Identification of the Synthetic Cannabinoid R()WIN55,212-2 as a Novel Regulator of IFN Regulatory Factor 3 Activation and IFN- Expression

Beta Interferons (IFN-βs) represent one of the first line treatments for relapsing remitting multiple sclerosis (RRMS), slowing disease progression whilst reducing the frequency of relapses. Despite this, more effective, well tolerated therapeutic strategies are needed. Cannabinoids palliate experimental autoimmune encephalomyelitis (EAE) symptoms and have therapeutic potential in MS patients although the precise molecular mechanism for these effects is not understood. Toll-like receptor (TLR) signaling controls innate immune responses and TLRs are implicated in MS. Here we demonstrate that the synthetic cannabinoid R(+)WIN55,212-2 is a novel regulator of TLR3 and TLR4 signaling by inhibiting the pro-inflammatory signaling axis triggered by TLR3 and TLR4 whilst selectively augmenting TLR3-induced activation of IFN regulatory factor 3 (IRF3) and expression of IFN-β. We present evidence that R(+)WIN55,212-2 strongly promotes the nuclear localization of IRF3. The potentiation of IFN-β expression by R(+)WIN55,212-2 is critical for manifesting its protective effects in the murine MS model EAE as evidenced by its reduced therapeutic efficacy in the presence of an anti-IFN-β antibody. R(+)WIN55,212-2 also induces IFN-β expression in MS patient peripheral blood mononuclear cells (PBMCs), whilst downregulating inflammatory signaling in these cells. These findings identify R(+)WIN55,212-2 as a novel regulator of TLR3 signaling to IRF3 activation and IFN-β expression and highlights a new mechanism that may be open to exploitation in the development of new therapeutics for the treatment of MS

Identification of the Synthetic Cannabinoid R()WIN55,212-2 as a Novel Regulator of IFN Regulatory Factor 3 Activation and IFN- Expression

Beta Interferons (IFN-βs) represent one of the first line treatments for relapsing remitting multiple sclerosis (RRMS), slowing disease progression whilst reducing the frequency of relapses. Despite this, more effective, well tolerated therapeutic strategies are needed. Cannabinoids palliate experimental autoimmune encephalomyelitis (EAE) symptoms and have therapeutic potential in MS patients although the precise molecular mechanism for these effects is not understood. Toll-like receptor (TLR) signaling controls innate immune responses and TLRs are implicated in MS. Here we demonstrate that the synthetic cannabinoid R(+)WIN55,212-2 is a novel regulator of TLR3 and TLR4 signaling by inhibiting the pro-inflammatory signaling axis triggered by TLR3 and TLR4 whilst selectively augmenting TLR3-induced activation of IFN regulatory factor 3 (IRF3) and expression of IFN-β. We present evidence that R(+)WIN55,212-2 strongly promotes the nuclear localization of IRF3. The potentiation of IFN-β expression by R(+)WIN55,212-2 is critical for manifesting its protective effects in the murine MS model EAE as evidenced by its reduced therapeutic efficacy in the presence of an anti-IFN-β antibody. R(+)WIN55,212-2 also induces IFN-β expression in MS patient peripheral blood mononuclear cells (PBMCs), whilst downregulating inflammatory signaling in these cells. These findings identify R(+)WIN55,212-2 as a novel regulator of TLR3 signaling to IRF3 activation and IFN-β expression and highlights a new mechanism that may be open to exploitation in the development of new therapeutics for the treatment of MS

A nucleotide-specific polymerase chain reaction assay to differentiate rabies virus biotypes in South Africa

Antigenic and nucleotide sequence analyses have shown that two distinct biotypes of rabies virus are circulating in South Africa. One of these typically infects members of the family Canidae, while the other comprises a heterogeneous group of apparently indigenous viruses, infecting members of the Viverridae family. In recent times, it has become evident that a considerable amount of crossinfection may occur and the manifestation of viverrid rabies in non-viverrid animals in particular appears to have become more commonplace. Consequently, the need to rapidly distinguish between rabies virus biotypes has become increasingly important in efforts to monitor the epidemiology of rabies in the southern African region. In this study, a nested polymerase chain reaction (PCR) assay was developed to distinguish between these two groups of rabies viruses. Consensus oligonucleotides were used to amplify the cytoplasmic domain of the rabies virus glycoprotein and the adjacent intergenic region. The resultant amplicon was subsequently used as template in a second round heminested PCR in the presence of type-specific primers, thereby successfully generating amplicons of characteristic size for each biotype.The articles have been scanned in colour with a HP Scanjet 5590; 600dpi. Adobe Acrobat X Pro was used to OCR the text and also for the merging and conversion to the final presentation PDF-format

