[Study on transcriptome characteristics of respiratory syncytial virus bronchiolitis in children by RNA sequencing].

To explore the biological characteristics related to the pathogenesis and severity of respiratory syncytial virus (RSV) bronchiolitis by RNA sequencing of white blood cells in children with RSV bronchiolitis. This study is a case-control study. A total of 87 children diagnosed with bronchiolitis and RSV antigen positive and/or RSV nucleic acid positive in the pediatric respiratory department of the Second Affiliated Hospital of Wenzhou Medical University from October 2019 to April 2022 were selected as the case group. The case group was divided into three groups based on the condition: mild, moderate, and severe, and there were two groups according to the presence or absence of atopic symptoms: the atopic group and the non-atopic group, forty healthy children in the same period were selected as the control group. The whole blood leukocyte RNA of the children in the case group and the control group was extracted for RNA sequencing, and the data were analyzed to obtain differentially expressed genes (DEGs). Then, the immunobiological pathways and genes related to the pathogenesis, disease condition, and atopy were screened through Gene Ontology (GO) annotation, Kyoto Gene and Genome Encyclopedia (KEGG) annotation, and protein interaction network (PPI) construction methods. Construct the weighted gene co-expression network analysis (WGCNA) module to identify potential biological indicators related to disease severity.Compared with the control group, the case group had a total of 1 782 DEGs, including 1 586 upregulated genes and 196 downregulated genes. The GO pathway enrichment of DEGs is mainly enriched in molecular functions such as peroxidase activity and oxidoreductase activity. In the cytological components, it is mainly enriched in cytoplasmic vesicle lumen and secretory granule lumen. In biological processes, it is mainly enriched in processes such as neutrophil activation involved in immune responses, neutrophil degranulation, and neutrophil activation. KEGG analysis is mainly concentrated in the signal pathway of the viral protein interaction with cytokine and cytokine receptor. A PPI network was constructed to screen four genes at the core position, including CCL2, IL-10, MMP9 and JUN. The DEGs obtained by comparing different disease groups with the control group are mainly enriched in retrograde endocannabinoid signaling and cell apoptosis pathways. WGCNA analysis showed that the brown module related to oxygen saturation was most closely related to the disease, and its gene was mainly enriched in the RNA helicase retinoic acid inducible gene-I (RIG-I) like receptor signal pathway. There are 230 specific DEGs in the atopic group and 444 in the non-atopic group. KEGG enrichment analysis results show that both groups are enriched to NF-κB signaling pathway, the characteristic does not cause significant changes in immune response and transcriptome characteristics in children with RSV bronchiolitis. In conclusion, neutrophil activation, degranulation pathway and signal pathway of interaction between viral protein and cytokine and cytokine receptor are involved in the immune response of RSV bronchiolitis host. CCL2, IL-10, MMP9 and JUN genes may be associated with the pathogenesis. They might be potential biomarkers related to disease severity in RIG-I like receptors, cell apoptosis, and endogenous cannabinoid related signaling pathways.

Pericoronary adipose tissue attenuation predicts outcome of percutaneous intervention for chronic total occlusion.

AIM: To investigate the association between pericoronary adipose tissue (PCAT) attenuation (PCATA) and outcomes of chronic total occlusion (CTO) after percutaneous coronary intervention (PCI), and to establish a clinical model that can be easily generalised to predict the outcomes of PCI-CTO. MATERIALS AND METHODS: Between September 2015 and September 2019, patients from two centres were enrolled retrospectively. The primary endpoint was a procedural success (defined as achieving residual stenosis of <30% and a grade 3 thrombolysis in myocardial infarction [TIMI] flow). The new predictive model was generated by factors that were determined by multivariate analysis. The PCATA of CTO (PCATA-CTO) score was developed by assigning 1 point for each independent predictor, and then summing all points accrued. In addition, the predictive efficacy and interobserver and intraobserver agreement of PCATA-CTO and other scoring systems based on coronary computed tomography angiography (CCTA) were compared. RESULTS: A total of 201 patients (mean age 58.9 ± 10.8 years, 85% male) were enrolled. The PCI success was achieved in 76% of the lesions. PCAT was higher in the PCI success group (-72.44 ± 10.45HU versus -76.76 ± 10.54 HU, p<0.05). Multivariable analysis yielded severe calcification, lesion length ≥15 mm, and perivascular fat attenuation index (FAI) ≤-69.5HU as independent negative predictors for procedural success. The area under the receiver operating characteristic curves for the PCATA-CTO score was 0.72. Comparing the PCATA-CTO score with other predictive scores, the PCATA-CTO score showed the highest interobserver (kappa = 0.74) and intraobserver agreement (kappa = 0.90, all p<0.01). CONCLUSION: FAI ≤-69.5HU is an independent negative predictor of procedural success. The PCATA-CTO score improved the reliability of the prediction model. Its potential for clinical implementation requires evaluation.

