxidative stress and depletion of ATP. Thus, it may be associated with a variety of disease including fibrosis in several organs. Recently, mitochonic acid 5 (MA-5) was synthesized and its therapeutic potential for mitochondrial dysfunction in kidney disease models has been reported. In this study, we investigated the effect of MA-5 for peritoneal fibrosis model in mice. Method Peritoneal fibrosis was induced by intraperitoneal injection of chlorhexidine gluconate (CG) every other day for 3 weeks in C57/BL6 mice. MA-5 was administered at 2 mg/kg by gavage every day from the initiation of CG injection. Control mice received only a vehicle (distilled water). After 3 weeks of treatment, the animals were sacrificed and the peritoneal tissues were collected. The peritoneal sections were stained with Masson’s trichrome for light microscopic examination and the fibrotic thickening of parietal peritoneum was measured on the randomly selected different regions on each section. The expressions of F4/80, which is a marker of macrophages, monocyte chemotactic protein 1 (MCP1), α-smooth muscle actin (α-SMA), and transforming growth factor-β (TGF-β) were evaluated by immunohistochemistry. Results The fibrotic thickening of parietal peritoneum was significantly attenuated in MA-5 treated mice compared with control mice (the thickness of submesothelial area: 100.24 +/- 13.67 vs 54.78 +/- 7.43 μm (p Conclusion These results suggest that MA-5 may have a therapeutic potential in the progression of peritoneal fibrosis as well as kidney disease models.

For a better understanding of epigenetic regulation of cell differentiation, it is important to analyze DNA methylation at a specific site. In this study, we examined changes in the methylation level of CCGG and GATCG sites during mouse folliculogenesis in paraffin-embedded sections of mouse ovaries. For the purpose, we used a new method, histo endonuclease-linked detection of methylation sites of DNA (HELMET), designed to detect methylation sites of DNA with a specific sequence in a tissue section. Unlike the global level of DNA methylation, which was no change in immunohistochemical staining of 5-methylcytosine throughout folliculogenesis, we found that there were hypermethylation of CCGG and GATCG sites in most of the granulosa cells of tertiary follicles compared to that of primary and secondary follicles. Interestingly, TUNEL-positive granulosa cells, which were frequent in mammalian folliculogenesis, became markedly Hpa II-reactive and Sau3A I-reactive, indicating that the CCGG and GATCG sites may be preferentially demethylated during apoptosis.

The purpose of this study was to examine the time-dependent changes in the development of joint capsule fibrosis and in the number of myofibroblasts in the joint capsule after immobilization, using a rat knee contracture model. Both knee joints were fixed in full flexion for 1, 2, and 4 weeks (immobilization group). Untreated rats were bred for each immobilization period (control group). Histological analysis was performed to evaluate changes in the amount and density of collagen in the joint capsule. The changes in type I and III collagen mRNA were examined by in situ hybridization. The number of myofibroblasts in the joint capsule was assessed by immunohistochemical methods. In the immobilization group, the amount of collagen increased within 1 week and the density of collagen increased within 2 weeks, as compared with that in the control group. Type I collagen mRNA-positive cell numbers in the immobilization group increased at all time points. However, type III collagen mRNA-positive cell numbers did not increase. Myofibroblasts in the immobilization group significantly increased compared with those in the control group at all time points, and they increased significantly with the period of immobilization. These results suggest that joint capsule fibrosis with overexpression of type I collagen occurs and progresses within 1 week after immobilization, and an increase in myofibroblasts is related to the mechanism of joint capsule fibrosis. The findings suggest the need for a treatment targeting accumulation of type I collagen associated with an increase in myofibroblasts. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1998-2006, 2017.

Background Although histone acetylation, an epigenetic modification, has been reported to be related to the progression of various diseases, its involvement in nephrosclerosis is unclear. Methods Dahl salt-sensitive rats were used as a model of nephrosclerosis in this study. The rats were divided into three groups: (i) normal-salt diet group, (ii) high-salt diet group (HS), and (iii) HS administered daily with curcumin, a histone acetyltransferase inhibitor (HS+C). At 6 weeks after the treatment, the kidneys were dissected. Morphologic changes were assessed by Masson's trichrome staining. The number of macrophages, fibroblasts and the cells expressing acetylated histone H3 at Lys 9 (H3K9) were assessed by immunohistochemistry. Results Although both HS and HS+C rats revealed a marked increase in systolic blood pressure, serum creatinine was increased only in HS rats at 6 weeks. In the HS rats, nephrosclerosis was induced, accompanying a significant accumulation of macrophages and fibroblasts. The inflammation and fibrosis was markedly suppressed in the HS+C group. The level of histone acetylation at Lys 9 was enhanced in the HS rats, whereas curcumin administration suppressed the histone acetylation. Moreover, in the HS rats, interleukin-6 gene expression was associated with acetylated H3K9, as revealed by chromatin immunoprecipitation assay. Conclusions Our results suggested that curcumin ameliorates nephrosclerosis via suppression of histone acetylation, independently of hypertension.

