Characteristics and Regulation of Human Eosinophil ETosis In Vitro.

Cytolytic ETosis is a type of programmed cell death distinct from apoptosis and necrosis and plays a major role in the innate immune system and disease progression. Through the process of ETosis, cells release their chromatin with diverse antimicrobial proteins into the extracellular milieu, forming extracellular traps (ETs). Although ETosis has been reported in several leukocyte types, few studies have compared ETosis and the component proteins of ETs in leukocytes. The aim of this study was to better understand the characteristics of eosinophil ETosis (EETosis) compared with other leukocytes. We isolated human blood eosinophils, neutrophils, basophils, monocytes, and lymphocytes and stimulated them with known ETosis inducers, a protein kinase C activator PMA, or a calcium ionophore A23187. Both stimuli induced eosinophil cell death and ET release after 180 minutes of stimulation in a NADPH-oxidase-dependent manner. PMA also induced NADPH-oxidase-dependent ETosis in neutrophils, whereas little or no significant ETosis was observed in basophils, monocytes, or lymphocytes at 180 minutes. Mass spectrometry-based proteomic analysis of eosinophil- and neutrophil-derived ETs identified 997 and 1415 proteins, respectively. Among the physiological stimuli tested, immobilized IgA and IgG induced EETosis. C-C motif chemokine ligand 11 (CCL11) and interleukin 5 (IL-5) were weak inducers of EETosis, but co-stimulation significantly induced rapid EETosis. Under high serum or albumin conditions, co-stimulation with CCL11 and IL-5 paradoxically prolonged cell survival by preventing spontaneous apoptosis. This study provides an in-depth characterization of EETosis and highlights the precise regulation of eosinophil survival and cell death pathways.

MGMT protein expression is a reliable predictive biomarker for temozolomide-containing chemotherapy in osteosarcoma.

The prognosis of patients with osteosarcoma who experience recurrence or progression (R/P) is extremely poor, and more effective and less toxic therapies are needed. In the current study, the clinical data of osteosarcoma patients who experienced R/P were retrospectively analyzed to verify the reliability of O-6-methylguanine-DNA methyltransferase (MGMT) protein expression or MGMT promoter methylation for predicting the response to off-label temozolomide (TMZ)-containing chemotherapy. Of the 30 evaluable patients, 9 (30%) showed no/low MGMT protein expression, whereas all 16 evaluable patients had unmethylated MGMT promoter irrespective of MGMT protein expression levels. Twenty-three patients received TMZ-containing chemotherapy for measurable lesions (n = 14) or as adjuvant therapy following resection of recurrent lesions (n = 9). Among 14 patients with radiologically measurable lesions, the objective response rate was higher in the MGMT no/low-expression group (50.0%) than in the MGMT intermediate/high-expression group with borderline significance (0%, p = 0.066). The 6-month progression-free survival (PFS) rate in patients with radiologically measurable lesions was significantly higher in the MGMT no/low-expression group (50.0%) than in the MGMT intermediate/high-expression group (0%, p = 0.036). In the multivariate analysis of the 23 patients receiving TMZ-containing chemotherapy, MGMT expression and disease status before TMZ-containing chemotherapy were significantly associated with PFS. No severe adverse effects were observed during TMZ-containing chemotherapy. MGMT protein expression, but not MGMT promoter methylation, could predict a favorable outcome in patients receiving TMZ-containing chemotherapy.

Quantitative evaluation of endometrium-expressed mRNAs for the purpose of discriminating between menstruation and traumatic vaginal injury in sexual assault cases.

