Development of an in vitro microfluidic model to study the role of microenvironmental cells in oral cancer metastasis.

Metastasis occurs when cancer cells leave the primary tumour and travel to a secondary site to form a new lesion. The tumour microenvironment (TME) is recognised to greatly influence this process, with for instance the vascular system enabling the dissemination of the cells into other tissues. However, understanding the exact role of these microenvironmental cells during metastasis has proven challenging. Indeed, in vitro models often appear too simplistic, and the study of the interactions between different cell types in a 3D space is limited. On the other hand, even though in vivo models incorporate the TME, observing cells in real-time to understand their exact role is difficult. Horizontal compartmentalised microfluidic models are a promising new platform for metastasis studies. These devices, composed of adjacent microchannels, can incorporate multiple cell types within a 3D space. Furthermore, the transparency and thickness of these models also enables high quality real-time imaging to be performed. This paper demonstrates how these devices can be successfully used for oral squamous cell carcinoma (OSCC) metastasis studies, focusing on the role of the vascular system in this process. Conditions for co-culture of OSCC cells and endothelial cells have been determined and staining protocols optimised. Furthermore, several imaging analysis techniques for these models are described, enabling precise segmentation of the different cell types on the images as well as accurate assessment of their phenotype. These methods can be applied to any study aiming to understand the role of microenvironmental cell types in cancer metastatic dissemination, and overcome several challenges encountered with current in vitro and in vivo models. Hence, this new in vitro model capable of recapitulating important aspects of the cellular complexity of human metastatic dissemination can ultimately contribute to replacing animal studies in this field

Evaluation of the Sysmex XN-550, a Novel Compact Haematology analyser from the XN-L (R) series, compared to the XN-20 system

INTRODUCTION: The XN-550 is a new, automated, compact, haematology analyser designed to generate a full blood count with a standard five-part white blood cell differential and an immature granulocyte count, as well as an optional reticulocyte and optical platelet (PLT) counts. The aim of the study was to evaluate the performance of the XN-550 and compare it to the established XN-20 system. METHODS: We evaluated the basic parameter and special measurement channels of the XN-550, using the XN-20 (which has a similar operating system), as a reference analyser. Precision, carry-over and throughput evaluations were performed. In addition, a total of 202 samples including normal controls and various pathological samples were studied for comparability. RESULTS: Good correlations with the reference analyser were obtained for all parameters except basophils. The XN-550 offers impedance and optical PLT counts and the latter showed a better correlation and less scatter than the impedance count and was comparable to the XN-20 fluorescent count at PLT counts ≤40×109/L. Precision was good, and no significant carry-over was detected. CONCLUSIONS: The XN-550 was simple and easy to use, while maintaining the good diagnostic sensitivity seen with high-range systems such as the XN-20, making this compact device suitable for near-patient services and smaller satellite laboratories

Analysis of Spontaneous Reports of Hypoglycemia and Hyperglycemia associated with marketed systemic fluoroquinolones made to the Canadian adverse drug reaction monitoring program

Hypoglycemia, an adverse effect that may develop rapidly and progress to cause potentially serious consequences over a short period of time, is difficult to monitor in both outpatients and inpatients, and may be associated with serious central nervous system sequelae. Four recently published cases of severe acute hypoglycemia with gatifloxacin stimulated a review of the published literature and spontaneous adverse drug reaction reports made in Canada on fluoroquinolone-induced hypoglycemia or hyperglycemia. A search of the English literature for published reports of hypoglycemia associated with ciprofloxacin, gatifloxacin, levofloxacin, and moxifloxacin revealed 2 published case reports of hypoglycemia attributed to the potential drug–drug interaction of an oral hypoglycemic agent with ciprofloxacin; 4 such reports with gatifloxacin; and no reports with either levofloxacin or moxifloxacin. All spontaneously reported adverse drug reactions made to the Canadian Adverse Drug Reaction Monitoring Program (CADRMP) listed under the Metabolic and Nutritional Disorders category for the 3 marketed respiratory fluoroquinolones (gatifloxacin, levofloxacin, and moxifloxacin) were then obtained. Altogether, 25 (93%) of 27 reports in this category were due to either hypoglycemia or hyperglycemia with gatifloxacin; 4 (11%) of 35 reports, with levofloxacin; and 1 (10%) of 10 reports, with moxifloxacin. The number of case reports for hypoglycemia (x2 = 24; p < 0.001), hyperglycemia (x2 = 8; p < 0.05), and total (hypoglycemia, hyperglycemia, and both hypoglycemia and hyperglycemia) (x2 = 46; p < 0.001) was significantly higher for gatifloxacin than for either levofloxacin or moxifloxacin. The CADRMP reports for hypoglycemia or hyperglycemia with the respiratory fluoroquinolones may have identified a safety signal for gatifloxacin. A systematic analysis to determine causality, risk factors, and incidence of hypoglycemia or hyperglycemia may be warranted

