Toll-like receptor-ligand induced thymic stromal lymphopoietin expression in primary equine keratinocytes.

BACKGROUND Thymic stromal lymphopoietin (TSLP) plays a key role in the development of allergic inflammation. Little is known about possible triggers of equine TSLP expression. HYPOTHESIS/OBJECTIVES To investigate TSLP expression in equine insect bite hypersensitivity (IBH) skin lesions. The capacity of TLR 1-8 ligands (L) and of atopic cytokine milieu as potential triggers of TSLP and of interleukin (IL)-6 as a downstream effector molecule of TLR signalling, were examined in primary equine keratinocyte cultures. ANIMALS Lesional skin from IBH-affected and healthy skin from control-horses (n = 9 each group) was sampled. METHODS AND MATERIALS Keratinocyte cultures were established from six healthy horses and stimulated with TLR 1-8-L, and with IL-4 and tumor necrosis factor-α, to mimic an atopic inflammation cytokine milieu. TSLP and IL-6 gene expression was assessed by quantitative real-time PCR. RESULTS Expression of TSLP was significantly greater in IBH lesions compared to healthy skin. TLR 1-8-L significantly upregulated TSLP expression in keratinocytes. The strongest upregulation was induced by TLR 1/2-L and TLR 3-L. Combination of atopic cytokine milieu and TLR 1/2-L or TLR 3-L further increased TSLP expression. TLR-L 1-5 stimulation significantly upregulated IL-6 expression. CONCLUSIONS AND CLINICAL IMPORTANCE The data herein suggest that the upregulation of TSLP expression in lesional skin of IBH-affected horses might play a role in IBH development. Moreover, TSLP expression is induced by TLR-L, in particular by TLR 1/2-L and TLR 3-L, and is further increased by atopic cytokine milieu, indicating a mechanism for TSLP-mediated exacerbation of IBH

A single residue substitution in the receptor-binding domain of H5N1 hemagglutinin is critical for packaging into pseudotyped lentiviral particles

© 2012 Tang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Background: Serological studies for influenza infection and vaccine response often involve microneutralization and hemagglutination inhibition assays to evaluate neutralizing antibodies against human and avian influenza viruses, including H5N1. We have previously characterized lentiviral particles pseudotyped with H5-HA (H5pp) and validated an H5pp-based assay as a safe alternative for high-throughput serological studies in BSL-2 facilities. Here we show that H5-HAs from different clades do not always give rise to efficient production of H5pp and the underlying mechanisms are addressed. Methodology/Findings: We have carried out mutational analysis to delineate the molecular determinants responsible for efficient packaging of HA from A/Cambodia/40808/2005 (H5Cam) and A/Anhui/1/2005 (H5Anh) into H5pp. Our results demonstrate that a single A134V mutation in the 130-loop of the receptor binding domain is sufficient to render H5Anh the ability to generate H5Anh-pp efficiently, whereas the reverse V134A mutation greatly hampers production of H5Cam-pp. Although protein expression in total cell lysates is similar for H5Anh and H5Cam, cell surface expression of H5Cam is detected at a significantly higher level than that of H5Anh. We further demonstrate by several independent lines of evidence that the behaviour of H5Anh can be explained by a stronger binding to sialic acid receptors implicating residue 134. Conclusions: We have identified a single A134V mutation as the molecular determinant in H5-HA for efficient incorporation into H5pp envelope and delineated the underlying mechanism. The reduced binding to sialic acid receptors as a result of the A134V mutation not only exerts a critical influence in pseudotyping efficiency of H5-HA, but has also an impact at the whole virus level. Because A134V substitution has been reported as a naturally occurring mutation in human host, our results may have implications for the understanding of human host adaptation of avian influenza H5N1 virusesThis work was supported by grants from the Research Fund for the Control of Infectious Diseases of Hong Kong (RFCID#08070972), the Area of Excellence Scheme of the University Grants Committee (grant AoE/M-12/-06 of the Hong Kong Special Administrative Region, China), the French Ministry of Health, and the RESPARI project of the Institut Pasteur International Network

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Research progress on inhibition of ocular neovascularization by traditional Chinese medicine monomers

Overview of the status application of traditional Chinese medicine monomers in ocular neovascular diseases. Through retrieving related domestic and overseas research state for more than ten years, summarizing, analyzing and summarizing, overviewing the research progress of traditional Chinese medicine monomer in inhibiting intraocular neovascularization, and expounding the potential advantages of traditional Chinese medicine in the field of intraocular angiogenesis research, providing information for treating and researching of neovascular diseases

Treatment Outcomes and Risk Factors for Incomplete Treatment after Definitive Chemoradiotherapy for Non-Resectable or Metastatic Esophageal Cancer

Among patients with unresectable or metastatic esophageal cancer who receive definitive chemotherapy or chemoradiotherapy, the rates of treatment-related adverse events and incomplete treatment remain high. We conducted this study to investigate survival after definitive treatments and identify predicting factors for incomplete treatment. The data of patients who received definitive chemotherapy or chemoradiotherapy for esophageal cancer were retrospectively examined. The patients were assigned to Group 1: incomplete definitive treatment; Group 2: complete definitive treatment; or Group 3: complete definitive treatment with additional salvage surgery. The data of 273 patients (90, 166, and 17 in Groups 1, 2, and 3, respectively) were analyzed. In the survival analysis, the median overall survival of Groups 1, 2, and 3 were 2.6, 10.3, and 29.5 months, respectively. A significant difference in 3-year overall survival was observed among the groups (2.2%, 12.4%, and 48.5%, p p = 0.001), bone metastasis (HR: 2.18, p = 0.024), airway invasion (HR: 2.90, p = 0.001), and liver cirrhosis (HR: 3.20, p = 0.026). Incomplete definitive treatment is associated with a far worse prognosis. Poor performance, bone metastasis, airway invasion, and liver cirrhosis are risk factors for incomplete treatment