The implantation of biomaterials induces a granulomatous reaction accompanied by foreign body giant cells (FBGCs). The characterization of multinucleated giant cells (MNGCs) around bone substitutes implanted in bone defects is more complicated because of healing with bone admixed with residual bone substitutes and their hybrid, and the appearance of two kinds of MNGCs, osteoclasts and FBGCs. Furthermore, the clinical significance of osteoclasts and FBGCs in the healing of implanted regions remains unclear. The aim of the present study was to characterize MNGCs around bone substitutes using an extraskeletal implantation model and evaluate the clinical significance of osteoclasts and FBGCs. Beta-tricalcium phosphate (β-TCP) granules were implanted into rat subcutaneous tissue with or without bone marrow mesenchymal cells (BMMCs), which include osteogenic progenitor cells. We also compared the biological significance of plasma and purified fibrin, which were used as binders for implants. Twelve weeks after implantation, osteogenesis was only detected in specimens implanted with BMMCs. The expression of two typical osteoclast markers, tartrate-resistant acid phosphatase (TRAP) and cathepsin-K (CTSK), was analyzed, and TRAP-positive and CTSK-positive osteoclasts were only detected beside bone. In contrast, most of the MNGCs in specimens without the implantation of BMMCs were FBGCs that were negative for TRAP, whereas the degradation of β-TCP was detected. In the region implanted with β-TCP granules with plasma, FBGCs tested positive for CTSK, and when β-TCP granules were implanted with purified fibrin, FBGCs tested negative for CTSK. These results showed that osteogenesis was essential to osteoclastogenesis, two kinds of FBGCs, CTSK-positive and CTSK-negative, were induced, and the expression of CTSK was plasma-dependent. In addition, the implantation of BMMCs was suggested to contribute to osteogenesis and the replacement of implanted β-TCP granules to bone.

Smoking frequently leads to epigenetic alterations, including DNA methylation and histone modifications. The effect that smoking has on the DNA methylation levels at CCGG sites, the expression of trimethylation of histone H3 at lysine 27 (H3K27me3) and enhancer of zeste homolog 2 (EZH2), and their interactions in patients with non-small cell lung cancer (NSCLC) were analyzed. There were a total of 42 patients with NSCLC, 22 with adenocarcinomas and 20 with squamous cell carcinomas enrolled in the present study. Expression of H3K27me3, EZH2 and proliferating cellular nuclear antigen (PCNA) were immunohistochemically detected. DNA methylation at CCGG sites was evaluated via histoendonuclease-linked detection of DNA methylation sites. The apoptotic index of cancerous tissues obtained from patients of different smoking statuses was evaluated via the terminal deoxynucleotidyl-transferase-mediated dUTP-biotin nick end labeling method. The association with clinicopathological data was calculated relative to different smoking statuses. Compared with the non-smokers, smokers with NSCLC exhibited a significantly lower apoptotic index (P

Objective To investigate whether human T lymphotropic virus type I (HTLV-I) directly infects salivary gland epithelial cells (SGECs) and induces the niche of the salivary glands in patients with Sjogren's syndrome (SS). Methods SGECs were cultured with the HTLV-I–producing CD4+ T cell line HCT-5 or with Jurkat cells. Antibody arrays, immunofluorescence analysis, and enzyme-linked immunosorbent assay (ELISA) were used to determine the profiles of inflammation-related molecules, and the profiles of apoptosis-related molecules were determined by antibody array and immunofluorescence analysis. The presence of HTLV-I–related molecules was assessed by immunofluorescence analysis and in situ polymerase chain reaction. Apoptosis of SGECs was evaluated by TUNEL staining. Results Among the SGECs, 7.8 ± 1.3% (mean ± SD) were positive for HTLV-I–related proteins after 96-hour coculture with HCT-5 cells. Nuclear NF-κB p65 was also detected in 10% of the SGECs. The presence of HTLV-I proviral DNA in SGECs after coculture with HCT-5 cells was detected by in situ polymerase chain reaction. After coculture of SGECs with HCT-5, the expression of cytokines and chemokines, including soluble intercellular adhesion molecule 1, RANTES, and interferon γ–induced protein 10 kd (IP-10/CXCL10) was increased in a time-dependent manner. The expression of proapoptotic molecules (e.g., cytochrome c and Fas) and antiapoptotic molecules (e.g., Bcl-2, Heme oxygenase 2, and Hsp27) was increased in the SGECs cocultured with HCT-5, showing that apoptosis of SGECs was not detected after coculture with HCT-5 or Jurkat cells. Conclusion HTLV-I is thought to infect SGECs and alter their cellular functions. These changes may induce the niche of SS and contribute to the development of SS in anti–HTLV-I antibody–positive individuals.

We demonstrated that repression of keratinocyte growth factor (KGF) receptor (KGFR) could be a potentially useful strategy in the conservative treatment of middle ear cholesteatoma.Recently, the use of a selective inhibitor of the KGFR, SU5402, in an in vitro experiment resulted in the inhibition of the differentiation and proliferation of epithelial cells through KGF secretion by fibroblasts isolated from the cholesteatoma. In this study, we investigated the effects of the KGFR inhibitor during middle ear cholesteatoma formation in vivo.Based on the role of KGF in the development of cholesteatoma, Flag-hKGF cDNA driven by CMV14 promoter was transfected through electroporation into the external auditory canal of rats five times on every fourth day. Ears transfected with empty vector were used as controls. KGFR selective inhibitor (SU5402) or MEK inhibitor (PD0325901) was administered in the right ear of five rats after vector transfection. In the control, 2% DMSO in PBS was administered in the other ears after vector transfection.The use of a selective KGFR inhibitor, SU5402, completely prevented middle ear cholesteatoma formation in the rats.