[Effects of tensile force on the vascular lumen formation in three-dimensional printed tissue].

Objective: To explore the effects of tensile force on vascular lumen formation in three-dimensional printed tissue. Methods: The experimental research method was used. Human umbilical vein endothelial cells (HUVECs) were extracted from discarded umbilical cord tissue of 3 healthy women (aged 22 to 35 years) who gave birth in the Department of Gynaecology and Obstetrics of Suzhou Ruihua Orthopaedic Hospital from September 2020 to May 2021. Human skin fibroblasts (HSFs) were extracted from discarded normal skin tissue of 10 male patients (aged 20 to 45 years) who underwent wound repair in the Department of Hand Surgery of Suzhou Ruihua Orthopaedic Hospital from September 2020 to September 2022. After identification of the two kinds of cells, the 4th to 6th passage of cells were taken for the follow-up experiments. HUVECs and HSFs were used as seed cells, and polycaprolactone, gelatin, hyaluronic acid, and fibrin were used as scaffold materials, and the three-dimensional printed vascularized tissue was created by three-dimensional bioprinting technology. The printed tissue with polycaprolactone scaffold of 6 and 10 mm spacing, and without polycaprolactone scaffold were set as 6 mm spacing polycaprolactone group, 10 mm spacing polycaprolactone group, and non-polycaprolactone group, respectively. After 4 days of culture, the printed tissue in 10 mm spacing polycaprolactone group was selected to detect the cell survival by cell viability detection kit, and the cell survival rate was calculated. After 14 days of culture, the printed tissue in three groups were taken, and the shape change of tissue was observed by naked eyes; immunofluorescence staining was performed to observe the arrangement of filamentous actin, and lumen diameter, total length, and number of branches of vessel in the tissue. The tissue with micro-spring structure in the above-mentioned three groups was designed, printed, and cultured for 9 days, and the tensile force applied in the printed tissue was measured according to the force-displacement curve. The number of samples was all 3 in the above experiments. Data were statistically analyzed with one-way analysis of variance and Tukey test. Results: After 4 days of culture, the cell survival rate in printed tissue in 10 mm spacing polycaprolactone group was (91.3±2.2)%. After 14 days of culture, the shape change of printed tissue in non-polycaprolactone group was not obvious, while the shape changes of printed tissue in 6 mm spacing polycaprolactone group and 10 mm spacing polycaprolactone group were obvious. After 14 days of culture, the arrangement of filamentous actin in the printed tissue in non-polycaprolactone group had no specific direction, while the arrangement of filamentous actin in the printed tissue in 6 mm spacing polycaprolactone group and 10 mm spacing polycaprolactone group had a specific direction. After 14 days of culture, The vascular lumen diameters of the printed tissue in 6 mm spacing polycaprolactone group and 10 mm spacing polycaprolactone group were (6.0±1.3) and (10.8±1.3) µm, respectively, which were significantly larger than 0 µm in non-polycaprolactone group (P<0.05), and the vascular lumen diameter of printed tissue in 10 mm spacing polycaprolactone group was significantly larger than that in 6 mm spacing polycaprolactone group (P<0.05); the total length and number of branches of blood vessel in the printed tissue in 6 mm spacing polycaprolactone group and 10 mm spacing polycaprolactone group were significantly shorter or less than those in non-polycaprolactone group (P<0.05), and the total length and number of branches of blood vessel in the printed tissue in 10 mm spacing polycaprolactone group were significantly shorter or less than those in 6 mm spacing polycaprolactone group. After 9 days of culture, the tensile forces applied in the printed tissue in 6 mm spacing polycaprolactone group and 10 mm spacing polycaprolactone group were (2 340±59) and (4 284±538) µN, respectively, which were significantly higher than 0 µN in non-polycaprolactone group (P<0.05), and the tensile force applied in the printed tissue in 10 mm spacing polycaprolactone group was significantly higher than that in 6 mm spacing polycaprolactone group (P<0.05). Conclusions: The three-dimensional printed scaffold structure can exert different tensile force in the printed tissue, and the vascular lumen diameter of the printed tissue can be regulated by adjusting the tensile force.

