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Immunoglobulin G (IgG) comprises a significant portion of the protein corona that forms on biomaterial surfaces and holds a pivotal role in modulating host immune responses. To shed light on the important relationship between biomaterial surface functionality, IgG adsorption, and innate immune responses, we prepared, using plasma deposition, four surface coatings with specific chemistries, wettability, and charge. We found that nitrogen-containing coatings such as these deposited from allylamine (AM) and 2-methyl-2-oxazoline (POX) cause the greatest IgG unfolding, while hydrophilic acrylic acid (AC) surfaces allowed for the retention of the protein structure. Structural changes in IgG significantly modulated macrophage attachment, migration, polarization, and the expression of pro- and anti-inflammatory cytokines. Unfolded IgG on the POX and AM surfaces enhanced macrophage attachment, migration, extracellular trap release, and pro-inflammatory factors production such as IL-6 and TNF-α. Retention of IgG structure on the AC surface downregulated inflammatory responses. The findings of this study demonstrate that the retention of protein structure is an essential factor that must be taken into consideration when designing biomaterial surfaces. Our study indicates that using hydrophilic surface coatings could be a promising strategy for designing immune-modulatory biomaterials for clinical applications.

The ongoing coronavirus disease 2019 (COVID-19) pandemic perpetuated by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants has highlighted the continued need for broadly protective vaccines that elicit robust and durable protection. Here, the vaccinia virus-based, replication-defective Sementis Copenhagen Vector (SCV) was used to develop a first-generation COVID-19 vaccine encoding the spike glycoprotein (SCV-S). Vaccination of mice rapidly induced polyfunctional CD8 T cells with cytotoxic activity and robust type 1 T helper-biased, spike-specific antibodies, which are significantly increased following a second vaccination, and contained neutralizing activity against the alpha and beta variants of concern. Longitudinal studies indicated that neutralizing antibody activity was maintained up to 9 months after vaccination in both young and middle-aged mice, with durable immune memory evident even in the presence of pre-existing vector immunity. Therefore, SCV-S vaccination has a positive immunogenicity profile, with potential to expand protection generated by current vaccines in a heterologous boost format and presents a solid basis for second-generation SCV-based COVID-19 vaccine candidates incorporating additional SARS-CoV-2 immunogens., (© 2022 The Authors. Immunology & Cell Biology published by John Wiley & Sons Australia, Ltd on behalf of Australian and New Zealand Society for Immunology, Inc.)

CDK8 regulates transcription either by phosphorylation of transcription factors or, as part of a four-subunit kinase module, through a reversible association of the kinase module with the Mediator complex, a highly conserved transcriptional coactivator. Deregulation of CDK8 has been found in various types of human cancer, while the role of CDK8 in supressing anti-cancer response of natural killer cells is being understood. Currently, CDK8-targeting cancer drugs are highly sought-after. Herein we detail the discovery of a series of novel pyridine-derived CDK8 inhibitors. Medicinal chemistry optimisation gave rise to 38 (AU1-100), a potent CDK8 inhibitor with oral bioavailability. The compound inhibited the proliferation of MV4-11 acute myeloid leukaemia cells with the kinase activity of cellular CDK8 dampened. No systemic toxicology was observed in the mice treated with 38. These results warrant further pre-clinical studies of 38 as an anti-cancer agent., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Poxvirus systems have been extensively used as vaccine vectors. Herein a RNA-Seq analysis of intramuscular injection sites provided detailed insights into host innate immune responses, as well as expression of vector and recombinant immunogen genes, after vaccination with a new multiplication defective, vaccinia-based vector, Sementis Copenhagen Vector. Chikungunya and Zika virus immunogen mRNA and protein expression was associated with necrosing skeletal muscle cells surrounded by mixed cellular infiltrates. The multiple adjuvant signatures at 12 hours post-vaccination were dominated by TLR3, 4 and 9, STING, MAVS, PKR and the inflammasome. Th1 cytokine signatures were dominated by IFNγ, TNF and IL1β, and chemokine signatures by CCL5 and CXCL12. Multiple signatures associated with dendritic cell stimulation were evident. By day seven, vaccine transcripts were absent, and cell death, neutrophil, macrophage and inflammation annotations had abated. No compelling arthritis signatures were identified. Such injection site vaccinology approaches should inform refinements in poxvirus-based vector design., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: NAP, LL and JDH own Sementis shares. JDH is the current CSO of Sementis. AS was a consultant for Sementis. PMH was the previous CEO/CSO of Sementis. LL and NAP have had, and/or currently have, salary and/or project support from funds provided, whole or in part, via Sementis. Sementis had no role in the design and interpretation of the study, or in preparation of the manuscript.