We reported previously that keratinocyte growth factor (KGF), a mesenchymal cell-derived paracrine growth factor, plays an important role in middle ear cholesteatoma formation, which is characterized by marked proliferation of epithelial cells. Here, we investigated whether KGF, the main factor that induces cholesteatoma, overexpression in vivo results in the formation of cholesteatoma. Flag-hKGF cDNA driven by CMV14 promoter was transfected through electroporation into the external auditory canal (EAC) of rats once (short-term model) or five times on every fourth day (long-term model). Ears transfected with empty vector were used as controls. Successful transfection of plasmids into epithelial and stromal cells was confirmed by Flag immunohistochemistry. In the short-term model, the intensity of KGF protein was the strongest in hKGF transfected ear at day 4. KGF expression induced epithelial cell proliferation, reaching a peak level at day 4 and then decreased later, while in the long-term model, KGF expression in the EAC led to middle ear cholesteatoma formation. In conclusion, we described here a new experimental model of human middle ear cholesteatoma, and demonstrated that KGF and KGF receptor paracrine action play an essential role in middle ear cholesteatoma formation in an in vivo model.

Encapsulating peritoneal sclerosis (EPS) is a fatal complication that can occur in patients undergoing long-term peritoneal dialysis. It is characterized by bowel obstruction and marked sclerotic thickening of the peritoneal membrane. Although the mechanisms underlying the development of EPS are complex, angiogenesis, inflammation, and peritoneal fibrosis are known to be essential factors. Now, several animal models that exhibit EPS have pathophysiology similar to that of human EPS and have been proposed for use in research to provide insights into it. Recent histochemical methods also help us to understand the pathophysiology of EPS. Advances in basic research based on the findings in those animal models have enabled the development of several strategies for the prevention and treatment of EPS. We describe here interventional studies in some animal models for peritoneal fibrosis, one of the histological disorders findings characteristic to EPS, and we highlight the need for a sophisticated animal model that closely resembles human conditions.

Agiopoieten regulates vascular angiogenesis and stabilization, and is reported to promote bone formation by facilitating angiogenesis. To estimate the role of Ang1 in odontogenesis, we explored the distribution of Ang1 and the receptor, Tie2 in the mouse developing and mature first molar of the mandible. At embryonic day 18, when differentiation of odontoblasts begins, immunosignals for Ang1 were intensely detected in the basement membrane and the distal side, which faced the basement membrane of odontoblasts. In situ hybridization revealed that Ang1 was expressed in odontoblasts and ameloblasts facing the basement membrane. Tie2 was localized in the distal side of odontoblasts. After birth, Ang1 was detected in the predentin, whereas both Ang1 and Tie2 were colocalized in odontoblasts and odontoblast processes. These distributions were retained up to 8 weeks. In contrast to odontoblasts, ameloblasts, cementoblasts and osteoblasts expressed Ang1 but did not express Tie2. Colocalization of Ang1 and Tie2 in odontoblasts and selective expression of Tie2 in odontoblasts among cells responsible for calcified tissue formation suggested the involvement of autocrine signals of Ang1-Tie2 in dentinogenesis., ACTA HISTOCHEMICA ET CYTOCHEMICA, 47(1), pp.19-25; 2014

Cell sheet engineering has been noted as a new and valuable approach in the tissue-engineering field. The objective of this study was to explore a procedure to induce hepatic progenitor cells and biliary duct structures in the liver. Sprague-Dawley rat dermal fibroblast (DF) sheets were transplanted into the incised surface of the liver of F344 nude rats. In the control group, an incision was made without transplantation of the DF sheets. Bile duct (BD)-like structures and immature hepatocyte-like cells were observed in the DF sheet transplant sites. These BD-like structures were cytokeratin-8-positive, while the hepatocyte-like cells were both OV-6-positive and α-fetoprotein-positive as well. The proliferation and differentiation of liver progenitor cells were not influenced by hepatectomy. We also transplanted DF sheets transfected with a plasmid encoding the enhanced yellow fluorescent protein target to mitochondria (pEYFP-Mito) by electroporation, and found that the new structures were pEYFP-Mito-negative. We observed new BD-like structures and immature hepatocytes after transplantation of DF sheets onto incised liver surfaces, and clarified that the origin of these BD-like structures and hepatocyte-like cells was the recipient liver. The present study described an aspect of the hepatic differentiation process induced at the site of liver injury.

The purpose of this study was to examine the time-dependent changes in the development of joint capsule fibrosis and in the number of myofibroblasts in the joint capsule after immobilization, using a rat knee contracture model. Both knee joints were fixed in full flexion for 1, 2, and 4 weeks (immobilization group). Untreated rats were bred for each immobilization period (control group). Histological analysis was performed to evaluate changes in the amount and density of collagen in the joint capsule. The changes in type I and III collagen mRNA were examined by in situ hybridization. The number of myofibroblasts in the joint capsule was assessed by immunohistochemical methods. In the immobilization group, the amount of collagen increased within 1 week and the density of collagen increased within 2 weeks, as compared with that in the control group. Type I collagen mRNA-positive cell numbers in the immobilization group increased at all time points. However, type III collagen mRNA-positive cell numbers did not increase. Myofibroblasts in the immobilization group significantly increased compared with those in the control group at all time points, and they increased significantly with the period of immobilization. These results suggest that joint capsule fibrosis with overexpression of type I collagen occurs and progresses within 1 week after immobilization, and an increase in myofibroblasts is related to the mechanism of joint capsule fibrosis. The findings suggest the need for a treatment targeting accumulation of type I collagen associated with an increase in myofibroblasts. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1998-2006, 2017.

