Attenuation of intrinsic ageing of the skin via elimination of senescent dermal fibroblasts with senolytic drugs.

BACKGROUND: Skin ageing is caused by numerous factors that result in structural and functional changes in cutaneous components. Research has shown that senescent cells are known to accumulate in skin ageing, however, the role of senescent cells in skin ageing has not been defined. OBJECTIVES: To elucidate the role of the senescent cell in skin ageing, we evaluated the effect of known senolytic drugs on senescent dermal fibroblasts. METHODS: Primary human dermal fibroblasts (HDFs) were induced to senescence by long-term passaging, UV irradiation, and H2 O2 treatment. Cell viability was measured after treatment of ABT-263 and ABT-737 on HDFs. Young and aged hairless mice were intradermally injected with drugs or vehicle on the dorsal skin for 10 days. Skin specimens were obtained and reverse-transcription quantitative PCR, western blotting, and histological analysis were performed. RESULTS: We found that ABT-263 and ABT-737 induced selective clearance of senescent dermal fibroblasts, regardless of the method of senescence induction. Aged mouse skin treated with ABT-263 or ABT-737 showed increased collagen density, epidermal thickness, and proliferation of keratinocytes, as well as decreased senescence-associated secretory phenotypes, such as MMP-1 and IL-6. CONCLUSIONS: Taken together, our results indicate that selective clearance of senescent skin cells can attenuate and improve skin ageing phenotypes and that senolytic drugs may be of potential use as new therapeutic agents for treating ageing of the skin.

Towards uterus tissue engineering: a comparative study of sheep uterus decellularisation.

Uterus tissue engineering may dismantle limitations in current uterus transplantation protocols. A uterine biomaterial populated with patient-derived cells could potentially serve as a graft to circumvent complicated surgery of live donors, immunosuppressive medication and rejection episodes. Repeated uterine bioengineering studies on rodents have shown promising results using decellularised scaffolds to restore fertility in a partially impaired uterus and now mandate experiments on larger and more human-like animal models. The aim of the presented studies was therefore to establish adequate protocols for scaffold generation and prepare for future in vivo sheep uterus bioengineering experiments. Three decellularisation protocols were developed using vascular perfusion through the uterine artery of whole sheep uteri obtained from slaughterhouse material. Decellularisation solutions used were based on 0.5% sodium dodecyl sulphate (Protocol 1) or 2% sodium deoxycholate (Protocol 2) or with a sequential perfusion of 2% sodium deoxycholate and 1% Triton X-100 (Protocol 3). The scaffolds were examined by histology, extracellular matrix quantification, evaluation of mechanical properties and the ability to support foetal sheep stem cells after recellularisation. We showed that a sheep uterus can successfully be decellularised while maintaining a high integrity of the extracellular components. Uteri perfused with sodium deoxycholate (Protocol 2) were the most favourable treatment in our study based on quantifications. However, all scaffolds supported stem cells for 2 weeks in vitro and showed no cytotoxicity signs. Cells continued to express markers for proliferation and maintained their undifferentiated phenotype. Hence, this study reports three valuable decellularisation protocols for future in vivo sheep uterus bioengineering experiments.

3D Engineering of Ocular Tissues for Disease Modeling and Drug Testing.

The success rate from investigational new drug filing to drug approval has remained low for decades despite major scientific and technological advances, and a steady increase of funding and investment. The failure to demonstrate drug efficacy has been the major reason that drug development does not progress beyond phase II and III clinical trials. The combination of two-dimensional (2D) cellular in vitro and animal models has been the gold standard for basic science research and preclinical drug development studies. However, most findings from these systems fail to translate into human trials because these models only partly recapitulate human physiology and pathology. The lack of a dynamic three-dimensional microenvironment in 2D cellular models reduces the physiological relevance, and for these reasons, 3D and microfluidic model systems are now being developed as more native-like biological assay platforms. 3D cellular in vitro systems, microfluidics, self-organized organoids, and 3D biofabrication are the most promising technologies to mimic human physiology because they provide mechanical cues and a 3D microenvironment to the multicellular components. With the advent of human-induced pluripotent stem cell (iPSC) technology, the 3D dynamic in vitro systems further enable extensive access to human-like tissue models. As increasingly complex 3D cellular systems are produced, the use of current visualization technologies is limited due to the thickness and opaqueness of 3D tissues. Tissue-clearing techniques improve light penetration deep into tissues by matching refractive indices among the 3D components. 3D segmentation enables quantitative measurements based on 3D tissue images. Using these state-of-the-art technologies, high-throughput screening (HTS) of thousands of drug compounds in 3D tissue models is slowly becoming a reality. In order to screen thousands of compounds, machine learning will need to be applied to help maximize outcomes from the use of cheminformatics and phenotypic approaches to drug screening. In this chapter, we discuss the current 3D ocular models recapitulating physiology and pathology of the back of the eye and further discuss visualization and quantification techniques that can be implemented for drug screening in ocular diseases.

