A Toll for lupus

Toll-like receptor (TLR)-9 recognizes CpG motifs in microbial DNA. TLR9 signalling stimulates innate antimicrobial immunity and modulates adaptive immune responses including autoimmunity against chromatin, e.g., in systemic lupus erythematosus (SLE). This review summarizes the available data for a role of TLR9 signalling in lupus and discusses the following questions that arise from these observations: 1) Is CpG-DNA/TLR9 interaction involved in infection-induced disease activity of lupus? 2) What are the risks of CpG motifs in vaccine adjuvants for lupus patients? 3) Is TLR9 signalling involved in the pathogenesis of lupus by recognizing self DNA

A graphene field-effect transistor as a molecule-specific probe of DNA nucleobases

© 2015 Macmillan Publishers Limited. All rights reserved. Fast and reliable DNA sequencing is a long-standing target in biomedical research. Recent advances in graphene-based electrical sensors have demonstrated their unprecedented sensitivity to adsorbed molecules, which holds great promise for label-free DNA sequencing technology. To date, the proposed sequencing approaches rely on the ability of graphene electric devices to probe molecular-specific interactions with a graphene surface. Here we experimentally demonstrate the use of graphene field-effect transistors (GFETs) as probes of the presence of a layer of individual DNA nucleobases adsorbed on the graphene surface. We show that GFETs are able to measure distinct coverage-dependent conductance signatures upon adsorption of the four different DNA nucleobases; a result that can be attributed to the formation of an interface dipole field. Comparison between experimental GFET results and synchrotron-based material analysis allowed prediction of the ultimate device sensitivity, and assessment of the feasibility of single nucleobase sensing with graphene

B cells do not take up bacterial DNA: An essential role for antigen in exposure of DNA to toll-like receptor-9

Murine dendritic cells (DC) and macrophages respond to bacterial CpG DNA through toll-like receptor 9 (TLR9). Although it is frequently assumed that bacterial DNA is a direct stimulus for B cells, published work does not reliably show responses of purified B cells. Here we show that purified splenic B cells did not respond to Escherichia coli DNA with induction of CD86, despite readily responding to single-stranded (ss) phosphodiester CpG oligodeoxynucleotides (ODN). This was due to a combination of weak responses to both long and double-stranded (ds) DNA. B-cell DNA uptake was greatly reduced with increasing DNA length. This contrasts with macrophages where DNA uptake and subsequent responses were enhanced with increasing DNA length. However, when DNA was physically linked to hen egg lysozyme (HEL), HEL-specific B cells showed efficient uptake of DNA, and limited proliferation in response to the HEL-DNA complex. We propose that, in the absence of other signals, B cells have poor uptake and responses to long dsDNA to prevent polyclonal activation. Conversely, when DNA is physically linked to a B-cell receptor (BCR) ligand, its uptake is increased, allowing TLR9-dependent B-cell activation in an antigen-specific manner. We could not generate fragments of E. coli DNA by limited DNaseI digestion that could mimic the stimulatory effect of ss CpG ODN on naive B cells. We suggest that the frequently studied polyclonal B-cell responses to CpG ODN are relevant to therapeutic applications of phosphorothioate-modified CpG-containing ODN, but not to natural responses to foreign or host dsDNA. Immunology and Cell Biology (2011) 89, 517-525; doi:10.1038/icb.2010.112; published online 5 October 201

Bacterial membrane vesicles transport their DNA cargo into host cells

© 2017 The Author(s). Bacterial outer membrane vesicles (OMVs) are extracellular sacs containing biologically active products, such as proteins, cell wall components and toxins. OMVs are reported to contain DNA, however, little is known about the nature of this DNA, nor whether it can be transported into host cells. Our work demonstrates that chromosomal DNA is packaged into OMVs shed by bacteria during exponential phase. Most of this DNA was present on the external surfaces of OMVs, with smaller amounts located internally. The DNA within the internal compartments of Pseudomonas aeruginosa OMVs were consistently enriched in specific regions of the bacterial chromosome, encoding proteins involved in virulence, stress response, antibiotic resistance and metabolism. Furthermore, we demonstrated that OMVs carry DNA into eukaryotic cells, and this DNA was detectable by PCR in the nuclear fraction of cells. These findings suggest a role for OMV-associated DNA in bacterial-host cell interactions and have implications for OMV-based vaccines