TNF-α-induced E3 ligase, TRIM15 inhibits TNF-α-regulated NF-κB pathway by promoting turnover of K63 linked ubiquitination of TAK1

Ubiquitin E3-ligases are recruited at different steps of TNF-α-induced NF-κB activation; however, their role in temporal regulation of the pathway remains elusive. The study systematically identified TRIMs as potential feedback regulators of the TNF-α-induced NF-κB pathway. We further observed that TRIM15 is “late” response TNF-α-induced gene and inhibits the TNF-α-induced NF-κB pathway in several human cell lines. TRIM15 promotes turnover of K63-linked ubiquitin chains in a PRY/SPRY domain-dependent manner. TRIM15 interacts with TAK1 and inhibits its K63-linked ubiquitination, thus NF-κB activity. Further, TRIM15 interacts with TRIM8 and inhibits cytosolic translocation to antagonize TRIM8 modualted NF-κB. TRIM8 and TRIM15 also show functionally inverse correlation in psoriasis condition. In conclusion, TRIM15 is TNF-α-induced late response gene and inhibits TNF-α induced NF-κB pathway hence a feedback modulator to keep the proinflammatory NF-κB pathway under control

Cytosine-5 RNA Methylation Regulates Neural Stem Cell Differentiation and Motility

Loss-of-function mutations in the cytosine-5 RNA methylase NSUN2 cause neurodevelopmental disorders in humans, yet the underlying cellular processes leading to the symptoms that include microcephaly remain unclear. Here, we show that NSUN2 is expressed in early neuroepithelial progenitors of the developing human brain, and its expression is gradually reduced during differentiation of human neuroepithelial stem (NES) cells in vitro. In the developing $\textit{Nsun2}$$^{-/-}$ mouse cerebral cortex, intermediate progenitors accumulate and upper-layer neurons decrease. Loss of NSUN2-mediated methylation of tRNA increases their endonucleolytic cleavage by angiogenin, and 5' tRNA fragments accumulate in $\textit{Nsun2}$$^{-/-}$ brains. Neural differentiation of NES cells is impaired by both $\textit{NSUN2}$ depletion and the presence of angiogenin. Since repression of NSUN2 also inhibited neural cell migration toward the chemoattractant fibroblast growth factor 2, we conclude that the impaired differentiation capacity in the absence of NSUN2 may be driven by the inability to efficiently respond to growth factors.This work was funded by Cancer Research UK (CR-UK C10701/A15181 ), Worldwide Cancer Research ( 15-0168 ), the Medical Research Council (MRC MR/M01939X/1 ), the European Research Council (ERC 310360 ), and EMBO. Research in M.F.'s laboratory was supported by a core support grant from the Wellcome Trust and MRC to the Wellcome Trust-Medical Research Cambridge Stem Cell Institute

Interferon regulatory factor 8-deficiency determines massive neutrophil recruitment but T cell defect in fast growing granulomas during tuberculosis

Following Mycobacterium tuberculosis (Mtb) infection, immune cell recruitment in lungs is pivotal in establishing protective immunity through granuloma formation and neogenesis of lymphoid structures (LS). Interferon regulatory factor-8 (IRF-8) plays an important role in host defense against Mtb, although the mechanisms driving anti-mycobacterial immunity remain unclear. In this study, IRF-8 deficient mice (IRF-8−/−) were aerogenously infected with a low-dose Mtb Erdman virulent strain and the course of infection was compared with that induced in wild-type (WT-B6) counterparts. Tuberculosis (TB) progression was examined in both groups using pathological, microbiological and immunological parameters. Following Mtb exposure, the bacterial load in lungs and spleens progressed comparably in the two groups for two weeks, after which IRF-8−/− mice developed a fatal acute TB whereas in WT-B6 the disease reached a chronic stage. In lungs of IRF-8−/−, uncontrolled growth of pulmonary granulomas and impaired development of LS were observed, associated with unbalanced homeostatic chemokines, progressive loss of infiltrating T lymphocytes and massive prevalence of neutrophils at late infection stages. Our data define IRF-8 as an essential factor for the maintenance of proper immune cell recruitment in granulomas and LS required to restrain Mtb infection. Moreover, IRF-8−/− mice, relying on a common human and mouse genetic mutation linked to susceptibility/severity of mycobacterial diseases, represent a valuable model of acute TB for comparative studies with chronically-infected congenic WT-B6 for dissecting protective and pathological immune reactions