Fit-for-purpose characterization of air-liquid-interface (ALI) in vitro exposure systems for e-vapor aerosol.

Air-liquid-interface (ALI) exposure systems deliver aerosol to the apical surface of cells which mimics the in vivo inhalation exposure conditions. It is necessary, however, to quantify the delivered amount of aerosol for ALI-based in vitro toxicity assessment. In this study, we evaluated two commercially available ALI exposure systems, a Vitrocell® Ames 48 (Ames 48) and a Vitrocell® 24/48 (VC 24/48), and the Vitrocell® VC1/7 smoking machine using a cig-a-like cartridge-based e-vapor device with a prototype formulation (containing 4% nicotine by weight). We characterized aerosol particle-size distribution, aerosol mass, and major chemical components (nicotine, propylene glycol, and glycerol) at the generation source and verified the repeatability of the aerosol generation. We determined aerosol delivery at the ALI by gravimetric analysis of mass collected on Cambridge filter pads and analytical quantitation of the buffer medium which showed that both aerosol mass and nicotine to an exposure insert linearly increased up to 400 puffs. The delivered aerosol mass covered a wide range of 0.8-3.4 mg per insert in the Ames 48 with variability (relative standard deviation, RSD) up to 12% and 1.1-6.4 mg per insert in the VC 24/48 with variability up to 15%. The delivered nicotine ranged approximately up to 200 µg per insert in both exposure systems. These results provided operation and aerosol delivery information of these ALI exposure systems for subsequent in vitro testing of e-vapor aerosols.

Targeting GM-CSF for collagenase-induced osteoarthritis pain and disease in mice.

OBJECTIVES: Pharmacological options for treating osteoarthritis (OA) are limited and alternative treatments are required. Given the clinical data indicating that granulocyte macrophage-colony stimulating factor (GM-CSF) may be a therapeutic target in human OA, we evaluated different treatment regimens with a neutralizing anti-GM-CSF monoclonal antibody (mAb) in an experimental OA model to determine their effectiveness on amelioration of pain and disease. METHODS: The collagenase-induced osteoarthritis (CiOA) model was induced in C57BL/6 mice, followed by different treatment regimens of anti-GM-CSF mAb or isotype control. Anti-CCL17 mAb treatment was also administered continually during the late stage of CiOA. Pain-related behavior (change in weight distribution of hind limbs), and disease (cartilage damage and osteophyte size) were assessed. RESULTS: Blocking GM-CSF only during early synovitis in CiOA prevented pain and disease development. Once OA pain was established, regardless of the treatment regimen, anti-GM-CSF mAb treatment rapidly and efficiently ameliorated it; however, unless the treatment was continued, pain returned and disease progressed. Continual late stage blockade of GM-CSF was able to ameliorate pain (between-group difference: -6.567; 95% confidence interval (CI): -10.12, -3.011) and suppress cartilage damage (P = 0.0317, 95% CI: -1.75, -0.0556). Continual late stage blockade of CCL17 showed similar effects on pain and disease development. CONCLUSIONS: Early and short-term GM-CSF neutralization is effective at preventing CiOA pain and disease development but, once pain is evident, continual GM-CSF blockade is required to prevent pain from returning and to suppress disease progression in mice. These data reinforce the potential benefits of anti-GM-CSF (and anti-CCL17) mAb therapy in OA and should inform further clinical trials.

Incidence of psoriasiform diseases secondary to tumour necrosis factor antagonists in patients with inflammatory bowel disease: a nationwide population-based cohort study.