In sexual assault cases, it is crucial to discriminate between peripheral blood and menstrual blood to provide evidence for vaginal intercourse with traumatic injury. In this study, the menstrual blood mRNA markers progestagen-associated endometrial protein (PAEP), matrix metallopeptidase 7 (MMP7), and left-right determination factor 2 (LEFTY2) were evaluated by quantitative RT-PCR (RT-qPCR) for the discrimination of menstrual blood from peripheral blood and vaginal fluid. As a result, all markers with cutoff delta cycle quantification (ΔCq) values were specifically determined in menstrual blood among forensically relevant body fluids. Even though the changes in the expression levels of each marker differed during the menstrual cycle, all markers were determined to be positive in most of the randomly collected menstrual blood samples that were analyzed. Additionally, the markers with proposed cutoff ΔCq values could discriminate between menstrual blood and peripheral blood-mixed vaginal fluid samples. The determination of positive markers was less affected by storage temperature under dry conditions than under wet conditions, while PAEP was detectable in samples stored below room temperature under wet conditions. The detectability of PAEP was considered to be the result of its higher expression level compared with MMP7 and LEFTY2. In conclusion, menstrual blood markers for the RT-qPCR procedure evaluated in this study were highly specific for menstrual blood. The proposed procedure could be useful for discriminating between menstruation and traumatic bleeding in the female genital tract. In particular, PAEP is expected to be applicable to forensic casework samples because of its high specificity and robustness.

A case of novel NFKB2 variant with hypertensive emergency and nephrotic syndrome leading to CKD 5D.

Nuclear factor kappa B (NF-κB) family plays a central role in the human immune system. Heterozygous variants in NFKB2 typically cause immunodeficiency with various degrees of central adrenal insufficiency, autoimmunity, and ectodermal dysplasia. No reported case has presented kidney failure as an initial symptom. Moreover, documentation of kidney involvement of this disease is limited. CASE DIAGNOSIS: A 2-year-old female who presented with dyspnea and hypertensive emergency in the setting of new-onset nephrotic syndrome with acute-on chronic kidney injury with resultant chronic kidney disease (CKD) was found to have a novel heterozygous N-terminal variant in NFKB2 (c.880del: p. Tyr294Ilefs*4) with mild hypogammaglobulinemia, but no adrenal insufficiency or ectodermal dysplasia. She became dialysis-dependent during her initial hospitalization and developed CKD stage 5D, requiring continued peritoneal dialysis. She is currently awaiting kidney transplantation. CONCLUSIONS: Whether nephrotic syndrome or kidney injury or failure is the primary symptom of this variant or secondary to some event remains unknown. Further case accumulation is warranted.

Improved cerebral blood flow and hippocampal blood flow in stroke-free patients after catheter ablation of atrial fibrillation.

INTRODUCTION: Atrial fibrillation (AF) is a risk factor for reduced cerebral blood flow (CBF) and cognitive dysfunction, even in stroke-free patients. We aimed to test the hypothesis that CBF and hippocampal blood flow (HBF), measured with arterial spin labeling magnetic resonance imaging (MRI), improve after catheter ablation of AF to achieve sinus rhythm (SR). METHODS: A total of 84 stroke-free patients (63.1 ± 9.1 years; paroxysmal AF, n = 50; non-paroxysmal AF, n = 34) undergoing AF catheter ablation were included. MRI studies were done before, 3 months, and 12 months after the procedure with CBF and HBF measurements. RESULTS: Baseline CBF and HBF values in 50 paroxysmal AF patients were used as controls. Baseline CBF was higher in patients with paroxysmal AF than with non-paroxysmal AF (100 ± 32% vs. 86 ± 28%, p = .04). Patients with non-paroxysmal AF had increased CBF 3 months after AF ablation (86 ± 28% to 99 ± 34%, p = .03). Differences in CBF and HBF were greater in the group with AF restored to SR (p < .01). Both CBF and HBF levels at 12 months were unchanged from the 3 months level. Successful rhythm control by catheter ablation was an independent predictor of an increase in CBF > 17.5%. The Mini-Mental State Examination score improved after ablation (p = .02). CONCLUSION: SR restoration with catheter ablation was associated with improved CBF and HBF at 3 months, maintenance of blood flow, and improved cognitive function at 12 months.

A Case of Recurrent Infective Endocarditis Caused by Streptococcus Agalactiae.