Early development of de novo hepatocellular carcinoma after direct-acting agent therapy: Comparison with pegylated interferon-based therapy in chronic hepatitis C patients.

Patients with chronic hepatitis C who achieve a sustained viral response after pegylated interferon therapy have a reduced risk of hepatocellular carcinoma, but the risk after treatment with direct-acting antivirals is unclear. We compared the rates of early development of hepatocellular carcinoma after direct-acting antivirals and after pegylated interferon therapy. We retrospectively analysed 785 patients with chronic hepatitis C who had no history of hepatocellular carcinoma (211 treated with pegylated interferon, 574 with direct-acting antivirals) and were followed up for at least 24 weeks after antiviral treatment. De novo hepatocellular carcinoma developed in 6 of 574 patients receiving direct-acting antivirals and in 1 of 211 patients receiving pegylated interferon. The cumulative incidence of early hepatocellular carcinoma development did not differ between the treatment groups either for the whole cohort (1.05% vs 0.47%, P = .298) or for those patients with Child-Pugh Class A cirrhosis (3.73% vs 2.94%, P = .827). Multivariate analysis indicated that alpha-fetoprotein level >9.5 ng/mL at the time of end-of-treatment response was the only independent risk factor for early development of hepatocellular carcinoma in all patients (P < .0001, hazard ratio 176.174, 95% confidence interval 10.768-2882.473) and in patients treated with direct-acting agents (P < .0001, hazard ratio 128.402, 95% confidence interval 8.417-1958.680). In conclusion, the rate of early development of hepatocellular carcinoma did not differ between patients treated with pegylated interferon and those treated with direct-acting antivirals and was associated with the serum alpha-fetoprotein level at the time of end-of-treatment response.

Local myogenic pulp-derived cell injection enhances craniofacial muscle regeneration in vivo.

OBJECTIVES: To enhance myogenic differentiation in pulp cells isolated from extracted premolars by epigenetic modification using a DNA demethylation agent, 5-aza-2'-deoxycytidine (5-Aza), and to evaluate the potent stimulatory effect of 5-Aza-treated pulp cell injection for craniofacial muscle regeneration in vivo. SETTING AND SAMPLE POPULATION: Pulp cells were isolated from premolars extracted for orthodontic purposes from four adults (age range, 18-22.1 years). MATERIAL AND METHODS: Levels of myogenic differentiation and functional contraction response in vitro were compared between pulp cells with or without pre-treatment of 5-Aza. Changes in muscle regeneration in response to green fluorescent protein (GFP)-labelled myogenic pulp cell injection in vivo were evaluated using a cardiotoxin (CTX)-induced muscle injury model of the gastrocnemius as well as the masseter muscle in mice. RESULTS: Pre-treatment of 5-Aza in pulp cells stimulated myotube formation, myogenic differentiation in terms of desmin and myogenin expression, and the level of collagen gel contraction. The local injection of 5-Aza pre-treated myogenic pulp cells was engrafted into the host tissue and indicated signs of enhanced muscle regeneration in both the gastrocnemius and the masseter muscles. CONCLUSION: The epigenetic modification of pulp cells from extracted premolars and the local injection of myogenic pulp cells may stimulate craniofacial muscles regeneration in vivo.

Impact of extranodal extension on prognosis in lymph node-positive gastric cancer.