Mammalian spermatogenesis is a progressive process comprising spermatogonial proliferation, spermatocytic meiosis, and later spermiogenesis, which is considered to be under the regulation of epigenetic parameters. To gain insights into the significance of DNA methylation in early spermatogenesis, 5-azadC was used as a molecular biological tool to mimic the level of DNA methylation in vivo. Since the drug is incorporated into DNA during the S-phase, spermatogonia and spermatocytes would be affected primarily in mouse spermatogenesis. Adult male ICR mice were intraperitoneally injected with 5-azadC at a dose of 0.25mg/kg/day for 10 consecutive days, allowing us to examine its maximum effect on the kinetics of spermatogonia and spermatocytes. In this short-term protocol, 5-azadC induced significant histological abnormalities, such as a marked increase in apoptosis of spermatogonia and spermatocytes, followed by severe loss of spermatids, while after termination of 5-azadC treatment, normal histology was restored in the testis within 35days. Quantification of the methylation level of CCGG sites as well as whole DNA showed spermatogonial hypomethylation, which correlated with increased apoptosis of spermatogonia. Interestingly, the hypomethylated cells were simultaneously positive for tri-methylated histone H3 at K4. On the other hand, no changes in methylation level were found in spermatocytes, but PCNA staining clearly showed disordered accumulation of S-phase spermatocytes, which increased their apoptosis in stage XII. In addition, different immunohistochemical staining pattern was found for DNA methyltransferases (DNMTs); DNMT1was expressed in the majority of all germ cells, but DNMT3a and b were only expressed in spermatogonia. Our results indicate that 5-azadC caused DNA hypomethylation in spermatogonia, but induced prolongation of S-phase in spermatocytes, resulting in the induction of apoptosis in both cases. Thus, 5-azadC affects spermatogenesis at more than one differentiation stage with different mechanisms, probably due to the specific usage of DNMTs.

Although iron overload is implicated in hepatocarcinogenesis, the precise mechanism was not known yet. In the present study, we investigated the effect of iron overload upon the induction of hepatocyte proliferation after 70% partial hepatectomy (PH) in rats fed with rat chow with 3% carbonyl iron for 3 months. In normal-diet rats, the increase in Ki-67 labeling index (LI) commenced at 24 h post-PH and the LIs of proliferating cell nuclear antigen (PCNA) incorporated 5-bromo-2'-deoxyuridine (BrdU) and phospho-histone H3 reached maximum values at 36 and 48 h after PH, respectively. In iron-overload rats, the above parameters occurred 12 h earlier compared to that of normal-diet rats, shortening the G0-G1 transition. Interestingly, nuclear staining for metallothionein (MT), which is essential for hepatocyte proliferation, was noted even at 0 h in iron-overload rats, while MT expression occurred at 6 h in the normal rats. Moreover, nuclear factor kappa B (NF-κB) expression, which is an essential early event leading to liver regeneration, was detected in Kupffer cells at 0 h in iron-overload rats. These results may indicate that overloaded iron, maybe through the induction of MT and NF-κB, may keep liver as a state ready to regenerate in response to PH, by bypassing signal transduction cascades involved in the initiation of liver regeneration.

Background Erythropoietin (EPO) has been found to provide cytoprotection against acute ischemic and toxic renal tubulointerstitial injury. This study aimed to elucidate the mechanism(s) underlying EPO protection while examining whether EPO provides tubulointerstitial protection in a mouse model with adriamycin (ADR)-induced tubulointerstitial injury. Methods Adriamycin nephropathy (AN) was induced by a single injection of ADR in the 2 experimental groups on day 0. The saline-control group and the AN-saline group were administered saline at days 7, 14, and 21, while the EPO-control group and the AN-EPO group were administered EPO at days 7, 14, and 21. Kidneys were harvested at days 14 and 28 after ADR injection to measure the expression levels of the EPO receptor (EPO-R), CD34, and phosphorylated Akt by immunohistochemistry; to determine the extent of apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) and active caspase-3 staining; and to map the hypoxic area by pimonidazole staining. Results EPO-R was detected in glomerular, tubular epithelial, and endothelial cells. EPO administration significantly improved tubulointerstitial injury, decreased the number of TUNEL-positive and active caspase-3-positive cells, and increased the phosphorylated-Akt-positive area in the tubulointerstitial area without increasing the hemoglobin or hematocrit levels. Conclusions EPO provides renoprotection against AN by reducing apoptotic cell death and preserving peritubular capillaries, possibly by exerting pleiotropic effects independently of its hemopoietic effects.

Experimental autoimmune orchitis (EAO) is characterized by T cell-dependent lymphocytic inflammation and seminiferous tubule damage, which can result in the death of germ cells. The aim of the present study is to investigate the roles of the Fas/Fas-L and Bax/Bcl-2 systems in the death of germ cells in mice with EAO that is induced by immunization with syngeneic testicular germ cells (TGC). The results using real-time reverse transcription-polymerase chain reaction and immunostaining show that many terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining germ cells were present in seminiferous tubules during the active inflammation stage, and these cells were persistently observed in the seminiferous epithelium until the postactive inflammation stage. Intratesticular mRNA expression levels of both Fas and Bax were increased during the active inflammation stage and were dramatically decreased during the post-active inflammation stage. In contrast, the intratesticular mRNA expression levels of both Fas-L and Bcl-2 did not show significant changes during the active inflammation stage but showed extreme increases during the post-active inflammation stage. Immunohistochemically, some Fas- and Bax-positive germ cells were detected during the active inflammation stage, but these were hardly found during the post-active inflammation stage. In contrast, some Fas-L- and Bcl-2-positive germ cells were found during the active inflammation stage, and many of these were also observed during the post-active inflammation stage. These results indicate that germ cell death during TGC-induced EAO is mediated by the Fas/Fas-L and Bax/Bcl-2 systems during the active inflammation stage but not during the post-active inflammation stage.