A 53-year-old man was admitted to hospital with a high fever and shivering. He had undergone aortic valve replacement 4 years previously due to infective endocarditis caused by Streptococcus agalactiae. 18F-fluorodeoxyglucose positron emission tomography/CT (18F-FDG PET/CT) showed increased uptake in the tissue surrounding the prosthetic valve. S. agalactiae was detected in blood cultures after admission. We made a diagnosis of prosthetic valve endocarditis due to an S. agalactiae infection relapse. After 6 weeks of antibacterial treatment, the inflammatory findings successfully improved. However, reexamination with 18F-FDG PET/CT suggested the possibility of persistent prosthetic valve infection. Therefore, we decided to continue the oral antibiotic treatment after discharge.

Highly Sensitive Carbon Monoxide Sensor Element with Wide-Range Humidity Resistance by Loading Pd Nanoparticles on SnO2 Surface.

To develop a highly sensitive carbon monoxide (CO) sensor with a wide range of humidity resistance, we focused on the Pd loading method on SnO2 nanoparticles and the thickness of the sensing layer. The Pd nanoparticles were loaded on the SnO2 surface using the surface immobilization method (SI-Pd/SnO2) and the colloidal protection method (CP-Pd/SnO2). The XPS analysis indicated that the Pd nanoparticles were a composite of PdO and Pd, regardless of the loading method. According to the evaluation of the electrical properties at 350 °C, the CO response in a humid atmosphere and the resistance toward humidity change using CP-Pd/SnO2 were higher than those using SI-Pd/SnO2, even though the Pd loading amount of SI-Pd/SnO2 was slightly larger than that of CP-Pd/SnO2. In addition, Pd/SnO2 prepared via the CP method with a thinner sensing layer showed a higher sensor response and greater stability to humidity changes at 300 °C, even though the humidity change influenced the CO response at 250 and 350 °C. Thus, the overall design of the surface Pd, including size, dispersity, and oxidation state, and the sensor fabrication, that is, the thickness of the sensing layer, offer a high-performance semiconductor-type CO gas sensor with a wide range of humidity resistance.

Effects of once-yearly zoledronic acid on bone density and incident vertebral fractures in nonmetastatic castration-sensitive prostate cancer patients with osteoporosis.

BACKGROUND: Androgen deprivation therapy (ADT) is the effective treating prostate cancer but is often accompanied by cancer treatment-induced bone loss (CTIBL), which impairs the patient's quality of life. In patients with nonmetastatic castration-sensitive prostate cancer (M0CSPC) who already have osteoporosis before starting ADT, appropriate bone-modifying agent intervention must be performed in parallel, as the patient has a high risk of future fracture. However, little is known about therapeutic interventions aimed at preventing the progression of CTIBL and new fractures. The present study explored the effect of once-yearly zoledronic acid 5 mg (ZOL 5 mg) on bone mineral density (BMD) and new vertebral fractures (VFs) in M0CSPC patients with coexisting osteoporosis before starting ADT. METHODS: We conducted a retrospective, multi-institutional, cohort study involving 42 M0CSPC patients with osteoporosis who had undergone ADT with/without a single intravenous infusion of ZOL 5 mg at the start of ADT (ZOL 5 mg group, n = 26; control group, n = 16). The association of the ZOL 5 mg with changes in the BMD from baseline to 12 months and the incidence of VFs were evaluated. RESULTS: Prevalent VFs were found in 47.6% of all patients at baseline. ZOL 5 mg significantly increased the lumbar spine BMD (LS-BMD) (mean rate of change: + 4.02%, p < 0.0001) and significantly decreased the TRACP-5b (mean rate of change: - 52.1%, p < 0.0001) at 12 months after starting ADT. Incident VFs were identified in 19.0% of all patients at 12 months after starting ADT. After adjusting for the age, BMI, and changes in the LS-BMD, ZOL 5 mg was not significantly associated with incident VFs (odds ratio 0.66, 95% confidence interval 0.04-11.3, p = 0.7774). CONCLUSION: ZOL 5 mg significantly increased the LS-BMD 12 months after starting ADT, and our short-term results showed that ZOL 5 mg was not significantly correlated with the suppression of incident vertebral fractures.

Secondary endpoints analysis in patients with estrogen receptor-positive metastatic breast cancer treated with everolimus and exemestane enrolled in Oral Care-BC.