Resource utilization and costs before and after total joint arthroplasty

<p>Abstract</p> <p>Background</p> <p>The purpose of this study was to compare pre- and post-surgical healthcare costs in commercially insured total joint arthroplasty (TJA) patients with osteoarthritis (OA) in the United States (U.S.).</p> <p>Methods</p> <p>Using a large healthcare claims database, we identified patients over age 39 with hip or knee OA who underwent unilateral primary TJA (hip or knee) between 1/1/2006 and 9/30/2007. Utilization of healthcare services and costs were aggregated into three periods: 12 months "pre-surgery," 91 days "peri-operative," and 3 to 15 month "follow-up," Mean total pre-surgery costs were compared with follow-up costs using Wilcoxon signed-rank test.</p> <p>Results</p> <p>14,912 patients met inclusion criteria for the study. The mean total number of outpatient visits declined from pre-surgery to follow-up (18.0 visits vs 17.1), while the percentage of patients hospitalized increased (from 7.5% to 9.8%) (both <it>p </it>< 0.01). Mean total costs during the follow-up period were 18% higher than during pre-surgery ($11,043 vs.$9,632, <it>p </it>< 0.01), largely due to an increase in the costs of inpatient care associated with hospital readmissions ($3,300 vs.$1,817, p < 0.01). Pharmacotherapy costs were similar for both periods ($2013 [follow-up] vs.$1922 [pre-surgery], p = 0.33); outpatient care costs were slightly lower in the follow-up period ($4338 vs.$4571, <it>p </it>< 0.01). Mean total costs for the peri-operative period were $36,553.</p> <p>Conclusions</p> <p>Mean total utilization of outpatient healthcare services declined slightly in the first year following TJA (exclusive of the peri-operative period), while mean total healthcare costs increased during the same time period, largely due to increased costs associated with hospital readmissions. Further study is necessary to determine whether healthcare costs decrease in subsequent years.</p

Academic doctors' views of complementary and alternative medicine (CAM) and its role within the NHS: an exploratory qualitative study

<p>Abstract</p> <p>Background</p> <p>There has been a marked increase in the use of complementary and alternative medicine (CAM) in the UK population in recent years. Surveys of doctors' perspectives on CAM have identified a variety of views and potential information needs. While these are useful for describing the proportions of doctors who hold particular attitudes towards CAM, they are less helpful for understanding why. In addition, while the views of non-academic doctors have begun to be studied, the perspective and rationales of academic doctors remains under-researched. It seems important to investigate the views of those with a research-orientation, given the emphasis on the need for more scientific evidence in recent debates on CAM.</p> <p>Methods</p> <p>This exploratory study used qualitative methods to explore academic doctors' views of CAM and the rationales they provided for their views. A purposeful sampling strategy was used to identify doctors with a dual clinical and academic role in the Bristol area, with an anticipated variety of views on CAM. Semi-structured interviews were conducted with nine doctors. The data were analysed thematically, drawing on the Framework Approach.</p> <p>Results</p> <p>The doctors expressed a spectrum of views on CAM, falling into three broad groups: the 'enthusiasts', the 'sceptics' and the 'undecided'. Scepticism or uncertainty about the value of CAM was prominent, except among those practising a form of CAM. A variety of rationales underpinned their perspectives on CAM, a key recurring rationale being their perspective on the scientific evidence base. The main themes arising included: the role of doctors' professional experiences of conventional medicine and CAM in shaping their attitudes towards CAM, doctor-patient communication about CAM and patient disclosure, whether there is a need for training and education in CAM for doctors, a hierarchy of acceptability of CAM and the nature of evidence; and the role of CAM within the NHS.</p> <p>Conclusion</p> <p>Despite the caution or scepticism towards CAM expressed by doctors in this study, more open doctor-patient communication about CAM may enable doctors' potential concerns about CAM to be addressed, or at least enhance their knowledge of what treatments or therapies their patients are using. Offering CAM to patients may serve to enhance patients' treatment choices and even increase doctors' fulfilment in their practice. However, given the recurring concerns about lack of scientific evidence expressed by the doctors in this study, perceptions of the evidence base may remain a significant barrier to greater integration of CAM within the NHS.</p