Thalidomide is clinically recognized as a therapeutic agent for multiple myeloma and has been known to exert anti-angiogenic actions. Recent studies have suggested the involvement of angiogenesis in the progression of peritoneal fibrosis. The present study investigated the effects of thalidomide on the development of peritoneal fibrosis induced by injection of chlorhexidine gluconate (CG) into the mouse peritoneal cavity every other day for 3 weeks. Thalidomide was given orally every day. Peritoneal tissues were dissected out 21 days after CG injection. Expression of CD31 (as a marker of endothelial cells), proliferating cell nuclear antigen (PCNA), vascular endothelial growth factor (VEGF), α-smooth muscle actin (as a marker of myofibroblasts), type III collagen and transforming growth factor (TGF)-β was examined using immunohistochemistry. CG group showed thickening of the submesothelial zone and increased numbers of vessels and myofibroblasts. Large numbers of VEGF-, PCNA-, and TGF-β-positive cells were observed in the submesothelial area. Thalidomide treatment significantly ameliorated submesothelial thickening and angiogenesis, and decreased numbers of PCNA- and VEGF-expressing cells, myofibroblasts, and TGF-β-positive cells. Moreover, thalidomide attenuated peritoneal permeability for creatinine, compared to the CG group. Our results indicate the potential utility of thalidomide for preventing peritoneal fibrosis., Acta Histochemica et Cytochemica, 44(2), pp.51-60; 2011

Middle ear acquired cholesteatoma is a pathological condition associated with otitis media, which may be associated with temporal bone resorption, otorrhea and hearing loss, and occasionally various other complications. Cholesteatoma is characterized by the enhanced proliferation of epithelial cells with aberrant morphologic characteristics. Unfortunately, our understanding of the mechanism underlying its pathogenesis is limited. To investigate its pathogenesis, different animal models have been used. This paper provides a brief overview of the current status of research in the field of pathogenesis of middle ear acquired cholesteatoma, four types of animal models previously reported on, up-to-date cholesteatoma research using these animal models, our current studies of the local hybrid ear model, and the future prospect of new animal models of middle ear cholesteatoma., Journal of Biomedicine & Biotechnology, 2011, article no.394241

INTRODUCTION The purpose of the study was to test a hypothesis that the keratinocyte growth factor (KGF) is a key factor in the pathologic difference between cholesteatomatous (C-COM) and noncholesteatomatous chronic otitis media (NC-COM). We compared the expression levels of KGF and its receptor (KGFR) and the proliferation activity of epithelial cells between NC-COM and C-COM. METHODS The epithelial lesion was surgically excised with subepithelial tissue from 18 patients with NC-COM and 70 patients with C-COM, and was processed for immunohistochemistry for KGF and KGFR. We also examined the proportion of proliferating epithelial cells using Ki-67 and the extent of infiltrating B and T cells. RESULTS Keratinocyte growth factor was positive in 5 of 18 (28%) NC-COM specimens and in 61 of 69 (88%) C-COM specimens (p < 0.0001). Furthermore, 37 (60%) C-COM specimens were positive for KGFR, but none of NC-COM were positive (0%; p < 0.01). The Ki-67 labeling index (LI) was significantly smaller in NC-COM than in C-COM (p < 0.001). B-Cell LI was almost similar in the 2 groups. T-Cell LI was significantly higher in C-COM than in NC-COM (p < 0.0001). Interestingly, T-cell LI in NC-COM was higher in KGF-positive tissues than in KGF-negative tissues (p < 0.05). CONCLUSION The results indicated that coexpression of KGF and KGFR seems to explain the pathologic difference between C-COM and NC-COM, and that KGF may play an important role in the development of cholesteatoma.

Middle ear cholesteatoma is characterized by enhanced proliferation of epithelial cells with aberrant morphological characteristics. To investigate the origin of the cholesteatoma cells, we analyzed spontaneously occurring cholesteatomas associated with a new transplantation model in Mongolian gerbils (gerbils). Cholesteatomas were induced in gerbils with a transplanted tympanic membrane by using the external auditory canal (EAC) ligation method. After the pars flaccida of the tympanic membranes were completely removed from male gerbils, corresponding portions of tympanic membranes of female gerbils were transplanted to the area of defect, and then we ligated the EAC (hybrid-model group). As a control group, the EAC of normal male and female gerbils was ligated without myringoplasty. In all ears of each group, the induced cholesteatomas were seen. In situ PCR was then performed to detect the mouse X chromosome-linked phosphoglycerate kinase-1 (pgk-1) gene on the paraffin sections. One pgk-1 spot in the epithelial nuclei was detected in male cholesteatoma, and two pgk-1 spots were detected in female cholesteatoma, respectively. On the other hand, in the hybrid-model group, we detected not only one but also two pgk-1 spots in the epithelial nuclei of cholesteatoma. These results strengthened the evidence that the origin of epithelial cells in cholesteatoma is the tympanic membrane in this model, but not the residential middle ear epithelial cells or the skin of the EAC.

Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that has been shown to play a major role in the processing and/or secretion of procollagen. However, the knowledge on which cells are actually synthesizing HSP47 in the lung parenchyma in pulmonary fibrosis was only limited. The aim of the present study was to investigate the localization of HSP47 messenger ribonucleic acid (mRNA) in normal lung and in the lungs of mice in bleomycin-induced pulmonary fibrosis, using in situ hybridization. For the purpose, ICR mice were intravenously injected with 10 mg/kg per day of bleomycin for five consecutive days. The lung cells expressing HSP47 mRNA were identified in control (saline alone) and bleomycin-treated mice by in situ hybridization. The signal for HSP47 mRNA was markedly increased in bleomycin-treated lungs compared with that of controls. HSP47 mRNA was localized in alpha-smooth-muscle-actin-positive myofibroblasts, surfactant-protein-A-positive type II pneumocytes, and F4/80-positive macrophages in the active fibrotic areas. These results suggest that these cells may synthesize procollagen in the fibrotic process of bleomycin-treated lungs through upregulation of HSP47 mRNA and play an important role in fibrogenesis., Virchows Archiv, 456(3), pp.309-315; 2010

Background: The mitotic activity of the epithelial cells of odontogenic keratocysts (OKCs) is greater than that of other odontogenic jaw cysts, and the mitotic activity of the epithelial cells decreases after marsupialization. Keratinocyte growth factor (KGF) interacts with its specific receptor (KGFR), and elicits the proliferation and/or differentiation of the various types of epithelial cells. The aim of this study was to investigate the expression of KGF/KGFR in OKCs before and after marsupialization. Methods: The expression of KGF was immunohistochemically detected in the specimens of 16 OKCs and 11 dentigerous cysts before and after marsupialization. The expression of KGF mRNA was measured in the fibroblasts isolated from OKCs by real-time PCR. Results: KGF was expressed in the epithelial cells and fibroblasts of 12 and seven of 16 OKC specimens, respectively. The intensity of the KGF expression in both the epithelial cells and the fibroblasts significantly decreased after marsupialization. KGFR was expressed throughout the epithelium in 15 of 16 OKC specimens, but the intensity of the KGFR expression did not change after marsupialization. The expression of KGF was detected in the epithelium of two of 11 dentigerous cyst specimens, but not in the fibroblasts before marsupialization. Real-time PCR revealed that recombinant human interleukin (IL)-1α increased the expression of KGF mRNA in the fibroblasts isolated from OKCs. Conclusion: KGF/KGFR signaling may play a crucial role in the epithelial cells of OKCs. Furthermore, the expression of KGF in the fibroblasts of OKCs is regulated by IL-1α.

In order to better understand the biology of adult T cell leukemia (ATL), we aimed to establish a novel method, which allows the primary growth of ATL cells using a co-culture system with murine bone marrow-derived stromal cells, MS-5. ATL cells grew in close contact with MS-5 layers and formed so-called "cobblestone areas" (CAs) without the addition of IL-2. In clinical samples, eight of ten (80.0%) cases of acute or lymphoma type ATL cells formed CAs. The frequency of CA forming cells in ATL cells ranged from 0.03 to 1.04%. The morphology, immunophenotyping, and DNA analysis indicated that cells composing CA were compatible with ATL cells, and clonally identical to primary CD4-positive ATL cells. Furthermore, in ATL cells composing CA, the expression of p40Tax was down-regulated in transcriptional and translational level, while that of HTLV-I basic leucine zipper factor (HBZ) gene was comparable to the level of primary ATL cells, resembling expression pattern of proviral genes in in vivo ATL cells. By microarray analysis, several genes which coded products involved in cell-cell interaction, and cellular survival and proliferation, were differentially expressed in ATL cells composing CA compared with primary samples. In conclusion, our co-culture system allows for the first time the growth of primary ATL cells in vitro, and might be useful as an in vitro assay for biological and clinical studies to develop molecular targeting drugs against ATL.

Neovasculogenesis is essential in tissue remodeling. Endothelial progenitor cells (EPCs) mobilize from bone marrow (BM) and participate in neovasculogenesis. This study examined the role of EPCs in a model of reversible glomerulonephritis induced by habu snake venom (HSV). Lethally irradiated FVB/N wild-type mice were transplanted with BM cells from donor transgenic mice expressing beta-galactosidase gene under the control of endothelial-specific tie-2 promoter. HSV or saline was injected intravenously after BM transplantation (BMT). The kidneys were removed before injection and at days 1, 7, 28, and 56 after injection. beta-Galactosidase-expressing cells were identified by X-gal staining. The expressions of CD31 (endothelial cell marker) and vascular endothelial cell growth factor (VEGF) in renal tissues were examined by immunohistochemistry. In BMT mice injected with saline, few X-gal-positive cells were detected in glomeruli. In HSV-injected mice, X-gal-positive EPCs were increased in damaged glomeruli, reaching maximum at day 28. Recovery of glomeruli was observed at day 56 in association with reduction of X-gal-positive EPCs. VEGF overexpression was detected in glomerular epithelial and endothelial cells, mesangial cells, and EPCs. Our results indicated that EPCs were mobilized into the damaged glomeruli, suggesting EPCs participation in glomerular capillary repair of damaged glomeruli in HSV-induced glomerulonephritis.

To assess whether azelnidipine (AZN) exerts renoprotective effects, 20-week-old adult male stroke-prone spontaneously hypertensive rats (SHRsp) were treated with AZN 10 mg/kg/d (n=6), olmesartan (OLM) 3 mg/kg/d (n=4), hydralazine (HYD) 20 mg/kg/d (n=3), or water (control; n=5). Each test agent was administered by oral gavage for 12 weeks. Systolic blood pressure (SBP) was measured every 2 weeks and urinary protein excretion (UproV) every 3 weeks. At the age of 32 weeks, the rats were sacrificed and blood and kidneys collected for biochemical, histological, and immunohistochemical studies. All drug treatments significantly (p, Biological & Pharmaceutical Bulletin, 31(12), pp.2237-2244; 2008