BACKGROUND: The Oral Care BC-trial reported that professional oral care (POC) reduces the incidence and severity of oral mucositis in patients receiving everolimus (EVE) and exemestane (EXE). However, the effect of POC on clinical response among patients receiving EVE and EXE was not established. We compared outcomes for estrogen receptor-positive metastatic breast cancer patients who received POC to those who had not, and evaluated clinical prognostic factors. All patients simultaneously received EVE and EXE. METHODS: Between May 2015 and Dec 2017, 174 eligible patients were enrolled in the Oral Care-BC trial. The primary endpoint was the comparative incidence of grade 1 or worse oral mucositis, as evaluated for both the groups over 8 weeks by an oncologist. The secondary endpoints were progression-free survival (PFS) and overall survival (OS). Data were collected after a follow-up period of 13.9 months. RESULTS: There were no significant differences in PFS between the POC and Control Groups (P = 0.801). A BMI <  25 mg/m2 and non-visceral metastasis were associated with longer PFS (P = 0.018 and P = 0.003, respectively) and the use of bone modifying agents (BMA) was associated with shorter PFS (P = 0.028). The PFS and OS between the POC and control groups were not significantly different in the Oral-Care BC trial. CONCLUSIONS: POC did not influence the prognosis of estrogen receptor-positive metastatic breast cancer patients. Patients with non-visceral metastasis, a BMI <  25 mg/m2, and who did not receive BMA while receiving EVE and EXE may have better prognoses. TRIAL REGISTRATION: The study protocol was registered online at the University Hospital Medical Information Network (UMIN), Japan (protocol ID 000016109), on January 5, 2015 and at ClinicalTrials.gov ( NCT02376985 ).

Deficiency of sphingomyelin synthase 2 prolongs survival by the inhibition of lymphoma infiltration through ICAM-1 reduction.

The tumor microenvironment (TME) formation involving host cells and cancer cells through cell adhesion molecules (CAMs) is essential for the multiple steps of cancer metastasis and growth. Sphingomyelin synthase 2 (SMS2) is involved in inflammatory diseases such as obesity and diabetes mellitus by regulation of the SM/ceramide balance. However, the involvement of SMS2 in TME formation and metastasis is largely unknown. Here, we report that SMS2-deficient (SMS2-KO) mice show suppressed the EL4 cell infiltration to liver and prolonged survival time. ICAM-1 was identified as a candidate for the inhibition of TME formation in immortalized mouse embryonic fibroblasts (tMEFs) from mRNA array analysis for CAMs. Reduced SM/ceramide balance in SMS2-KO tMEFs suppressed the attachment of EL4 cells through transcriptional reduction of ICAM-1 by the inhibition of NF-κB activation. TNF-α-induced NF-κB activation and subsequent induction of ICAM-1 were suppressed in SMS2-KO tMEFs but restored by SMS2 re-introduction. In the EL4 cell infiltration mouse model, EL4 injection increased ICAM-1 expression in WT liver but not in SMS2-KO mouse liver. Therefore, inhibition of SMS2 may be a therapeutic target to suppress the infiltration of malignant lymphoma.

Impact of pre-operative coronary artery disease on the clinical outcomes of patients with aortic aneurysms.

Aortic aneurysm is an increasingly important public health problem with high morbidity and mortality. It is associated with coronary artery disease (CAD), which is a comorbidity of high incidence that is reported to worsen perioperative complications and long-term clinical outcomes in patients with an aortic aneurysm. Patients with significant coronary artery stenosis may require coronary revascularization and/or optimal medical therapy in the perioperative period of aneurysm surgery. However, the prognostic impact of non-significant coronary artery stenosis not indicated for coronary revascularization on clinical outcomes of patients with aortic aneurysms remains unclear. We performed coronary angiography on 239 consecutive patients with thoracic and abdominal aortic aneurysms before endovascular aortic repair or surgical repair. The patients were divided into the following 3 groups according to the severity of stenosis of major coronary arteries: non-CAD group (with < 25% stenosis), non-significant CAD group (with ≥ 25% but < 75% stenosis), and significant CAD group (with ≥ 75% stenosis). CAD was diagnosed in 133 (56%) patients consisting of 48 (20%) patients with non-significant CAD and 85 (36%) patients with significant CAD. Thirty-nine major adverse cardiovascular and cerebrovascular events (MACCEs) occurred in a median follow-up period of 723 days. Kaplan-Meier analysis revealed that the risk of MACCEs was higher in the significant and non-significant CAD groups than in the non-CAD group. Multivariate Cox proportional hazard regression analysis showed that the risk of MACCEs was equally high in the non-significant CAD and significant CAD groups compared to that in the non-CAD group after adjustment for confounding factors. CAD is significantly associated with poor outcomes in patients with aortic aneurysms, irrespective of the significance of CAD.