Interleukin-17D and Nrf2 mediate initial innate immune cell recruitment and restrict MCMV infection.

Innate immune cells quickly infiltrate the site of pathogen entry and not only stave off infection but also initiate antigen presentation and promote adaptive immunity. The recruitment of innate leukocytes has been well studied in the context of extracellular bacterial and fungal infection but less during viral infections. We have recently shown that the understudied cytokine Interleukin (IL)-17D can mediate neutrophil, natural killer (NK) cell and monocyte infiltration in sterile inflammation and cancer. Herein, we show that early immune cell accumulation at the peritoneal site of infection by mouse cytomegalovirus (MCMV) is mediated by IL-17D. Mice deficient in IL-17D or the transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf2), an inducer of IL-17D, featured an early decreased number of innate immune cells at the point of viral entry and were more susceptible to MCMV infection. Interestingly, we were able to artificially induce innate leukocyte infiltration by applying the Nrf2 activator tert-butylhydroquinone (tBHQ), which rendered mice less susceptible to MCMV infection. Our results implicate the Nrf2/IL-17D axis as a sensor of viral infection and suggest therapeutic benefit in boosting this pathway to promote innate antiviral responses

CpG DNA modulates interleukin 1β-induced interleukin-8 expression in human bronchial epithelial (16HBE14o-) cells

BACKGROUND: Recognition of repeat unmethylated CpG motifs from bacterial DNA through Toll-like receptor (TLR-9) has been shown to induce interleukin (IL)-8 expression in immune cells. We sought to investigate the role of CpG oligodeoxynucleotides (ODN) on a human bronchial epithelial cells. METHODS: RT-PCR and Western blot analysis were used to determine expression of TLR-9 in human bronchial epithelial cells (16HBE14o-). Cells were treated with CpG ODN in the presence or absence of IL-1β and IL-8 protein was determined using ELISA. In some cases cells were pretreated with chloroquine, an inhibitor of TLR-9 signaling, or SB202190, an inhibitor of the mitogen activated protein kinase p38, prior to treatment with IL-1β and CpG. TLR9 siRNA was used to silence TLR9 prior to treatment with IL-1β and CpG. IκBα and p38 were assessed by Western blot, and EMSA's were performed to determine NF-κB activation. To investigate IL-8 mRNA stability, cells were treated with IL-1β in the absence or presence of CpG for 2 h and actinomycin D was added to induce transcriptional arrest. Cells were harvested at 15 min intervals and Northern blot analysis was performed. RESULTS: TLR-9 is expressed in 16HBE14o- cells. CpG synergistically increased IL-1β-induced IL-8 protein abundance, however treatment with CpG alone had no effect. CpC (a control ODN) had no effect on IL-1β-induced IL-8 levels. In addition, CpG synergistically upregulated TNFα-induced IL-8 expression. Silencing TLR9 using siRNA or pretreatment of cells with chloroquine had little effect on IL-1β-induced IL-8 levels, but abolished CpG-induced synergy. CpG ODN had no effect on NF-κB translocation or DNA binding in 16HBE14o- cells. Treatment with CpG increased phosphorylation of p38 and pretreatment with the p38 inhibitor SB202190 attenuated the synergistic increase in IL-8 protein levels. Analysis of the half-life of IL-8 mRNA revealed that IL-8 mRNA had a longer half-life following the co-treatment of CpG and IL-1β compared to treatment with IL-1β alone. CONCLUSION: Together, these data demonstrate that CpG modulates IL-8 synthesis in the presence of a pro-inflammatory mediator utilizing TLR9 and post-transcriptional mechanisms involving the activation of p38 and stabilization of IL-8 mRNA