BACKGROUND: The TNM classification system is used widely for tumour staging, and directs the treatment and prognosis of patients with cancer. The aim of this study was to assess the prognostic value of extranodal extension (ENE) in patients with early gastric cancer. METHODS: All patients who underwent gastrectomy with lymphadenectomy for primary gastric cancer with lymph node metastases between January 2003 and June 2006 were reviewed. Histological slides of metastatic nodes were reviewed by two gastrointestinal pathologists. The association of ENE with clinicopathological characteristics was assessed. The disease-specific survival rate was calculated by the Kaplan-Meier method, and a multivariable Cox regression model was used to identify independent prognostic factors. RESULTS: Some 1143 patients were included. ENE was associated with advanced pT and pN category, larger tumour size and lymphovascular/perineural invasion. In multivariable analysis, pT category, pN category, ENE, lymphovascular invasion and perineural invasion were found to be independent prognostic factors in node-positive gastric carcinoma. The 5-year survival rate of patients with ENE was 48·1 per cent, compared with 78·2 per cent for patients without ENE (P < 0·001). In the subgroup of patients with early gastric cancer, ENE was associated with a worse 5-year survival rate in patients with early (T1) gastric cancer: 75 per cent in patients with ENE versus 96·9 per cent in those without (P < 0·001). CONCLUSION: ENE is an independent prognostic factor in patients with early and advanced gastric cancer.

Transection of the obturator nerve by an electrosurgical instrument and its immediate repair during laparoscopic pelvic lymphadenectomy: a case report.

Obturator nerve injury seldom occurs in gynecologic surgery. However, gynecologic oncologic surgery, including pelvic lymph node dissection, increases the risk of this type of injury. Microsurgical techniques are usually performed for the repair of the nerve injury. Herein the authors report a case of obturator nerve injury caused by an electrosurgical instrument during laparoscopic pelvic lymphadenectomy, and its prompt repair by laparoscopic procedure in a 44-year-old patient with cervical cancer.

Association of KIR genes and HLA-C alleles with HPV-related uterine cervical disease in Korean women.

This study investigated whether killer-cell immunoglobulin-like receptor (KIR) genes and human leukocyte antigen (HLA)-C alleles, receptors and ligands of natural killer cells are associated with the development of human papillomavirus (HPV)-related cervical disease in Korean women. Blood samples from 132 women with HPV-related cervical disease and 159 women without HPV infection were collected for genotyping of KIR genes and HLA-C alleles. Although no relationship was found between KIR genes and HPV-related cervical disease, a significant relationship was found between HLA-C alleles as ligands of KIR and HPV-related cervical disease. Women with HPV-related cervical disease were found to be significantly more likely to carry HLA-C*0303, particularly those with HPV 16 or 18 infection, and less likely to carry HLA-C*01 compared to women without HPV infection. HLA-C*0303 was found to confer susceptibility to HPV-related cervical disease, whereas HLA-C*01 was found to confer a protective effect against HPV-related cervical disease.

Diagnostic value of CA125 as a predictor of recurrence in advanced ovarian cancer.

PURPOSE: The aim of this study was to establish the guidelines for detecting early recurrences of advanced epithelial ovarian cancer by use of the CA-125 level. MATERIALS AND METHODS: Eighty-five of the patients who met the inclusion criteria were enrolled in this study. The authors examined 25 incremental changes of CA125 from one to 25 IU/ml, and compared the CA-125 value with other prognostic factors. Increases in the CA-125 level from the nadir level were expressed as CA-125- increments. RESULTS: Among the 25 increments, a CA-125-8 (eight IU/ml) was selected as the predictor that was the most efficient and time-effective. CA-125-8 had a sensitivity of 91.5%, a specificity of 84.6%, a positive predictive value of 93.1%, a negative predictive value of 81.5%, an efficiency of 89.4%. and a median lead-time of 68.5 days (p <0.0001). CONCLUSION: The authors suggest the incremented CA-125-8 as a predictor of recurrent advanced ovarian cancer.

A case of bilateral ovarian synchronous tumors (left ovarian serous papillary adenocarcinoma and right ovarian malignant mixed Müllerian tumor).

Synchronous bilateral ovarian cancer is extremely rare and there is no established guideline for management. A case of a 58-year-old multiparous woman with bilateral ovarian synchronous malignant tumors is presented. The clinical consideration and treatment of related cases are discussed.

Parovarian tumors of borderline malignancy.