RETINAL SURFACE WRINKLING AS AN INDICATOR FOR INTERNAL LIMITING MEMBRANE PEELING DURING VITRECTOMY FOR RETINAL DETACHMENT.

PURPOSE: To assess the validity of retinal surface wrinkling (RSW) as an indicator to select patients relevant for internal limiting membrane peeling during vitrectomy for rhegmatogenous retinal detachment, to prevent postoperative visual decline due to epiretinal membrane growth. METHODS: This was a prospective, interventional case series of 78 consecutive eyes that underwent initial vitrectomy to repair rhegmatogenous retinal detachments and were followed for 6 months. The presence/absence of RSW was evaluated presurgically on en face optical coherence tomographic images. The internal limiting membrane was peeled if RSW was identified. The main outcome measure was the prevalence of postsurgical epiretinal membrane growth that caused a visual decline of 0.2 or more in logarithm of the minimum angle of resolution unit. RESULTS: The internal limiting membrane was peeled for RSW appearance in 22 eyes (28.2%). Mild epiretinal membranes developed in 8 of the 56 internal limiting membrane-unpeeled eyes (10.3% of total, 6 eyes at stage 1 in the classification of Govetto); however, visual decline occurred in none of them with the mean visual acuity of these 8 eyes maintained at -0.08 ± 0.11 in logarithm of the minimum angle of resolution (≈20/16). CONCLUSION: Visual decline due to epiretinal membrane growth after rhegmatogenous retinal detachment repair was entirely prevented by peeling the internal limiting membrane in about 30% of cases selected for the presence of RSW.

New cell delivery system CellSaic with adipose-derived stromal cells promotes functional angiogenesis in critical limb ischemia model mice.

Current therapies for patients with critical limb ischemia have not reduced amputation risk owing to poor cell engraftment. The recombinant peptide Cellnest increases the engraftment rate of administered cells by forming a complex with the cells (CellSaic). We hypothesized that CellSaic containing adipose-derived stromal cells (ADSCs) could improve lower limb blood flow better than ADSCs alone, resulting in better transplanted cell engraftment. ADSCs were extracted from 8-week-old C57BL/6N mice. Thirty-two critical limb ischemia model mice were established by ligating femoral arteries. They were divided into CellSaic (n = 11), ADSC (n = 10), saline (n = 9), and Cellnest (n = 9) groups. Blood flow rate (affected side blood flow / healthy side blood flow × 100%) was evaluated using a laser Doppler blood flow meter every week. Mice were euthanized on day 28 for histological evaluation. Compared with the ADSC group (54.5 ± 17.2%), treated side blood flow rate of the CellSaic group (78.0 ± 24.9%) showed significant improvement on day 28 after administration (p < 0.05). CD31 staining showed significantly higher number of capillary vessels in the CellSaic group (53.0 ± 8.9 cells/mm3) than in the ADSC group (43.0 ± 6.8 cells/mm3) (p < 0.05). Fluorescent staining showed significantly higher number of arterioles containing both CD31 and αSMA double-positive cells in the CellSaic group than in the ADSC group (p < 0.05). CellSaic containing ADSCs exhibited superiority to ADSC transplantation alone in promoting functional angiogenesis, suggesting its potential in improving clinical outcomes of angiogenic therapy for ischemic limbs.

Equine nonneoplastic abnormal ovary in a draft mare with high serum anti-Müllerian hormone: a case study.