BACKGROUND: There are increasing reports of paradoxical psoriasiform diseases secondary to anti-tumour necrosis factor (TNF) agents. AIMS: To determine the risks of paradoxical psoriasiform diseases secondary to anti-TNF agents in patients with inflammatory bowel disease (IBD). METHODS: A nationwide population study was performed using the Korea National Health Insurance Claim Data. A total of 50 502 patients with IBD were identified between 2007 and 2016. We compared 5428 patients who were treated with any anti-TNF agent for more than 6 months (anti-TNF group) and 10 856 matched controls who had never taken anti-TNF agents (control group). RESULTS: Incidence of psoriasis was significantly higher in the anti-TNF group (36.8 per 10 000 person-years) compared to the control group (14.5 per 10 000 person-years) (hazard ratio [HR] 2.357, 95% confidence interval [CI] 1.668-3.331). Palmoplantar pustulosis (HR 9.355, 95% CI 2.754-31.780) and psoriatic arthritis (HR 2.926, 95% CI 1.640-5.218) also showed higher risks in the anti-TNF group. In subgroup analyses, HRs for psoriasis by IBD subtype were 2.549 (95% CI 1.658-3.920) in Crohn's disease and 2.105 (95% CI 1.155-3.836) in ulcerative colitis. Interestingly, men and younger (10-39 years) patients have significantly higher risks of palmoplantar pustulosis (HR 19.682 [95% CI 3.867-100.169] and HR 14.318 [95% CI 2.915-70.315], respectively), whereas women and older (≥40 years) patients showed similar rates between the two groups. CONCLUSIONS: The risks of psoriasiform diseases are increased by anti-TNF agents in patients with IBD. Among psoriasiform diseases, the risk of palmoplantar pustulosis shows the biggest increase particularly in male and younger patients.

Homer2 within the central nucleus of the amygdala modulates withdrawal-induced anxiety in a mouse model of binge-drinking.

A history of binge-drinking decreases protein expression of the glutamate-related scaffolding protein Homer2 within the central nucleus of the amygdala (CEA), coinciding with behavioral signs of negative affect. To assess the functional relevance of this protein change for withdrawal-induced hyper-anxiety, adult (PND 56) and adolescent (PND 28) male C57BL/6J mice were administered an intra-CEA infusion of an adeno-associated viral vector (AAV) carrying either cDNA to express Homer2 (H2-cDNA) or GFP as control. Mice underwent 14 days of binge-drinking under multi-bottle, limited-access conditions and were assayed for behavioral signs of negative affect during withdrawal using the light-dark box, marble burying, and forced swim tests (FST). Following behavioral testing, all animals experienced 5 days of drinking to evaluate the effects of prior alcohol experience and Homer2 manipulation on subsequent alcohol consumption. During protracted (4 weeks) withdrawal, adolescent alcohol-experienced GFP controls showed increased signs of negative affect across all 3 assays, compared to water-drinking GFP animals, and also showed elevated alcohol consumption during the subsequent drinking period. Homer2-cDNA infusion in adolescent-onset alcohol-drinking animals was anxiolytic and reduced subsequent alcohol consumption. Conversely, Homer2-cDNA was anxiogenic and increased drinking in water-drinking adolescents. Unfortunately, the data from adult-onset alcohol-drinking animals were confounded by low alcohol consumption and negligible behavioral signs of anxiety. Nevertheless, the present results provide novel cause-effect evidence supporting a role for CEA Homer2 in the regulation of both basal anxiety and the time-dependent intensification of negative affective states in individuals with a history of binge-drinking during adolescence.

Risk factors of falls among inpatients with cancer.

AIM: To investigate the risk factors and predictors of falls according to the general characteristics, conscious state, physical condition and treatment of hospitalized patients with cancer. BACKGROUND: Inpatients with cancer experience falls more frequently than those without cancer, and the degree of injuries is more severe among inpatients with cancer. A specific fall prevention strategy is needed for each patient. Prevention of falls in patients with cancer is very important for improving the quality of nursing care. METHODS: This retrospective study included matched case-control patients. We evaluated patients between January 1, 2013, and December 31, 2014. A total of 356 patients (fall group, 178; non-fall group, 178) were included. For fall prediction, logistic regression was performed on the variables that were statistically significant in the univariate analysis. RESULTS: The variables that were significant predictors of falls were the use of an assistive device, history of falls and fatigue. DISCUSSION: The predictors of falls in patients with cancer include physical conditions and general characteristics. Fall prevention strategies in patients with cancer should be planned individually with multifaceted aspects, including physical symptom management. LIMITATIONS: The study was conducted at a single cancer center in Korea; thus, our results cannot be generalized. Additionally, in Korea, it is common to have family members or private caregivers for patient care, and this might have influenced the results. CONCLUSION AND IMPLICATIONS FOR NURSING AND HEALTH POLICY: The predictive factors for falls reflect the nature of the patient's environment, culture and disease. Falls have a negative effect on patient safety and can significantly influence quality of life. Policies for patient safety need more specialized and customized approaches.