Christian hermeneutics in South Africa

Hermeneutics remains a divisive and polarizing topic within scholarly and ecclesiastical communities in South Africa. These tensions are not limited to theoretical differences but often crystallize on a grassroots level when local churches and church assemblies have to make important decisions on controversial ethical topics such as ordaining women in church offices, assessing the ethics of gay marriages, and taking a stance on the land debate in South Africa. This book makes a unique contribution in two ways: firstly, it focuses on the uniquely South African hermeneutical landscape; secondly, it relates theories to practical ethical application. The unique scholarly contribution of this consists in it relating hermeneutics to ethics within the South African landscape. A diverse group of scholars have been invited to partake in the project and the views expressed are often quite diverse. This allows readers to develop an understanding and sensitivity of the various angles employed and the interests at stake in addressing difficult societal problems

A case study of rabies diagnosis from formalin-fixed brain material

Rabies is caused by several Lyssavirus species, a group of negative sense RNA viruses. Although rabies is preventable, it is often neglected particularly in developing countries in the face of many competing public and veterinary health priorities. Epidemiological information based on laboratory-based surveillance data is critical to adequately strategise control and prevention plans. In this regard the fluorescent antibody test for rabies virus antigen in brain tissues is still considered the basic requirement for laboratory confirmation of animal cases. Occasionally brain tissues from suspected rabid animals are still submitted in formalin, although this has been discouraged for a number of years. Immunohistochemical testing or a modified fluorescent antibody technique can be performed on such samples. However, this method is cumbersome and cannot distinguish between different Lyssavirus species. Owing to RNA degradation in formalin-fixed tissues, conventional RT-PCR methodologies have also been proven to be unreliable. This report is concerned with a rabies case in a domestic dog from an area in South Africa where rabies is not common. Typing of the virus involved was therefore important, but the only available sample was submitted as a formalin-fixed specimen. A real-time RT-PCR method was therefore applied and it was possible to confirmrabies and obtain phylogenetic information that indicated a close relationship between this virus and the canid rabies virus variants from another province (KwaZulu-Natal) in South Africa.http://www.journals.co.za/ej/ejour_savet.htmlnf201

Christian hermeneutics in South Africa

Hermeneutics remains a divisive and polarizing topic within scholarly and ecclesiastical communities in South Africa. These tensions are not limited to theoretical differences but often crystallize on a grassroots level when local churches and church assemblies have to make important decisions on controversial ethical topics such as ordaining women in church offices, assessing the ethics of gay marriages, and taking a stance on the land debate in South Africa. This book makes a unique contribution in two ways: firstly, it focuses on the uniquely South African hermeneutical landscape; secondly, it relates theories to practical ethical application. The unique scholarly contribution of this consists in it relating hermeneutics to ethics within the South African landscape. A diverse group of scholars have been invited to partake in the project and the views expressed are often quite diverse. This allows readers to develop an understanding and sensitivity of the various angles employed and the interests at stake in addressing difficult societal problems

Participation of MyD88 and interleukin-33 as innate drivers of Th2 immunity to Trichinella spiralis

Trichinella spiralis is a highly destructive parasitic nematode that invades and destroys intestinal epithelial cells, injures many different tissues during its migratory phase, and occupies and transforms myotubes during the final phase of its life cycle. We set out to investigate the role in immunity of innate receptors for potential pathogen- or danger-associated molecular patterns (PAMPs or DAMPs). Focusing on the MyD88-dependent receptors, which include Toll-like receptors (TLRs) and interleukin-1 (IL-1) family members, we found that MyD88-deficient mice expelled worms normally, while TLR2/4-deficient mice showed accelerated worm expulsion, suggesting that MyD88 was active in signaling pathways for more than one receptor during intestinal immunity. A direct role for PAMPs in TLR activation was not supported in a transactivation assay involving a panel of murine and human TLRs. Mice deficient in the IL-1 family receptor for the DAMP, IL-33 (called ST2), displayed reduced intestinal Th2 responses and impaired mast cell activation. IL-33 was constitutively expressed in intestinal epithelial cells, where it became concentrated in nuclei within 2 days of infection. Nuclear localization was an innate response to infection that occurred in intestinal regions where worms were actively migrating. Th2 responses were also compromised in the lymph nodes draining the skeletal muscles of ST2-deficient mice, and this correlated with increased larval burdens in muscle. Our results support a mechanism in which the immune system recognizes and responds to tissue injury in a way that promotes Th2 responses

Enhancement of chemokine function as an immunomodulatory strategy employed by human herpesviruses