We performed a standing hand-assisted laparoscopic ovariectomy in a draft mare that presented with high serum anti-Müllerian hormone (AMH) level and had an enlarged single cystic ovary. Histopathological examination revealed no tumor cell proliferation in the ovary, but the presence of a large ovarian cyst was confirmed. In the diagnosis of abnormal ovaries in mares, a comprehensive assessment should be performed, including the monitoring of ovarian morphology and biomarkers over time, to determine the disease prognosis and treatment plan. The case of this mare with a nonneoplastic abnormal ovary and increased serum AMH level was rare. We suggest that standing hand-assisted laparoscopic ovariectomy is useful for the removal of large ovaries in draft mares.

3D quantitative analysis of diffusion-weighted imaging for predicting the malignant potential of intraductal papillary mucinous neoplasms of the pancreas.

PURPOSE: To investigate the predictors of intraductal papillary mucinous neoplasms of the pancreas (IPMNs) with high-grade dysplasia, using 2-dimensional (2D) analysis and 3-dimensional (3D) volume-of-interest-based apparent diffusion coefficient (ADC) histogram analysis. MATERIAL AND METHODS: The data of 45 patients with histopathologically confirmed IPMNs with high-grade or low-grade dysplasia were retrospectively assessed. The 2D analysis included lesion-to-spinal cord signal intensity ratio (LSR), minimum ADC value (ADCmin), and mean ADC value (ADCmean). The 3D analysis included the overall mean (ADCoverall mean), mean of the bottom 10th percentile (ADCmean0-10), mean of the bottom 10-25th percentile (ADCmean10-25), mean of the bottom 25-50th percentile (ADCmean25-50), skewness (ADCskewness), kurtosis (ADCkurtosis), and entropy (ADCentropy). Diagnostic performance was compared by analysing the area under the receiver operating characteristic curve (AUC). Inter-rater reliability was assessed by blinded evaluation using the intraclass correlation coefficient. RESULTS: There were 16 and 29 IPMNs with high- and low-grade dysplasia, respectively. The LSR, ADCoverall mean, ADCmean0-10, ADCmean10-25, ADCmean25-50, and ADCentropy showed significant between-group differences (AUC = 72-93%; p < 0.05). Inter-rater reliability assessment showed almost perfect agreement for LSR and substantial agreement for ADCoverall mean and ADCentropy. Multivariate logistic regression showed that ADCoverall mean and ADCentropy were significant independent predictors of malignancy (p < 0.05), with diagnostic accuracies of 80% and 73%, respectively. CONCLUSION: ADCoverall mean and ADCentropy from 3D analysis may assist in predicting IPMNs with high-grade dysplasia.

Oral Care Evaluation to Prevent Oral Mucositis in Estrogen Receptor-Positive Metastatic Breast Cancer Patients Treated with Everolimus (Oral Care-BC): A Randomized Controlled Phase III Trial.

BACKGROUND: The incidence of oral mucositis (any grade) after everolimus treatment is 58% in the general population and 81% in Asian patients. This study hypothesized that professional oral care (POC) before everolimus treatment could reduce the incidence of everolimus-induced oral mucositis. MATERIALS AND METHODS: This randomized, multicenter, open-label, phase III study evaluated the efficacy of POC in preventing everolimus-induced mucositis. Patients were randomized into POC and control groups (1:1 ratio) and received everolimus with exemestane. Patients in the POC group underwent teeth surface cleaning, scaling, and tongue cleaning before everolimus initiation and continued to receive weekly POC throughout the 8-week treatment period. Patients in the control group brushed their own teeth and gargled with 0.9% sodium chloride solution or water. The primary endpoint was the incidence of all grades of oral mucositis. We targeted acquisition of 200 patients with a 2-sided type I error rate of 5% and 80% power to detect 25% risk reduction. RESULTS: Between March 2015 and December 2017, we enrolled 175 women from 31 institutions, of which five did not receive the protocol treatment and were excluded. Over the 8 weeks, the incidence of grade 1 oral mucositis was significantly different between the POC group (76.5%, 62 of 82 patients) and control group (89.7%, 78 of 87 patients; p = .034). The incidence of grade 2 (severe) oral mucositis was also significantly different between the POC group (34.6%, 28 of 82 patients) and control group (54%, 47 of 87 patients; p = .015). As a result of oral mucositis, 18 (22.0%) patients in the POC group and 28 (32.2%) in the control group had to undergo everolimus dose reduction. CONCLUSION: POC reduced the incidence and severity of oral mucositis in patients receiving everolimus and exemestane. This might be considered as a treatment option of oral care for patients undergoing this treatment. Clinical trial identification number: NCT02069093. IMPLICATIONS FOR PRACTICE: The Oral Care-BC trial that prophylactically used professional oral care (POC), available worldwide, did not show a greater than 25% difference in mucositis. The 12% difference in grade 1 or higher mucositis and especially the ∼20% difference in grade 2 mucositis are likely clinically meaningful to patients. POC before treatment should be considered as a treatment option of oral care for postmenopausal patients who are receiving everolimus and exemestane for treatment of hormone receptor-positive, HER2-negative advanced breast cancer and metastatic breast cancer. However, POC was not adequate for prophylactic oral mucositis in these patients, and dexamethasone mouthwash prophylaxis is standard treatment before everolimus.