The Post-Venous Thromboembolism Functional Status Scale: From Call to Action to Application in Research, Extension to COVID-19 Patients, and Its Use in Clinical Practice.

A broad spectrum of long-term sequelae may be present in venous thromboembolism (VTE) survivors, affecting their quality of life and functioning. To monitor recovery and improve the prognosis of patients with persistent functional limitations, the development of a new outcome measure that could better capture the consequences of VTE was an unmet need. Starting as a call to action, the Post-VTE Functional Status (PVFS) scale was developed to meet this need. The PVFS scale is an easy-to-use clinical tool to measure and quantify functional outcomes after VTE by focusing on key aspects of daily life. As the scale was considered useful in coronavirus disease 2019 (COVID-19) patients as well, the Post-COVID-19 Functional Status (PCFS) scale was introduced early in the pandemic after slight adaptation. The scale has been well incorporated into both the VTE and COVID-19 research communities, contributing to the shift of focus toward patient-relevant functional outcomes. Psychometric properties have been evaluated, mainly for the PCFS scale but recently also for the PVFS scale, including validation studies of translations, showing adequate validity and reliability. In addition to serving as outcome measure in studies, guidelines and position papers recommend using the PVFS and PCFS scale in clinical practice. As broad use of the PVFS and PCFS scale in clinical practice is valuable to capture what matters most to patients, widespread implementation is a crucial next step. In this review, we discuss the development of the PVFS scale and introduction in VTE and COVID-19 care, the incorporation of the scale in research, and its application in clinical practice.

Prevalence and characteristics of gastroesophageal reflux disease in pregnant women.

INTRODUCTION AND AIMS: The prevalence of gastroesophageal reflux disease (GERD) has been reported to be increasing in recent years. However, there have been few reports on the prevalence of GERD during pregnancy in the Asian population. The aim of our study was to evaluate the prevalence and characteristics of GERD in Vietnamese pregnant women. MATERIALS AND METHODS: This cross-sectional study was conducted at the antenatal clinic of the Nhan Dan Gia Dinh Hospital, Ho Chi Minh, Vietnam. Four hundred females, at various stages of pregnancy, were enrolled. GERD was diagnosed if there was troublesome heartburn and/or acid regurgitation, at least once a week, during the current pregnancy. RESULTS: The overall prevalence of GERD in pregnancy was 38.5% (154/400). The prevalence of GERD in the third trimester was significantly higher than that in the second trimester (46.8% vs. 30.7%, P=0.008) and tended to be higher than its prevalence in the first trimester (46.8% vs. 35.4%, P=0.051). In the pregnant women with GERD, the frequency of regurgitation was significantly higher than that of heartburn (92.9% vs. 30.5%, P<0.001). Those typical symptoms were more prevalent in the daytime, compared with nighttime. CONCLUSION: Our study showed that GERD was prevalent during pregnancy in Vietnam. In the pregnant women with GERD, regurgitation was much more common than heartburn, and those typical reflux symptoms occurred more frequently in the daytime, compared with nighttime.

[Diagnosis and treatment of pneumatosis cystoides intestinalis].