The incidence of a parovarian tumor is 10-20% of all uterine adnexal masses, however, it is benign in most cases, and a borderline or malignant tumor is extremely rare. The classification of disease stage and treatment is still controversial owing to its scarcity. We have managed one mucinous and two serous cystadenomas of borderline malignancy originating from paraovarian cysts in our institute over ten year. We report and discuss the cases herein.

Clinicopathologic analysis of extramammary Paget's disease.

PURPOSE: To retrospectively analyze the clinicopathologic characteristics of patients with extramammary Paget's disease who were surgically treated in a single institution. METHOD: The charts of 14 patients with extramammary Paget's disease were retrospectively reviewed, and the clinicopathologic data were collected and analyzed. RESULTS: From January 1990 to July 2009, 14 patients were treated at our institution. Most patients (11/14 patients) had delayed diagnosis. Two patients (14.3%) had associated malignant neoplasms. Eight of 14 patients (57.1%) had positive surgical margins; of these patients, five patients had no evidence of recurrence. In the six patients with negative surgical margins, two patients (33.3%) developed recurrence. CONCLUSIONS: The diagnosis of extramammary Paget's disease is commonly delayed. Because of the possible association with other malignancies before or after the diagnosis of extramammary Paget's disease, thorough examinations are recommended. Disease recurrence is common regardless of the surgical margin status, so long-term monitoring of patients is recommended.

Surface-tuned hierarchical ɤ-Fe2O3-N-rGO nanohydrogel for efficient catalytic removal and electrochemical sensing of toxic nitro compounds.

4- Nitrophenol (4-NP) is a top rated hazardous environmental pollutant and secondary explosive chemicals. For the sake of ecology and environment safety, the catalytic reduction and detection of 4-NP is highly important. In this work, ɤ-Fe2O3-nitrogen doped rGO (ɤ-Fe2O3-N-rGO) nanohydrogel was synthesized by green hydrothermal method. The morphology and phase purity of prepared ɤ-Fe2O3-N-rGO nanohydrogel were confirmed by various analytical (SEM, TEM, XRD, and XPS) and electrochemical techniques. The morphological structure of ɤ-Fe2O3-N-rGO nanohydrogel confirmed that the nanocrystals are well covered over the 2D N-rGO layer. Further, ɤ-Fe2O3-N-rGO nanohydrogel was applied for the catalytic reduction and electrochemical detection of ecotoxic 4-NP. A low cost, ɤ-Fe2O3-N-rGO nanohydrogel displayed an excellent catalytic activity, high recyclability (>5 cycles) and high conversion efficiency of 4-NP to 4-Aminophenol (4-AP). In addition, ɤ-Fe2O3-N-rGO nanohydrogel modified GCE displayed a wide linear sensing range (0.1-1000 µM), and a low detection limit (LOD) of 0.1 µM with excellent sensitivity, high selectivity (<1.2%) and good stability (>4 weeks). The developed sensor electrode shows the low reduction potential of -0.3 V and -0.60 V for the determination of 4-NP. The proposed ɤ-Fe2O3-N-rGO nanohydrogel is promising catalyst for the detection and removal of toxic aromatic nitro compounds in real site applications.

The ultrastructure of patch-clamped membranes: a study using high voltage electron microscopy.

We have developed techniques for studying patch-clamped membranes inside glass pipettes using high voltage electron microscopy (HVEM). To preserve the patch structure with the least possible distortion, we rapidly froze and freeze dried the pipette tip. The pipette is transparent for more than 50 microns from the tip. HVEM images of patches confirm light microscopy observations that the patch is not a bare bilayer, but a membrane-covered bleb of cytoplasm that may include organelles and cytoskeleton. The membrane that spans the pipette is commonly tens of micrometers from the tip of the pipette and occasionally as far as 100 microns. The structure of patches taken from a single cell type is variable but there are consistent differences between patches made from different cell types. With suction applied to the pipette before seal formation, we have seen in the light microscope vesicles swept from the plasmalemma up the pipette. These vesicles are visible in electron micrographs, particularly those made from chick cardiac muscle. Colloidal gold labeling of the patch permitted identification of lectin-binding sites and acetylcholine receptors. In young cultures of Xenopus myocytes, the receptors were diffuse. In 1-wk-old cultures, the receptors formed densely packed arrays. The patch pipette can serve, not only as a recording device, but as a tool for sampling discrete regions of the cell surface. Because the pipette has a constant path length for axial rotation, it is a unique specimen holder for microtomography. We have made preliminary tomographic reconstructions of a patch from Xenopus oocyte.