Chronic Inflammatory and Proliferative Lesions of the Gallbladder in Aged Pigs.

Primary epithelial tumors of the gallbladder are rarely reported in animals. In this study, 9 aged pigs (6-12 years old) were histopathologically examined for gallbladder proliferative lesions. At necropsy, a large gallstone occupied the lumen of the gallbladder of 3 pigs. Histopathological examination revealed chronic cholecystitis in all 9 pigs, mucosal hyperplasia in 2 pigs, adenoma in 1 pig, and adenocarcinoma in 2 pigs. Bacilli were detected in the gallbladder lumen of 6 pigs by Warthin-Starry stain. Mucosal hyperplasia, adenoma, and adenocarcinoma were characterized by papillary projections of the mucosa with occasional acinar structures. Tumor invasion of the surrounding tissue was observed in the cases of adenocarcinoma. On Alcian blue and periodic acid-Schiff double-stained sections, the acinar structure of gallbladder mucosa in chronic cholecystitis and mucosal hyperplasia was stained in a mosaic pattern, indicating pyloric gland metaplasia. The results of immunohistochemistry revealed a CD10-positive epithelial brush border and mucin (MUC) 2-positive goblet cells in chronic cholecystitis, adenoma, and adenocarcinomas, indicating intestinal metaplasia. Immunoreactivity of MUC5 AC and cytokeratin 19 was weaker in adenoma and adenocarcinomas compared with the normal and hyperplastic gallbladder mucosa. The number of p53-positive nuclei and the Ki-67 index were higher in adenocarcinomas compared with benign lesions. These results suggest that chronic cholecystitis associated with gallstones and/or bacterial infections may contribute to metaplastic changes and development of gallbladder tumors in aged pigs. Alteration of mucin, cytokeratin, and p53 profiles in gallbladder proliferative lesions in pigs were similar to that in humans, suggesting a common pathogenesis in tumor development.

The Evaluation of Fetal Cardiac Remote Screening in the Second Trimester of Pregnancy Using the Spatio-Temporal Image Correlation Method.

We prospectively performed remote fetal cardiac screening using the spatio-temporal image correlation (STIC), and examined the usefulness and problems of remote screening. We performed heart screening for all pregnant women at four obstetrics clinics over the three years from 2009 to 2014. The STIC data from 15,404 examinations in normal pregnancies (16-27 weeks, median 25 weeks) were analyzed. Obstetricians and sonographer collected STIC data from four-chamber view images. Eight pediatric cardiologists analyzed the images offline. A normal heart was diagnosed in 14,002 cases (90.9%), an abnormal heart was diagnosed in 457 cases (3.0%), and poor images were obtained in 945 cases (6.1%). 138 cases had congenital heart disease (CHD) after birth, and severe CHD necessitating hospitalization occurred in 36 cases. We were not able to detect CHD by screening in 12 cases. The sensitivity and specificity of STIC in CHD screening was 50% and 99.5%, respectively. The sensitivity and specificity of STIC in screening for severe CHD was 82% and 99.9%, respectively. The STIC method was useful in fetal remote screening for CHD. However, the fact that > 10% of images that could not be analyzed by this method was a problem.