Mathematical model of a telomerase transcriptional regulatory network developed by cell-based screening: analysis of inhibitor effects and telomerase expression mechanisms

Cancer cells depend on transcription of telomerase reverse transcriptase (TERT). Many transcription factors affect TERT, though regulation occurs in context of a broader network. Network effects on telomerase regulation have not been investigated, though deeper understanding of TERT transcription requires a systems view. However, control over individual interactions in complex networks is not easily achievable. Mathematical modelling provides an attractive approach for analysis of complex systems and some models may prove useful in systems pharmacology approaches to drug discovery. In this report, we used transfection screening to test interactions among 14 TERT regulatory transcription factors and their respective promoters in ovarian cancer cells. The results were used to generate a network model of TERT transcription and to implement a dynamic Boolean model whose steady states were analysed. Modelled effects of signal transduction inhibitors successfully predicted TERT repression by Src-family inhibitor SU6656 and lack of repression by ERK inhibitor FR180204, results confirmed by RT-QPCR analysis of endogenous TERT expression in treated cells. Modelled effects of GSK3 inhibitor 6-bromoindirubin-3′-oxime (BIO) predicted unstable TERT repression dependent on noise and expression of JUN, corresponding with observations from a previous study. MYC expression is critical in TERT activation in the model, consistent with its well known function in endogenous TERT regulation. Loss of MYC caused complete TERT suppression in our model, substantially rescued only by co-suppression of AR. Interestingly expression was easily rescued under modelled Ets-factor gain of function, as occurs in TERT promoter mutation. RNAi targeting AR, JUN, MXD1, SP3, or TP53, showed that AR suppression does rescue endogenous TERT expression following MYC knockdown in these cells and SP3 or TP53 siRNA also cause partial recovery. The model therefore successfully predicted several aspects of TERT regulation including previously unknown mechanisms. An extrapolation suggests that a dominant stimulatory system may programme TERT for transcriptional stability

Colon cancer-derived oncogenic EGFR G724S mutant identified by whole genome sequence analysis is dependent on asymmetric dimerization and sensitive to cetuximab

Background: Inhibition of the activated epidermal growth factor receptor (EGFR) with either enzymatic kinase inhibitors or anti-EGFR antibodies such as cetuximab, is an effective modality of treatment for multiple human cancers. Enzymatic EGFR inhibitors are effective for lung adenocarcinomas with somatic kinase domain EGFR mutations while, paradoxically, anti-EGFR antibodies are more effective in colon and head and neck cancers where EGFR mutations occur less frequently. In colorectal cancer, anti-EGFR antibodies are routinely used as second-line therapy of KRAS wild-type tumors. However, detailed mechanisms and genomic predictors for pharmacological response to these antibodies in colon cancer remain unclear. Findings: We describe a case of colorectal adenocarcinoma, which was found to harbor a kinase domain mutation, G724S, in EGFR through whole genome sequencing. We show that G724S mutant EGFR is oncogenic and that it differs from classic lung cancer derived EGFR mutants in that it is cetuximab responsive in vitro, yet relatively insensitive to small molecule kinase inhibitors. Through biochemical and cellular pharmacologic studies, we have determined that cells harboring the colon cancer-derived G719S and G724S mutants are responsive to cetuximab therapy in vitro and found that the requirement for asymmetric dimerization of these mutant EGFR to promote cellular transformation may explain their greater inhibition by cetuximab than small-molecule kinase inhibitors. Conclusion: The colon-cancer derived G719S and G724S mutants are oncogenic and sensitive in vitro to cetuximab. These data suggest that patients with these mutations may benefit from the use of anti-EGFR antibodies as part of the first-line therapy