The proto-oncoprotein KR-POK represses transcriptional activation of CDKN1A by MIZ-1 through competitive binding.

This corrects the article DOI: 10.1038/onc.2011.331.

The importance of the Crohn's disease activity index in surgery for small bowel Crohn's disease.

AIM OF THE STUDY: Compared with patients with other benign intestinal conditions, patients with CD are at increased risk of developing postoperative complications following intestinal resection. We searched for useful tools for predicting postoperative complication in patients with CD by comparing the relationship between postoperative morbidity in these patients as measured by three different scoring tools: general surgical risk (POSSUM score), disease activity (CDAI), and nutritional screening (nutritional prognostic index). METHODS: We performed a retrospective review of 50 patients with small bowel CD who underwent surgical resection and primary anastomosis between 1999 and 2014. RESULTS: This study enrolled 34 men and 16 women. The mean age was 38.4 years (range: 20-81 years). There was no postoperative mortality. The overall postoperative morbidity rate (33.7%) predicted by POSSUM was similar to the rate in the study patients (36.0%). Although POSSUM score predicted higher postoperative morbidity rates in patients who underwent emergency surgery (estimated morbidity: 52.8%), the actual postoperative morbidity rate in the emergency surgery group (26.7%) was smaller than in the elective surgery group (40.0%). In addition, neither preoperative nutritional status nor POSSUM score was related to the severity of postoperative complications. CDAI score was significantly related to the severity of postoperative complications (P=0.032). CONCLUSION: Based on the above results, a high preoperative CDAI score can predict negative postoperative outcomes. We believe that disease activity should be controlled using various treatment modalities, such as enteral or total parenteral nutrition as well as medication, before performing surgery in patients with CD.

ECM1 regulates tumor metastasis and CSC-like property through stabilization of ß-catenin.

Extracellular Matrix Protein 1 (ECM1) is a marker for tumorigenesis and is correlated with invasiveness and poor prognosis in various types of cancer. However, the functional role of ECM1 in cancer metastasis is unclear. Here, we detected high ECM1 level in breast cancer patient sera that was associated with recurrence of tumor. The modulation of ECM1 expression affected not only cell migration and invasion, but also sphere-forming ability and drug resistance in breast cancer cell lines. In addition, ECM1 regulated the gene expression associated with the epithelial to mesenchymal transition (EMT) progression and cancer stem cell (CSC) maintenance. Interestingly, ECM1 increased ß-catenin expression at the post-translational level through induction of MUC1, which was physically associated with ß-catenin. Indeed, the association between ß-catenin and the MUC1 cytoplasmic tail was increased by ECM1. Furthermore, forced expression of ß-catenin altered the gene expression that potentiated EMT progression and CSC phenotype maintenance in the cells. These data provide evidence that ECM1 has an important role in cancer metastasis through ß-catenin stabilization.

Cytokeratin19 induced by HER2/ERK binds and stabilizes HER2 on cell membranes.

Cytokeratin19 (KRT19) is widely used as a biomarker for the detection of disseminated tumors. Using an LC-MS/MS proteomics approach, we found that KRT19 was upregulated in HER2-overexpressing cells and tissues. KRT19 expression was induced by HER2-downstream ERK at the transcriptional level. Another HER2-downstream kinase, Akt, was found to phosphorylate KRT19 on Ser35 and induce membrane translocation of KRT19 and remodeling of KRT19 from filamentous to granulous form. KRT19 phosphorylated by Akt could bind HER2 on the plasma membrane and stabilized HER2 via inhibition of proteasome-mediated degradation of HER2. Silencing of KRT19 by shRNA resulted in increased ubiquitination and destabilization of HER2. Moreover, treatment of KRT19 antibody resulted in downregulation of HER2 and reduced cell viability. These data provide a new rationale for targeting HER2-positive breast cancers.

Effect of cryopreservation and cell passage number on cell preparations destined for autologous chondrocyte transplantation.