Deficiency of sphingomyelin synthase 1 but not sphingomyelin synthase 2 reduces bone formation due to impaired osteoblast differentiation.

BACKGROUND: There are two isoforms of sphingomyelin synthase (SMS): SMS1 and SMS2. SMS1 is located in the Golgi apparatus only while SMS2 is located in both the plasma membrane and the Golgi apparatus. SMS1 and SMS2 act similarly to generate sphingomyelin (SM). We have undertaken the experiments reported here on SMS and osteoblast differentiation in order to better understand the role SMS plays in skeletal development. METHODS: We analyzed the phenotype of a conditional knockout mouse, which was generated by mating a Sp7 promoter-driven Cre-expressing mouse with an SMS1-floxed SMS2-deficient mouse (Sp7-Cre;SMS1f/f;SMS2-/- mouse). RESULTS: When we compared Sp7-Cre;SMS1f/f;SMS2-/- mice with C57BL/6, SMS2-deficient mice (SMS1f/f;SMS2-/-) and SP7-Cre positive control mice (Sp7-Cre, Sp7-Cre;SMS1+/+;SMS2+/- and Sp7-Cre;SMS1+/+;SMS2-/-), we found that although cartilage formation is normal, Sp7-Cre;SMS1f/f;SMS2-/- mice showed reduced trabecular and cortical bone mass, had lower bone mineral density, and had a slower mineral apposition rate than control mice. Next, we have used a tamoxifen-inducible knockout system in vitro to show that SMS1 plays an important role in osteoblast differentiation. We cultured osteoblasts derived from ERT2-Cre;SMS1f/f SMS2-/- mice. We observed impaired differentiation of these cells in response to Smad1/5/8 and p38 that were induced by bone morphogenic protein 2 (BMP2). However, Erk1/2 phosphorylation was unaffected by inactivation of SMS1. CONCLUSIONS: These findings provide the first genetic evidence that SMS1 plays a role in bone development by regulating osteoblast development in cooperation with BMP2 signaling. Thus, SMS1 acts as an endogenous signaling component necessary for bone formation.

The Versican G1 Fragment and Serum-Derived Hyaluronan-Associated Proteins Interact and Form a Complex in Granulation Tissue of Pressure Ulcers.

The hyaluronan (HA)-rich extracellular matrix plays dynamic roles during tissue remodeling. Versican and serum-derived HA-associated protein (SHAP), corresponding to the heavy chains of inter-α-trypsin inhibitor, are major HA-binding molecules in remodeling processes, such as wound healing. Versican G1-domain fragment (VG1F) is generated by proteolysis and is present in either remodeling tissues or the mature dermis. However, the macrocomplex formation of VG1F has not been clarified. Therefore, we examined the VG1F-containing macrocomplex in pressure ulcers characterized by chronic refractory wounds. VG1F colocalized with SHAP-HA in specific regions of the granulation tissue but not with fibrillin-1. A unique VG1F-SHAP-HA complex was isolated from granulation tissues using gel filtration chromatography and subsequent cesium chloride-gradient ultracentrifugation under dissociating conditions. Consistent with this molecular composition, recombinant versican G1, but not versican G3, interacted with the two heavy chains of inter-α-trypsin inhibitor. The addition of recombinant VG1 in fibroblast cultures enhanced VG1F-SHAP-HA complex deposition in the pericellular extracellular matrix. Comparison with other VG1F-containing macrocomplexes, including dermal VG1F aggregates, versican-bound microfibrils, and intact versican, highlighted the tissue-specific organization of HA-rich extracellular matrix formation containing versican and SHAP. The VG1F-SHAP-HA complex was specifically detected in the edematous granulation tissues of human pressure ulcers and in inflamed stages in a mouse model of moist would healing, suggesting that the complex provides an HA-rich matrix suitable for inflammatory reactions.