MicroRNA-218 prevents lung injury in sepsis by inhibiting RUNX2.

OBJECTIVE: To explore the regulatory effects of microRNA-218 on lung tissue of rats with acute lung injury (ALI) and its underlying mechanism. MATERIALS AND METHODS: A total of 32 Sprague Dawley (SD) rats were randomly divided into control group, lung injury group and microRNA-218 treatment group. The in vitro lung injury model was established by injections of lipopolysaccharide (LPS), PGN (peptidoglycan) and IgG IC (immune complex). Polymerase Chain Reaction (PCR) was performed to detect the expression of microRNA-218 in lung tissues of ALI rats. Enzyme-Linked Immunosorbent Assay (ELISA) was used to measure the level of cytokine secretion in ALI rats. The activity and expression level of nuclear factor-kappa B (NF-κB) in MH-S and RA264.7 cells were determined by Luciferase activity assay and Western blot, respectively. RESULTS: MicroRNA-218 was significantly down-regulated in bleomycin and IgG IC-induced lung injury rat model, as well as in cells treated with LPS, PGN and IgG IC. Inflammatory factors, including TNF-α, IL-1b, and IL-6, also showed increased in vivo and in vitro expressions. Besides, the overexpression of microRNA-218 inhibited the secretion of inflammatory factors. PCR analysis and Luciferase activity assay indicated that the expressions of RUNX2 and BIRC3 were down-regulated by microRNA-218 in MH-S and RA264.7 cells. Subsequent studies on mechanisms demonstrated that microRNA-218 inhibited the activity of the NF-κB pathway. CONCLUSIONS: The expression of microRNA-218 markedly decreased in lung tissue of ALI rats, while the expression of inflammatory cytokines showed a remarkable increase, which might be related to the activation of RUNX2 and NF-κB.

Clinical features and treatment of nonalcoholic fatty liver disease across the Asia Pacific region-the GO ASIA initiative.

BACKGROUND: The Gut and Obesity Asia (GO ASIA) workgroup was formed to study the relationships between obesity and gastrointestinal diseases in the Asia Pacific region. AIM: To study factors associated with nonalcoholic steatohepatitis (NASH) and advanced fibrosis, and medical treatment of biopsy-proven nonalcoholic fatty liver disease (NAFLD) patients. METHODS: Retrospective study of biopsy-proven NAFLD patients from centres in the GO ASIA Workgroup. Independent factors associated with NASH and with advanced fibrosis on binary logistic regression analyses in a training cohort were used for the development of their corresponding risk score, which were validated in a validation cohort. RESULTS: We included 1008 patients from nine centres across eight countries (NASH 62.9%, advanced fibrosis 17.2%). Independent predictors of NASH were body mass index ≥30 kg/m2 , diabetes mellitus, dyslipidaemia, alanine aminotransferase ≥88 U/L and aspartate aminotransferase ≥38 U/L, constituting the Asia Pacific NASH risk score. A high score has a positive predictive value of 80%-83% for NASH. Independent predictors of advanced fibrosis were age ≥55 years, diabetes mellitus and platelet count <150 × 109 /L, constituting the Asia-Pacific NAFLD advanced fibrosis risk score. A low score has a negative predictive value of 95%-96% for advanced fibrosis. Only 1.7% of patients were referred for structured lifestyle program, 4.2% were on vitamin E, and 2.4% were on pioglitazone. CONCLUSIONS: More severe liver disease can be suspected or ruled out based on factors identified in this study. Utilisation of structured lifestyle program, vitamin E and pioglitazone was limited despite this being a cohort of biopsy-proven NAFLD patients with majority of patients having NASH.

Epigenetic regulation of RNA polymerase III transcription in early breast tumorigenesis.