Pneumatosis cystoides intestinalis (PCI) is a rare disease, which is characterized by the accumulation of gas cysts located in the submucosa or subserosa of the gastrointestinal tract. It can occur in the whole or part of the gastrointestinal tract from the esophagus to the rectum, but clinically the main involved sites are the colon and small intestine. PCI can also appear in other sites such as mesentery, the greater omentum and the hepatogastric ligament. In recent years, with the renewal of imaging method, the detection rate of PCI has been on the rise. Most patients with PCI have no obvious symptoms or only non-specific symptoms of the digestive tract like abdominal distension, abdominal pain, diarrhea, hematochezia, etc. The atypical clinical symptoms of PCI can easily lead to missed diagnosis or misdiagnosis. A small amount of patients would have complications like peritonitis and even perforation of the digestive tract. The therapeutic principle for these patients is different from that for patients with acute abdomen. The prognosis of PCI depends on its severity and comorbidities. In this article, a literature review would be conducted on the epidemiological characteristics, etiology and pathogenesis, clinical manifestations, diagnosis and treatment of PCI, which might help clinical doctors with diagnosis and treatment of the disease.

Comparing monovalent and combination hepatitis B vaccine outcomes in children delivered by mothers with chronic hepatitis B.

AIM: We compared the vaccine effectiveness of monovalent and combination hepatitis B vaccine regimens in infants born to chronic hepatitis B carrier mothers. METHODS: An observational cohort of neonates was recruited over 78 months from two public hospital maternity units in Singapore. We enrolled term infants, born to chronic hepatitis B surface antigen-positive mothers regardless of their hepatitis Be antigen status, who completed the hepatitis B virus (HBV) vaccination programme in Singapore. Infants born to mothers on antiviral therapy, or with concurrent hepatitis C or human immunodeficiency virus infection were excluded. All infants received hepatitis B immunoglobulin at birth. One group received three doses of monovalent hepatitis B vaccine (0, 1, 6 months) (regimen A). The other group received two doses of monovalent vaccine, followed by one dose combination vaccine DTaP-IPV-Hib-HBV (0, 1, 6 months) (regimen B). Vaccine effectiveness was determined by immunoprophylaxis failure leading to HBV vertical transmission. Immunogenicity was assessed by hepatitis B surface antibody (anti-HBs) levels at 9 months of age. RESULTS: Total of 177 term neonates received regimen A and 115 received regimen B. Immunoprophylaxis failure rate was low, 2.3 and 2.6% (P = 1.00) in regimen A and B, respectively. Mean anti-HBs titres were similar at 643 ± 374 and 561 ± 396 IU/L (P = 0.08) for regimen A and B, respectively. CONCLUSION: Hepatitis B vaccine regimens using monovalent or combination vaccine for the third dose showed similarly high vaccine effectiveness and low immunoprophylaxis failure rate in term infants born to chronic hepatitis B carrier mothers.

Modeling and predicting osteoarthritis progression: data from the osteoarthritis initiative.

OBJECTIVE: The goal of this study was to model the longitudinal progression of knee osteoarthritis (OA) and build a prognostic tool that uses data collected in 1 year to predict disease progression over 8 years. DESIGN: To model OA progression, we used a mixed-effects mixture model and 8-year data from the Osteoarthritis Initiative (OAI)-specifically, joint space width measurements from X-rays and pain scores from the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) questionnaire. We included 1243 subjects who at enrollment were classified as being at high risk of developing OA based on age, body mass index (BMI), and medical and occupational histories. After clustering subjects based on radiographic and pain progression, we used clinical variables collected within the first year to build least absolute shrinkage and selection (LASSO) regression models for predicting the probabilities of belonging to each cluster. Areas under the receiver operating characteristic curve (AUC) represent predictive performance on held-out data. RESULTS: Based on joint space narrowing, subjects clustered as progressing or non-progressing. Based on pain scores, they clustered as stable, improving, or worsening. Radiographic progression could be predicted with high accuracy (AUC = .86) using data from two visits spanning 1 year, whereas pain progression could be predicted with high accuracy (AUC = .95) using data from a single visit. Joint space narrowing and pain progression were not associated. CONCLUSION: Statistical models for characterizing and predicting OA progression promise to improve clinical trial design and OA prevention efforts in the future.

Establishment of a cell line with high transfection efficiency from zebrafish Danio rerio embryos and its susceptibility to fish viruses.