Background. Patients on long-term peritoneal dialysis develop progressive peritoneal fibrosis and loss of mesothelial layer. Regeneration of the mesothelium has been reported in the normal peritoneum but not the fibrotic peritoneum. Moreover, the origin of the regenerated mesothelial cells remains obscure. The aim of this study was to investigate mesothelial regeneration in fibrotic peritoneum induced by chlorhexidine gluconate. Methods. Peritoneal fibrosis was induced by injection of CG into the peritoneal cavity of Wistar rats. After injection, the abdomen was opened, and the parietal fibrotic peritoneum with mesothelial cells was stripped from the abdominal wall, and then the abdominal incision was closed. Rats were sacrificed, and peritoneal tissues were dissected out at 0, 1, 3, 5, or 7 days after the stripping procedure. Results. Spindle-shaped cells with microvilli appeared on the surface of stripped peritoneum at day 3 after denudation. Immunohistochemistry identified staining for vimentin, a marker of mesoderm cells, in the spindle-shaped cells at days 3, 5, and 7. Expression of α-SMA was observed in the same cells at days 3 and 5, but not 7. Expression of cytokeratin and HBME-1, markers for mesothelial cells, in these cells was delayed until day 7. Conclusions. Mesothelium can regenerate on the fibrotic peritoneum. The regenerated mesothelial cells seem to originate from vimentin-positive mesenchymal cells., This is an electronic version of an article published in Renal Failure, 30(1), pp.97-105; 2008. Renal Failure is available online at: http://www.informaworld.com/openurl?genre=article&id=doi:10.1080/08860220701741619., Renal Failure, 30(1), pp.97-105; 2008

Transcriptional initiation is a key step in the control of mRNA synthesis and is intimately related to chromatin structure and histone modification. Here, we show that the ubiquitylation of H2A (ubH2A) correlates with silent chromatin and regulates transcriptional initiation. The levels of ubH2A vary during hepatocyte regeneration, and based on microarray expression data from regenerating liver, we identified USP21, a ubiquitin-specific protease that catalyzes the hydrolysis of ubH2A. When chromatin is assembled in vitro, ubH2A, but not H2A, specifically represses the di- and trimethylation of H3K4. USP21 relieves this ubH2A-specific repression. In addition, in vitro transcription analysis revealed that ubH2A represses transcriptional initiation, but not transcriptional elongation, by inhibiting H3K4 methylation. Notably, ubH2A-mediated repression was not observed when H3 Lys 4 was changed to arginine. Furthermore, overexpression of USP21 in the liver up-regulates a gene that is normally down-regulated during hepatocyte regeneration. Our studies revealed a novel mode of trans-histone cross-talk, in which H2A ubiquitylation controls the di- and trimethylation of H3K4, resulting in regulation of transcriptional initiation.

Background/Aim: Unilateral ureteral obstruction (UUO) is a well-established model for tubulointerstitial fibrosis. During the progression of tubulointerstitial fibrosis, upregulation of collagen synthesis and subsequent accumulation of collagen were observed in the tubulointerstitial area. Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone and plays an essential role in regulating collagen synthesis. We designed small interfering RNA (siRNA) sequences for HSP47 mRNA to examine whether HSP47 is involved in the progression of renal tubulointerstitial fibrosis in a mouse UUO model. Methods: The HSP47 siRNA was injected once via the ureter at the time of UUO preparation. We also applied a new gene delivery system for siRNA using cationized gelatin microspheres. The kidneys were harvested 7 and 14 days after UUO. The HSP47 and type I, III, and IV collagen expression levels were analyzed by immunohistochemistry and Western blotting. Results: Seven days after UUO, the expression levels of HSP47 and type I, III, and IV collagens were markedly upregulated in obstructed kidneys or green fluorescent protein siRNA treated obstructed kidneys. HSP47 siRNA injection significantly reduced the protein expression levels and significantly diminished the accompanying interstitial fibrosis. Moreover, cationized gelatin microspheres as a delivery system enhanced and lengthened the antifibrotic effect of HSP47 siRNA. Conclusions: Our results indicate that HSP47 is a candidate target for the prevention of tubulointerstitial fibrosis and that selective blockade of the HSP47 expression by using siRNA could be a potentially useful therapeutic approach for patients with renal disease.

Background Early detection of vulnerable plaques at risk of causing thromboembolic events is very important, and many investigators report the usefulness of high-resolution MRI. The purpose of this study was to determine whether the detection of atherosclerotic carotid plaques can be enhanced after administration of contrast agents and, if so, to evaluate the potential for functional information. Methods We studied 9 patients (10 subjects) who underwent a high-resolution MRI examination using a gadolinium-based contrast agent before CEA. Pre- and postcontrast-enhanced T1-weighted images were reviewed, and their histopathologic characteristics evaluated in the corresponding tissue slices. Results Strong contrast enhancement patterns were found in 6 of 10 subjects. For 5 of 6 subjects, many microvessels with inflammatory cells or intraplaque hemorrhages were demonstrated in their corresponding tissue slices. Contrast enhancement patterns were noted to be focal, diffuse, and along the luminal surface or the vessel adventitial boundary. Moreover, some plaques were clearly demonstrated by using contrast agent, and others were clearly divided into fibrous and lipid regions. Conclusion Gadolinium-based contrast agent can penetrate human atherosclerotic carotid plaques. The extent or size of neovascularization and the endothelial permeability are likely related to the mechanism of enhancement, and contrast-enhanced MRI may be essential for the identification of plaque neovascularization which is an important factor of vulnerable plaques. In addition to morphologic information, with the functional information provided using various contrast agents, we may expect a more correct diagnosis of carotid plaques at risk of causing thromboembolic events.