Herpes simplex virus (HSV) types 1 and 2 are highly prevalent human neurotropic pathogens that cause a variety of diseases, including lethal encephalitis. The relationship between HSV and the host immune system is one of the main determinants of the infection outcome. Chemokines play relevant roles in antiviral response and immunopathology, but the modulation of chemokine function by HSV is not well understood. We have addressed the modulation of chemokine function mediated by HSV. By using surface plasmon resonance and crosslinking assays we show that secreted glycoprotein G (SgG) from both HSV-1 and HSV-2 binds chemokines with high affinity. Chemokine binding activity was also observed in the supernatant of HSV-2 infected cells and in the plasma membrane of cells infected with HSV-1 wild type but not with a gG deficient HSV-1 mutant. Cell-binding and competition experiments indicate that the interaction takes place through the glycosaminoglycan-binding domain of the chemokine. The functional relevance of the interaction was determined both in vitro, by performing transwell assays, time-lapse microscopy, and signal transduction experiments; and in vivo, using the air pouch model of inflammation. Interestingly, and in contrast to what has been observed for previously described viral chemokine binding proteins, HSV SgGs do not inhibit chemokine function. On the contrary, HSV SgGs enhance chemotaxis both in vitro and in vivo through increasing directionality, potency and receptor signaling. This is the first report, to our knowledge, of a viral chemokine binding protein from a human pathogen that increases chemokine function and points towards a previously undescribed strategy of immune modulation mediated by viruses

A microfluidic-SERSplatform for isolation and immuno-phenotyping of antigen specific T-cells

T-cells play a major role in host defense mechanisms against many diseases. With the current growth of immunotherapy approaches, there is a strong need for advanced technologies to detect and characterize these immune cells. Herein, we present a simple approach for the isolation of antigen specific T-cells from the complex biological sample based on T-cell receptor (TCR) and peptide major histocompatibility complex (pMHC) interaction. Subsequently, we characterize those antigen specific T-cells by profiling TCR expression heterogeneity. Our approach utilizes an alternating current electrohydrodynamic (ac-EHD) based microfluidic platform for isolation and surface enhanced Raman scattering (SERS) for TCR expression profiling. The use of ac-EHD enables specific isolation of T-cells by generating a nanoscopic shear force at the double layer of the sensing surface which enhances the frequency of pMHC and TCR interactions and consequently shears off the nonspecific targets. TCR expression profiling of the isolated T-cells was performed by encoding them with SERS-labelled pMHCs followed by SERS detection in bulk as well as in single T-Cell. In proof-of-concept experiments, 56.93 ± 7.31% of the total CD4+T-cells were captured from an excess amount of nonspecific cells (e.g., PBMCs) with high specificity and sensitivity (0.005%). Moreover, TCR analysis data using SERS shows the heterogeneity in the T-cell receptor expression which can inform on the activation status of T-cells and the patient’s response to immunotherapy. We believe that this approach may hold potential for numerous applications towards monitoring immune status, understanding therapeutic responses,and effective vaccine development

Regulation of Tolerogenic Features on Dexamethasone-Modulated MPLA-Activated Dendritic Cells by MYC

The potential of tolerogenic dendritic cells (tolDCs) to shape immune responses and restore tolerance has turn them into a promising therapeutic tool for cellular therapies directed toward immune regulation in autoimmunity. Although the cellular mechanisms by which these cells can exert their regulatory function are well-known, the mechanisms driving their differentiation and function are still poorly known, and the variety of stimuli and protocols applied to differentiate DCs toward a tolerogenic phenotype makes it even more complex to underpin the molecular features involved in their function. Through transcriptional profiling analysis of monocyte-derived tolDCs modulated with dexamethasone (Dex) and activated with monophosphoryl lipid A (MPLA), known as DM-DCs, we were able to identify MYC as one of the transcriptional regulators of several genes differentially expressed on DM-DCs compared to MPLA-matured DCs (M-DCs) and untreated/immature DCs (DCs) as revealed by Ingenuity Pathway Analysis (IPA) upstream regulators evaluation. Additionally, MYC was also amidst the most upregulated genes in DM-DCs, finding that was confirmed at a transcriptional as well as at a protein level. Blockade of transactivation of MYC target genes led to the downregulation of tolerance-related markers IDO1 and JAG1. MYC blockade also led to downregulation of PLZF and STAT3, transcription factors associated with immune regulation and inhibition of DC maturation, further supporting a role of MYC as an upstream regulator contributing to the regulatory phenotype of DM-DCs. On the other hand, we had previously shown that fatty acid oxidation, oxidative metabolism and zinc homeostasis are amongst the main biological functions represented in DM-DCs, and here we show that DM-DCs exhibit higher intracellular expression of ROS and Zinc compared to mature M-DCs and DCs. Taken together, these findings suggest that the regulatory profile of DM-DCs is partly shaped by the effect of the transcriptional regulation of tolerance-inducing genes by MYC and the modulation of oxidative metabolic processes and signaling mediators such as Zinc and ROS