A cell line ZBE3 isolated from a continuous cell culture derived from zebrafish Danio rerio blastomeres by clonal growth was characterized. ZBE3 cells had been subcultured for >120 passages since the initial primary culture of the blastomeres. The ZBE3 cells grow stably at temperature from 20 to 32° C with an optimum temperature of 28° C in ESM2 or ESM4 medium with 15% foetal bovine serum (FBS). The optimum FBS concentration for ZBE3 cell growth ranged from 15 to 20%. Cytogenetical analysis indicated that the modal chromosome number of ZBE3 cells was 50, the same as the diploid chromosome number of D. rerio. Significant cytopathic effect was observed in ZBE3 cells after infection with redspotted grouper nervous necrosis virus, Singapore grouper iridovirus and grass carp reovirus, and the viral replication in the cells was confirmed by real-time quantitative PCR and transmission electron microscopy, indicating the susceptibility of ZBE3 cells to the three fish viruses. After transfected with pEGFP-N3 plasmid, ZBE3 cells showed a transfection efficiency of about 40% which was indicated by the percentage of cells expressing green fluorescence protein. The stable growth, susceptibility to fish viruses as well as high transfection efficiency make ZBE3 cells be a useful tool in transgenic manipulation, fish virus-host cell interaction and immune response in fish.

[Exploration and practice in the construction of curriculum on epidemiology in preventive medicine].

Epidemiology is one of main courses for undergraduate students majoring in preventive medicine. There are some limitations in the traditional epidemiology teaching, which is usually characterized in indoctrinated education: "lectured by the teachers and listened by the students." In Zhejiang University, staff of the epidemiology division tried to explore a new teaching mode as 'student-centered, teacher-leading, question-based, and combining with literature discussion and course practice.' After practicing for two years, students were inspired in learning initiatives, with teaching effectiveness obviously improved.

[Correlation between high risk type human papillomavirus E6/E7 mRNA and cervical cancer].

OBJECTIVE: To investigate the correlation between the positive rate of high risk human papillomavirus(HPV)mRNA E6/E7 and cervical cancer, and provide evidence for the prevention and treatment of cervical cancer. METHODS: A total of 100 cervical cancer cases and 100 healthy controls were selected in our hospital from January 2015 to December 2015. The fluorescence quantitative PCR and pathological examination on HPV E6/E7 mRNA were carried out. The correlation between HPV E6/E7 mRNA and cervical squamous epithelial lesions were analyzed. RESULTS: In case group, the positive rate of HPV E6/E7 mRNA was 76.0%(76/100). In control group, the positive rate was 13.0%(13/100). The positive rate in case group was significantly higher than that in control group, and the difference was statistically significant(χ(2)=24.522, P<0.001). The positive predictive value and negative predictive value of the two groups were compared, and the difference was not significant(P>0.05). The positive rate of HPV E6/E7 mRNA was significantly higher than high-grade squamous intraepithelial lesion(SIL)rate(26.1%), low-grade SIL rate(17.6%)and atypical squamous cell hyperplasia rate(6.7%), the difference was statistically significant(χ(2)=7.615, P= 0.001; χ(2) =9.114, P=0.001; χ(2)=18.241, P<0.001). CONCLUSIONS: The detection rate of HPV E6/E7 mRNA in cervical cancer patients was high. And with the increased severity of cervical squamous epithelial lesions, the positive rate of HPV E6/E7 mRNA increased.

Impact of Si4+ Ions Doping on the Electrochemical Cycling Performance of NiTiO3 as Anodes for Li-Lon Batteries.

Pristine and Si4+ doped NiTiO3 are successfully synthesized by molten salt method and electrochemically characterized for its use as an anode material for Li-ion batteries. The X-ray diffraction (XRD) results enumerates that the lattice parameter and the cell volume decreases upon the addition of Si4+ due to its smaller ionic radius. The presence of Si4+ in NiTiO3 structure was also confirmed using FTIR analysis, which showed the stretching vibrations of Si-O at -1008 cm(-1). The SEM images reveal that the NiTiO3 particles are in micrometer range and the size of the particle is found to be decrease after Si4+ addition. The electrical studies infers an enhancement in the conductivity from 4.4 x 10(-7) S x cm(-1) to 1.7 x 10(-6) S x cm(-1) on dopant addition. The initial discharge capacity of NiTiO3 is found to be 1257 mA h g(-1) and there is a capacity fading on consecutive cycles. NiTi0.9Si0.1O3 enhances the cyclic performances and a constant capacity around 400 mA h g(-1) is maintained, a very good reversibility with almost 100% efficiency is observed elucidating the fact that almost all the Li ions intercalated are successfully de-intercalated during the discharge process.