RNA polymerase III (Pol III) transcribes medium-sized non-coding RNAs (collectively termed Pol III genes). Emerging diverse roles of Pol III genes suggest that individual Pol III genes are exquisitely regulated by transcription and epigenetic factors. Here we report global Pol III expression/methylation profiles and molecular mechanisms of Pol III regulation that have not been as extensively studied, using nc886 as a representative Pol III gene. In a human mammary epithelial cell system that recapitulates early breast tumorigenesis, the fraction of actively transcribed Pol III genes increases reaching a plateau during immortalization. Hyper-methylation of Pol III genes inhibits Pol III binding to DNA via inducing repressed chromatin and is a determinant for the Pol III repertoire. When Pol III genes are hypo-methylated, MYC amplifies their transcription, regardless of its recognition DNA motif. Thus, Pol III expression during tumorigenesis is delineated by methylation and magnified by MYC.

Local influence of cell viability on stretch-induced permeability of alveolar epithelial cell monolayers.

Ventilator induced lung injury (VILI), often attributed to over-distension of the alveolar epithelial cell layer, can trigger loss of barrier function. Alveolar epithelial cell monolayers can be used as an idealized in vitro model of the pulmonary epithelium, with cell death and tight junction disruption and permeability employed to estimate stretch-induced changes in barrier function. We adapted a method published for vascular endothelial permeability, compare its sensitivity with our previously published method, and determine the relationship between breeches in barrier properties after stretch and regions of cell death After 4-5 days in culture, primary rat alveolar epithelial cells seeded on plasma treated polydimethylsiloxane membrane coated with biotin-labeled fibronectin, or fibronectin alone were stretched in the presence of FITC-tagged streptavidin (biotin-labeled membrane) or BODIPY-ouabain. We found that the FITC-labeling method was a more sensitive indicator of permeability disruption, with significantly larger positively stained areas visible in the presence of stretch and with ATP production inhibitor Antimycin-A. Triple-stained images with Hoescht (nuclei), Ethidium Homodimer (EthD, damaged cell nuclei) and FITC (permeable regions) were used to determine that within permeable regions intact cells were positioned closer to damaged cells than in non-permeable regions. We concluded that local cell death may be an important contributor to barrier integrity.

Internalization of Ia molecules into Birbeck granule-like structures in murine dendritic cells.

Dendritic cells isolated from the draining lymph nodes of mice sensitized epicutaneously with hapten are potent antigen-presenting cells and contain Birbeck granules and cored tubules characteristic of antigen-activated epidermal Langerhans cells. We used immunogold labeling and transmission electron microscopy to follow the internalization of Ia molecules in these antigen-presenting cells. We found that Ia molecules were internalized into Birbeck granule-like structures in the antigen-activated dendritic cells. Computer reconstruction of serial sections of the dendritic cells demonstrated that these structures span the cytoplasm from the cell membrane to the nuclear membrane and are associated with lysosomes. The internalization of Ia molecules into these structures supports the hypothesis that the Birbeck granule-like structures are derived from the cell membrane and are involved in the antigen-processing/presenting function of the dendritic cells.

Sensory innervation in the inner conical body of the vibrissal follicle-sinus complex of the rat.

The innervation of the inner conical body of the vibrissal follicle-sinus complex of the rat was examined by high-voltage and conventional transmission electron microscopy of serial and semi-serial sections. The inner conical body is innervated by axons supplied almost exclusively by several superficial vibrissal nerves that arise from the infraorbital branch of the trigeminal nerve and converge upon the neck of the follicle-sinus complex. Each superficial vibrissal nerve contains a few A delta myelinated axons and several bundles of 20-30 unmyelinated axons. These axons enter the inner conical body and distribute circumferentially within 7-10 ring-like arrays that encircle the vibrissal follicle and are stacked through the superficial-to-deep extent of the inner conical body. Each ring consists of 1 or 2 myelinated axons and several small bundles of 2-15 unmyelinated axons enclosed in sheaves of parallel collagen fibrils. Myelinated axons provide exclusively lanceolate endings that may arise at the termination of the axon or at nodes of Ranvier. Within the small bundles, unmyelinated axons individually terminate in succession as abrupt cytoplasmic swellings referred to as cytoplasmic blebs, which contain mitochondria or clusters of clear or dense-core vesicles. Because of their affiliation with collagen fibrils and the proximity of myelinated axons, the blebbed endings may have been misinterpreted as Ruffini endings in previous studies. Their structure, distribution, and origin from unmyelinated axons suggest that the blebbed endings may constitute a unique array of low-threshold C-mechanoreceptors.