The aim of this study was to survey the effect of deposited iron on the cell kinetics of hepatitis C virus (HCV)-positive hepatocellular carcinoma (HCC) in Myanmar (Burmese) patients.Formalin-fixed and paraffin-embedded liver tissues from 34 Myanmar patients with HCC were used. To detect iron deposition, Prussian blue staining was performed. Cell proliferation and apoptosis were assessed by Ki-67 staining and by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) assay, respectively. HCV RNA was detected by in situ hybridization, and HCV protein, Fas and Fas ligand (FasL) were localized by immunohistochemistry. To identify the subtype of lymphocytes, CD8 was used as a surface marker.Iron deposition was found in 43% of the HCC cases, and was heavier in moderately differentiated HCC than in well-differentiated HCC. The Ki-67 labeling index (LI) in cancer cells was higher in Prussian blue-positive-HCC than in -negative HCC (3.8 +/- 2.2 vs 1.5 +/- 1.7, mean +/- SD; P=0.0067), whereas there was no significant difference between these groups in TUNEL LI. HCV protein was localized in cancer cells, and was found in 89% of the patients. In addition, Fas was expressed in HCC cells, and FasL was localized in HCC cells as well as in infiltrating CD8+ T lymphocytes. The frequency of apoptosis of HCC cells was correlated significantly with the population density of infiltrating CD8+ T lymphocytes.Our results indicated that, in Myanmar patients with HCC, iron deposition might accelerate hepatocarcinogenesis, by promoting cancer cell proliferation, without affecting the Fas/FasL apoptotic system.

Long-term peritoneal dialysis causes peritoneal fibrosis, and previous reports suggest that inflammation plays a critical role in peritoneal fibrosis. Chondroitin sulfate (CS) suppresses the inflammatory response by preventing activation of nuclear factor (NF)-κB. We examined the effect of CS on the peritoneal fibrosis induced by chlorhexidine gluconate (CG) in mice. CS or water was administered daily. We divided mice into four groups: administered vehicle and water (control); administered vehicle and CS (CS); administered CG and water (CG); and administered CG and CS (CG+CS). Morphologic changes were assessed by Masson’s trichrome staining. Inflammation- and fibrosis-associated factors were assessed by immunohistochemistry. Activation of NF-κB was examined by southwestern histochemistry. CS administration suppressed the progression of submesothelial thickening. The numbers of inflammation- and fibrosis-associated factors -positive cells were significantly decreased in the CG+CS group, compared to the CG group. Based on SWH, the CG+CS group contained significantly fewer NF-κB-activated cells than the CG group. Our results indicate that CS suppresses peritoneal fibrosis via suppression of NF-κB activation. These results suggest that CS has therapeutic potential for peritoneal fibrosis.

In the area of manufacturing surgical instruments, the ability to rapidly design, prototype and test surgical instruments is critical. This paper provides a simple case study of the rapid development of two bio-mechanism based surgical instruments which are ergonomic, aesthetic and were successfully designed, prototyped and conceptually tested in a very short period of time.

Long-term peritoneal dialysis therapy causes inflammation and histological changes in the peritoneal membrane. Inflammation generally activates fibroblasts and results in fibroblast-myofibroblast differentiation. Heat-shock protein 47 (HSP 47), a collagen-specific molecular chaperone, is localized in myofibroblasts and is involved in the progression of peritoneal fibrosis. Daikenchuto (DKT), a Kampo medicine, is used to prevent postoperative colon adhesion. It inhibits inflammation and HSP 47 expression in the gastrointestinal tract. We examined the effect of DKT on chlorhexidine gluconate (CG)-induced peritoneal fibrosis in mice injected with 0.1% CG dissolved in 15% ethanol. DKT was dissolved in the drinking water. Histological changes were assessed using Masson trichrome staining. Cells expressing α-smooth muscle actin (α-SMA), HSP 47, phospho-Smad 2/3, F4/80, and monocyte chemotactic protein-1 were examined immunohistochemically. Compared with the control group, the peritoneal tissues of the CG group were markedly thickened, and the number of cells expressing α-SMA, HSP 47, phospho-Smad 2/3, F4/80, and monocyte chemotactic protein-1 was significantly increased. However, these changes were inhibited in the DKT-treated group. These results indicate that DKT can prevent peritoneal fibrosis by inhibiting inflammation and HSP 47 expression.

BACKGROUND Although intraepithelial lymphocytes (IELs) in human oviductal epithelium have been implicated in the regulation of local immunity, the precise kinetics and mechanism of steroid regulation of IEL are largely unknown. METHODS We examined the localization of estrogen receptors (ERs) and progesterone receptors (PRs) in 41 human oviducts by immunohistochemistry. These tissues were obtained from various menstrual cycles, also from both post-menopausal women and tubal pregnancies. The expressions of ERbeta mRNA and membrane (m)PR mRNA were examined by in situ hybridization and RT-PCR, respectively. RESULTS Most of the IEL expressed ERbeta at both mRNA and protein levels. The number of ERbeta-positive IEL, which were identified as CD8-positive T lymphocytes and also were mPR positive, was increased in the late proliferative, the mid-secretory and late secretory phases in normally cycling women (P < 0.05). Interestingly, in tubal pregnancy, ERbeta-positive IELs were consistently abundant. In addition, we found a high Ki-67-labelling index for IEL, although ERalpha was entirely absent in the tubal pregnancy oviducts. CONCLUSIONS These results suggest that the number of IEL fluctuated because of estrogen and progesterone levels probably through ERbeta and mPR, respectively. ERbeta-positive IEL may be involved in regulating immune tolerance in tubal pregnancy oviducts.