Intra-operative 3D guidance and edema detection in prostate brachytherapy using a non-isocentric C-arm.

PURPOSE: Brachytherapy (radioactive seed insertion) has emerged as one of the most effective treatment options for patients with prostate cancer, with the added benefit of a convenient outpatient procedure. The main limitation in contemporary brachytherapy is faulty seed placement, predominantly due to the presence of intra-operative edema (tissue expansion). Though currently not available, the capability to intra-operatively monitor the seed distribution, can make a significant improvement in cancer control. We present such a system here. METHODS: Intra-operative measurement of edema in prostate brachytherapy requires localization of inserted radioactive seeds relative to the prostate. Seeds were reconstructed using a typical non-isocentric C-arm, and exported to a commercial brachytherapy treatment planning system. Technical obstacles for 3D reconstruction on a non-isocentric C-arm include pose-dependent C-arm calibration; distortion correction; pose estimation of C-arm images; seed reconstruction; and C-arm to TRUS registration. RESULTS: In precision-machined hard phantoms with 40-100 seeds and soft tissue phantoms with 45-87 seeds, we correctly reconstructed the seed implant shape with an average 3D precision of 0.35 mm and 0.24 mm, respectively. In a DoD Phase-1 clinical trial on six patients with 48-82 planned seeds, we achieved intra-operative monitoring of seed distribution and dosimetry, correcting for dose inhomogeneities by inserting an average of over four additional seeds in the six enrolled patients (minimum 1; maximum 9). Additionally, in each patient, the system automatically detected intra-operative seed migration induced due to edema (mean 3.84 mm, STD 2.13 mm, Max 16.19 mm). CONCLUSIONS: The proposed system is the first of a kind that makes intra-operative detection of edema (and subsequent re-optimization) possible on any typical non-isocentric C-arm, at negligible additional cost to the existing clinical installation. It achieves a significantly more homogeneous seed distribution, and has the potential to affect a paradigm shift in clinical practice. Large scale studies and commercialization are currently underway.

SU-E-T-385: Accelerated Beam Delivery with MLC Gaps in IMRT Fields.

PURPOSE: By default, the 4-mm wide leaf-pairs of the Elekta Beam Modulator MLC not used for field shaping are moved to under the primary collimator to minimize the leakage dose. The trade-off is the increased beam delivery time in the case of IMRT. This study examines the dosimetric impact and improved delivery efficiency by allowing the unused leaf-pair as 2mm gap to stay within the PTV aperture during beam-on. METHODS: The capability of the treatment planning system (Pinnacle) in modeling the central and off-axis dose through 2mm MLC gaps, respectively, was investigated. EBT film measurement performed in a solid water phantom was compared with the computed dose. For two representative IMRT cases, the dosimetric features of treatment plans generated with and without MLC gaps in the field were compared. Similarly, the delivery times for 13 IMRT cases with and without MLC gaps were compared. RESULTS: The peak dose of the leaf gap computed with Pinnacle was dependent on dose grid resolution. The difference between a 0.5mm and 2mm dose grid was about 16.5% for 6MV and 15MV beam respectively. Using a 0.5mm dose grid, the maximum difference between measured and calculated dose was < 1% for a central axis gap, and < 3% for an off-axis gap. The maximum dose through a 2mm×4mm leaf gap was estimated < 10cGy for a typical IMRT plan. DVH comparison showed an increase of target dose up to 2% for the IMRT plans with gap, with minimal dose change to all critical structures. For the 13 IMRT cases, the beam delivery times were reduced by 27+/-9.7%, from 8.7+/-4.5 minutes to 6.5+/-4.0 minutes. CONCLUSIONS: The beam delivery is accelerated by utilizing minimal gaps in the IMRT fields. The increased dose to the target is acceptable and is minimal to all critical structures.

Ischaemia-induced retinal neovascularisation and diabetic retinopathy in mice with conditional knockout of hypoxia-inducible factor-1 in retinal Müller cells.