Autologous chondrocyte transplantation (ACT) is an effective and safe therapy for repairing articular cartilage defects and requires cell preservation and subculture before transplantation. We compared the effects of cryopreservation and passaging on cell viability, proliferation, and maintenance of the function of chondrocytes and synovium-derived mesenchymal stem cells (MSCs) used as sources for ACT. These cells were isolated from the knee joints of rabbits and were cultured, passaged serially, and divided into 2 groups that were either cryopreserved or not. The morphology, viability, gene expression, and differentiation potential of the 2 groups were compared. Maintenance of the potential to undergo chondrogenic differentiation was determined with the use of a 3-dimensional culture method. Passaging and cryopreservation significantly affected the ability of chondrocytes to maintain their morphology, express chondrogenic genes, and differentiate. In contrast, synovium-derived cells were not affected by passaging and cryopreservation. Our results may serve as the foundation for the application of passaged and cryopreserved chondrocyte or other source cells of MSCs in ACT.

Artificial islets from hybrid spheroids of three pancreatic cell lines.

Pancreatic islets have been the focus of recent studies exploring the pathologic mechanisms of diabetes mellitus as well as more effective and radical treatments for this disease. Islet transplantation is a promising therapeutic strategy; however, isolation of pancreatic islets for this purpose has been challenging, because the technique is time consuming and technically difficult, and tissue handling can be variable. Pseudo-islets can be used as an alternative to naïve islets, but require cellular sources or artificial materials. In this study, pancreas-derived cells were used to generate pseudo-islets. Because the pancreas is composed of a variety of cell types, namely α cells, ß cells, δ cells, and other pancreatic cells that perform different functions, we used 3 different cell lines-NIT-1 (a ß-cell line), α TC1 clone 6 (an α-cell line), and TGP52 (a pancreatic epithelial-like cell line)-which we cocultured in nonadhesive culture plates to produce hybrid cellular spheroids. These pseudo-islets had an oval shape and were morphologically similar to naïve islets; additionally, they expressed and secreted the pancreatic hormones insulin, glucagon, and somatostatin, as confirmed by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. The results demonstrate that pseudo-islets that mimic naïve islets can be successfully generated by a coculture method. These artificial islets can potentially be used for in vitro tests related to diabetes mellitus, specifically, in drug discovery or for investigating pathology. Moreover, they can be useful for examining basic questions pertaining to cell-cell interactions and tissue development.

Experimental canine facial transplantation.

OBJECTIVE: Facial allotransplantation represents a novel frontier in the reconstruction of complex human facial defects. To develop more refined surgical techniques and yield fine results, it is required to make a suitable animal model. The development of a model of composite facial and scalp allograft in canines is more appealing: In large animals, canine facial anatomy is the most similar to humans; its facial nerve anatomy also resembles humans'; and canines possess the most similar facial vascular anatomy to humans. These factors led to the development of a canine composite facial allograft model. METHODS: Two operative teams performed simultaneously on both donor and recipient to harvest the hemifacial/scalp flap and to prepare the recipient to shorten operative time. The flap was harvested with the common carotid artery and external jugular vein and transferred to the recipient. After insetting of the cartilage, skin, and muscles, the anastomosis of the external jugular vein and anastomosis between the external carotid artery and lingual artery were performed. RESULTS: The total mean time of transplantation was 5 hours ± 30 minutes. All of the transplanted animals were wreated with FK-506 [tacrolimus, 2 mg/kg] for 7 days after surgery. Clinical rejection response was also identified by close monitoring. Most allografts survived with perfect viability without vascular problems in the early postoperative period. CONCLUSIONS: We documented that this model is well qualified in every aspect for use as a standard transplantation training model and future research work.

Adjuvant radiotherapy for endometrial cancer--a comparative review of radiotherapy technique with acute toxicity.