Prognostic Effect of the Dose of Radiation Therapy and Extent of Lymphadenectomy in Patients Receiving Neoadjuvant Chemoradiotherapy for Esophageal Squamous Carcinoma

Neoadjuvant chemoradiotherapy has been used for patients with locally advanced esophageal squamous cell carcinoma (ESCC). However, the optimal dose of radiation therapy and the effect of lymphadenectomy after neoadjuvant therapy on patient outcomes are uncertain. We retrospectively reviewed the data of patients who received neoadjuvant therapy followed by surgery for ESCC. Overall survival (OS), disease-free survival (DFS), and perioperative outcomes were compared between patients who received radiation doses of 45.0 Gy (PF4500) and 50.4 Gy (PF5040). Subgroup analysis was performed based on the number of lymph nodes removed through lymph node dissection (LND). Data from a total of 126 patients were analyzed. No significant differences were found in 3-year OS and DFS between the PF4500 and PF5040 groups (OS: 45% versus 54%, p = 0.218; DFS: 34% versus 37%, p = 0.506). In both groups, no significant differences were found in 3-year locoregional-specific DFS between patients with a total LND number ≤17 and >17 (PF4500, 35% versus 50%, p = 0.291; PF5040 group, 45% versus 46%, p = 0.866). The PF5040 and PF4500 groups were comparable in terms of survival outcomes and local control. Although no additional survival benefits were identified, the extent of LND should not be altered according to the radiation dose

Differential Effects of Tyrosine Kinase Inhibitors on Volume-sensitive Chloride Current in Human Atrial Myocytes

To determine whether protein tyrosine kinase (PTK) modulates volume-sensitive chloride current (I(Cl.vol)) in human atrial myocytes and to identify the PTKs involved, we studied the effects of broad-spectrum and selective PTK inhibitors and the protein tyrosine phosphatase (PTP) inhibitor orthovanadate (VO(4) (−3)). I(Cl.vol) evoked by hyposmotic bath solution (0.6-times isosmotic, 0.6T) was enhanced by genistein, a broad-spectrum PTK inhibitor, in a concentration-dependent manner (EC(50) = 22.4 μM); 100 μM genistein stimulated I(Cl.vol) by 122.4 ± 10.6%. The genistein-stimulated current was inhibited by DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid, 150 μM) and tamoxifen (20 μM), blockers of I(Cl.vol). Moreover, the current augmented by genistein was volume dependent; it was abolished by hyperosmotic shrinkage in 1.4T, and genistein did not activate Cl(−) current in 1T. In contrast to the stimulatory effects of genistein, 100 μM tyrphostin A23 (AG 18) and A25 (AG 82) inhibited I(Cl.vol) by 38.2 ± 4.9% and 40.9 ± 3.4%, respectively. The inactive analogs, daidzein and tyrphostin A63 (AG 43), did not alter I(Cl.vol). In addition, the PTP inhibitor VO(4) (−3) (1 mM) reduced I(Cl.vol) by 53.5 ± 4.5% (IC(50) = 249.6 μM). Pretreatment with VO(4) (−3) antagonized genistein-induced augmentation and A23- or A25-induced suppression of I(Cl.vol). Furthermore, the selective Src-family PTK inhibitor PP2 (5 μM) stimulated I(Cl.vol), mimicking genistein, whereas the selective EGFR (ErbB-1) kinase inhibitor tyrphostin B56 (AG 556, 25 μM) reduced I(Cl.vol), mimicking A23 and A25. The effects of both PP2 and B56 also were substantially antagonized by pretreatment with VO(4) (−3). The results suggest that I(Cl.vol) is regulated in part by the balance between PTK and PTP activity. Regulation is complex, however. Src and EGFR kinases, distinct soluble and receptor-mediated PTK families, have opposing effects on I(Cl.vol), and multiple target proteins are likely to be involved

Validation of the Taiwan Chinese version of the EORTC QLQ-CR29 to assess quality of life in colorectal cancer patients

Abstract Background The increasing incidence of colorectal cancer in Taiwan has generated a need for a disease-specific quality-of-life measuring instrument. We aimed to validate the Taiwan Chinese version of the European Organisation for Research and Treatment of Cancer (EORTC) QLQ-C30 and QLQ-CR29. Methods A total of 108 patients were interviewed. Convergent and discriminant validity, Cronbach’s alpha coefficient, test-retest reliability, and known-groups comparisons were used to examine the reliability and validity. Results We found good internal consistency reliability for multi-item scales of the QLQ-C30 and QLQ-CR29, except for the cognitive function and pain scale of the QLQ-C30. Patients in the active treatment group reported compromised functional scale scores (global health status/quality of life, QLQ-C30) and worse symptoms (blood and mucus in stool, QLQ-CR29) than those in the follow-up group. Similar results were found in comparisons based on Eastern Cooperative Oncology Group (ECOG) Performance Status and Bristol Stool Scale: higher physical function/sexual interest, less fatigue/urine frequency symptoms for patients with the lowest ECOG Performance Status (Grade 0), and borderline worse stool frequency scores from Types 5 and 6 patients on the Bristol Stool Scale. Conclusion The study validated the Taiwan Chinese version of the EORTC QLQ-C30 and QLQ-CR29. The clinical applicability warrants further studies with greater number of participants