AIMS/HYPOTHESIS: Retinal Müller cells are known to produce inflammatory and angiogenic cytokines, which play important roles in diabetic retinopathy. Hypoxia-inducible factor (HIF)-1 has been shown to play a crucial role in retinal inflammation and neovascularisation. We sought to determine the role of Müller cell-derived HIF-1 in oxygen-induced retinopathy (OIR) and diabetic retinopathy using conditional Hif-1α (also known as Hif1a) knockout (KO) mice. METHODS: Conditional Hif-1α KO mice were generated by crossing mice expressing cyclisation recombinase (cre, also known as P1_gp003) in Müller cells with floxed Hif-1α mice and used for OIR and streptozotocin-induced diabetes to induce retinal neovascularisation and inflammation, respectively. Abundance of HIF-1α and pro-angiogenic and pro-inflammatory factors was measured by immunoblotting and immunohistochemistry. Retinal neovascularisation was visualised by angiography and quantified by counting pre-retinal nuclei. Retinal inflammation was evaluated by leucostasis and vascular leakage. RESULTS: While the Hif-1α KO mice showed significantly decreased HIF-1α levels in the retina, they exhibited no apparent histological or visual functional abnormalities under normal conditions. Compared with wild-type counterparts, Hif-1α KO mice with OIR demonstrated attenuated overproduction of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule (ICAM)-1, reduced vascular leakage and alleviated neovascularisation in the retina. Under diabetes conditions, disruption of Hif-1α in Müller cells attenuated the increases of retinal vascular leakage and adherent leucocytes, as well as the overproduction of VEGF and ICAM-1. CONCLUSIONS/INTERPRETATION: Müller cell-derived HIF-1α is a key mediator of retinal neovascularisation, vascular leakage and inflammation, the major pathological changes in diabetic retinopathy. Müller cell-derived HIF-1α is therefore a promising therapeutic target for diabetic retinopathy.

TNF-α acutely upregulates amylin expression in murine pancreatic beta cells.

AIMS/HYPOTHESIS: Amylin, a secretory protein mainly produced by pancreatic beta cells, is elevated in the circulation of patients with diseases related to acute and chronic inflammation, including acute pancreatitis, pancreas graft rejection, obesity and insulin resistance. TNF-α is involved in these disorders. We investigated the effect of TNF-α on amylin levels and the underlying mechanisms, using murine pancreatic beta cell line MIN6 and pancreatic islets. METHODS: Amylin, proinsulin and prohormone convertase 1/3, 2 (Pc1/3, Pc2 [also known as Pcsk1/3 and Pcsk2, respectively]) mRNA levels, and amylin promoter and nuclear factor κB (NF-κB) activation were examined by real-time PCR and luciferase reporter assay, respectively. Amylin protein level and mitogen-activated protein kinase phosphorylation were detected by western blot. Activator protein 1 (AP1) activation was examined by electrophoretic mobility shift assay (EMSA). RESULTS: TNF-α acutely induced amylin expression at the transcriptional level and increased proamylin and the intermediate form of amylin in MIN6 cells and islets. However, it had no effect on proinsulin, Pc1/3 and Pc2 expression. Studies with (1) MIN6 cells treated with inhibitors of MEK1/2, c-Jun-N-terminal kinase (JNK) or protein kinase Cζ (PKC(ζ)), (2) MIN6 cells expressing a c-Jun-dominant negative construct and (3) islets from Fos knockout mice demonstrated that TNF-α induced amylin expression through the PKC(ζ)-extracellular signal-regulated kinase (ERK)/JNK pathways. EMSA showed that (PKC(ζ)), JNK and ERK1/2 were involved in TNF-α-induced AP1 activation, suggesting that TNF-α induces murine amylin expression through the (PKC(ζ)) - ERK1/2 - AP and PKC(ζ) - JNK - AP1 pathways. Further studies showed that TNF-α also induced murine amylin expression through the phosphatidylinositol 3 kinase-NF-κB signalling pathway and enhanced human amylin promoter activation through NF-κB and AP1. CONCLUSIONS/INTERPRETATION: TNF-α acutely induces amylin gene expression in beta cells through multiple signalling pathways, possibly contributing to amylin elevation in acute inflammation-related pancreatic disorders.