OBJECTIVES: The addition of pelvic radiotherapy to brachytherapy (EBRT-BT) in early-stage endometrial cancer is controversial and may cause unnecessary toxicity. The incidence of acute toxicity of EBRT-BT will have an impact on clinical decision and patient compliance but is currently poorly understood. This study compares the acute toxicities of EBRT-BT versus BT alone. MATERIALS AND METHODS: Seventy-nine patients with FIGO Stage IA-II endometrial cancer who underwent adjuvant radiotherapy, (EBRT-BT or BT alone) from 2001 to 2011 were included in the study. Medical records of these patients were reviewed retrospectively and toxicity graded according to the Common Terminology Criteria for Adverse Events (CTCAE) version 4.0. Patients were followed up for at least three months post-treatment to assess resolution of toxicity. RESULTS: The mean age of the study group was 60.6 years. Median follow-up was four years. Forty patients received EBRT-BT. There was a 37% increase in Grade 1-3 diarrhea with the addition of pelvic radiotherapy (OR 18.67, p < 0.0005) and a 34% increase in lethargy (p < 0.0005). There was also an increased occurrence of genitourinary and skin toxicities. Two patients in the EBRT-BT group required hospitalisation for severe diarrhea and three patients were unable to complete the treatment. All acute toxicities had resolved by three months post treatment. CONCLUSION: EBRT-BT causes significantly more acute toxicities compared to BT alone. Patients should be informed of this during counselling.

Chemoembolization with drug-eluting microspheres (DEM-TACE) for hepatocellular carcinoma: single-center review of safety and efficacy.

PURPOSE: This study examines the safety and efficacy of transarterial chemoembolization using doxorubicin-loaded 30-60 µm QuadraSphere microspheres (DEM-TACE) for the treatment of hepatocellular carcinoma. MATERIALS AND METHODS: Over 10 weeks, patients with hepatocellular carcinoma. (Child-Pugh A/B: 65%/35%) were embolized with 30-60 µm QuadraSphere microspheres. Excluded patients had previous locoregional therapy, macrovascular invasion, extrahepatic disease, Child-Pugh score >B7, ECOG performance status >0, and total bilirubin >3 mg/dL. Technical success, minor and major complications, 30-day hospital readmission rate, and 30-day mortality were assessed. α-Fetoprotein levels before and after treatment were compared. Local response was evaluated by radiologic tumor response per modified Response Evaluation Criteria in Solid Tumors 1 month after treatment. RESULTS: Thirty tumors (mean size, 2.3 cm; range, 1.0-4.9 cm) were treated in 20 patients (16 male and 4 female; mean age, 64.7 years). There were no major complications. Thirty-day mortality was 0%. Minor complications included postembolization syndrome in 16.7% of cases and transient rise in liver enzymes requiring no therapy. Mean α-fetoprotein levels trended down following treatment (71.8±201.9 ng/mL vs 53.4±116.7 ng/mL), but were not statistically significant. Complete response was achieved in 30% of patients, partial response in 35%, stable disease in 30%, and progression of disease in 5%. Overall objective response was 65%. Mean follow-up was 10.4 months (range, 2-16.4 months). CONCLUSION: DEM-TACE with doxorubicin-loaded 30-60 µm QuadraSpheres is feasible, well tolerated, and associated with promising tumor response in early and intermediate stage disease.

Attenuation of donor-reactive T cells allows effective control of allograft rejection using regulatory T cell therapy.

Regulatory T cells (Tregs) are essential for the establishment and maintenance of immune tolerance, suggesting a potential therapeutic role for Tregs in transplantation. However, Treg administration alone is insufficient in inducing long-term allograft survival in normal hosts, likely due to the high frequency of alloreactive T cells. We hypothesized that a targeted reduction of alloreactive T effector cells would allow a therapeutic window for Treg efficacy. Here we show that preconditioning recipient mice with donor-specific transfusion followed by cyclophosphamide treatment deleted 70-80% donor-reactive T cells, but failed to prolong islet allograft survival. However, infusion of either 5 × 10(6) Tregs with direct donor reactivity or 25 × 10(6) polyclonal Tregs led to indefinite survival of BALB/c islets in more than 70% of preconditioned C57BL/6 recipients. Notably, protection of C3H islets in autoimmune nonobese diabetic mice required islet autoantigen-specific Tregs together with polyclonal Tregs. Treg therapy led to significant reduction of CD8(+) T cells and concomitant increase in endogenous Tregs among graft-infiltrating cells early after transplantation. Together, these results demonstrate that reduction of the donor-reactive T cells will be an important component of Treg-based therapies in transplantation.

New culture medium concepts for cell transplantation.

BACKGROUND: Before cell or tissue transplantation, cells or tissues have to be maintained for a certain period in vitro using culture medium and methods. Most culture media contain substances such as pH indicators and buffers. It is not known whether some of these substances are safe for subsequent application in the transplantation of cells or tissues into the human body. We investigated culture media and methods with respect to the safety of the components in future transplantation applications. METHODS: A modified culture medium--medical fluid-based culture medium (FCM)--was designed by using various fluids and injectable drugs that are already currently permitted for use in clinical medicine. Medium components necessary for optimal cell growth were obtained from approved drugs. FCM was manufactured with adjusted final concentrations of the medium components similar to those in commercial Dulbecco's modified Eagle's medium (DMEM). In particular, 1029.40 mg/L amino acids, approximately 88.85 mg/L vitamins, 13,525.77 mg/L inorganic salts, and 4500 mg/L D-glucose comprise the high-glucose FCM. Next, human fat synovium-derived mesenchymal stem cells and rat H9c2 (2-1) cells were cultured under 2 conditions: (1) DMEM-high glucose (HG), an original commercial medium, and (2) optimized FCM-HG. We assessed the morphologies and proliferation rates of these cells. RESULTS: We observed that FCM-HG was able to induce the growth of FS-MSC and commercially available H9c2 cell. The morphologies and proliferation patterns of these cells cultured under FCM-HG showed no differences compared with cells grown in DMEM-HG. CONCLUSION: Our data suggest that FCM, which we developed for the first time according to the concept of drug repositioning, was a useful culture medium, especially in cultured cells intended for human cell transplantation.

Efficacy and toxicity of intensity-modulated radiation therapy for prostate cancer in Chinese patients.

OBJECTIVE: To report the treatment efficacy and toxicity profile of intensitymodulated radiation therapy in Chinese patients with clinically localised prostate cancer. DESIGN: Historical cohort study. SETTING: Oncology unit in a university teaching hospital in Hong Kong. PATIENTS: Patients with clinically localised prostate cancer undergoing intensity-modulated radiation therapy in our institution between May 2001 and November 2009 were reviewed. MAIN OUTCOME MEASURES: The 5-year biochemical failure­free survival, 5-year overall survival, as well as acute/late gastro-intestinal toxicities and genito-urinary toxicities. RESULTS: A total of 182 patients were treated with prostate intensitymodulated radiation therapy with or without whole-pelvic radiotherapy. The median follow-up was 44 months. The median patient age was 72 years. Overall survival of the cohort was 92% after 5 years. The favourable, intermediate, and unfavourable risk category distributions of the National Comprehensive Cancer Network were 21 (12%), 42 (23%), and 119 (65%), respectively. The 5-year actuarial biochemical failure­free survival rates for patients in these categories were 95%, 82%, and 80%, respectively. Multivariate analysis identified early tumour stage, low pre-treatment prostate-specific antigen levels, and the use of adjuvant androgen deprivation as independent prognostic factors for better biochemical failure­free survival. Grade 2 and 3 late gastro-intestinal/genito-urinary toxicities occurred in 8%/3% and 4%/3% of the patients, respectively. CONCLUSION: Intensity-modulated radiation therapy for prostate cancer is feasible and safe in the Chinese population. These data are consistent with the results of other series in Caucasian populations.

Late presentation of simple virilising 21-hydroxylase deficiency in a Chinese woman with Turner's syndrome.

Classical congenital adrenal hyperplasia due to 21-hydroxylase deficiency is a well-known disorder of sexual development (previously known as ambiguous genitalia) in genotypic female neonates. We report on a 66-year-old Chinese, brought up as male, with a simple virilising form of congenital adrenal hyperplasia associated with Turner's syndrome (karyotype 45,X/47,XXX/46,XX). His late presentation was recognised due to his exceptionally short stature and persistent sexual ambiguity. His condition was only brought to medical attention as he developed a huge abdominal mass, which later turned out to be a benign ovarian mucinous cyst. It is therefore important to look out for co-existing congenital adrenal hyperplasia in patients with Turner's syndrome and virilisation, after the presence of Y chromosome material has been excluded.