There are many unanswered questions surrounding the function of immune cells and how they interact with the reproductive system to support successful pregnancy or contribute to pregnancy pathologies. While the role of immune cells such as uterine natural killer and dendritic cells, and more recently regulatory T cells has been established, the role of another major immune cell population, the B cell, and particularly the regulatory B cells, is relatively poorly understood. This review outlines what is known about B-cell subsets in the context of pregnancy, what constitutes a regulatory B cell and what role they may play, particularly during early pregnancy. Lastly, we discuss why immunotherapies for the treatment of pregnancy disorders is not widely progressed clinically and speculate on the potential of functional regulatory B cells as the basis of novel immunotherapeutic approaches for the treatment of immune-based pregnancy pathologies., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Problem: Autism spectrum disorder (ASD)-like phenotypes in murine models are linked to elevated pro-inflammatory cytokine profiles caused by maternal immune activation (MIA), but whether MIA alters the immune response in the offspring remains unclear., Method of Study: Polyinosinic:polycytidylic acid (poly:[IC]) was used to induce MIA in immunocompetent and control TLR3-deficient pregnant mice, and cytokine levels were measured in maternal and foetal organs. Furthermore, cytokines and behaviour responses were tested after challenge with lipopolysaccharide in 7-day-old and adult mice., Results: MIA induced on E12 resulted in changes in the cytokine expression profile in maternal and foetal organs and correlated with TNFα and IL-18 dysregulation in immune organs and brains from neonatal mice born to MIA-induced dams. Such changes further correlated with altered behavioural responses in adulthood., Conclusion: MIA induced by pathogens during pregnancy can interfere with the development of the foetal immune and nervous systems leading to dysfunctional immune responses and behaviour in offspring., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

The Sementis Copenhagen Vector (SCV) is a new vaccinia virus-derived, multiplication-defective, vaccine technology assessed herein in non-human primates. Indian rhesus macaques ( Macaca mulatta ) were vaccinated with a multi-pathogen recombinant SCV vaccine encoding the structural polyproteins of both Zika virus (ZIKV) and chikungunya virus (CHIKV). After one vaccination, neutralising antibody responses to ZIKV and four strains of CHIKV, representative of distinct viral genotypes, were generated. A second vaccination resulted in significant boosting of neutralising antibody responses to ZIKV and CHIKV. Following challenge with ZIKV, SCV-ZIKA/CHIK-vaccinated animals showed significant reductions in viremias compared with animals that had received a control SCV vaccine. Two SCV vaccinations also generated neutralising and IgG ELISA antibody responses to vaccinia virus. These results demonstrate effective induction of immunity in non-human primates by a recombinant SCV vaccine and illustrates the utility of SCV as a multi-disease vaccine platform capable of delivering multiple large immunogens., Competing Interests: Competing interestsA.S., N.A.P., P.E., L.L., T.H.C. and J.D.H. own Sementis shares. J.D.H.’s research group conducts collaborative research with Sementis financially supported, whole or in part, by Sementis. A.S. was an unpaid Scientific Advisory Board member of Sementis (2011–2019) and was an occasional paid consultant for Sementis. P.M.H. was the CEO/CSO of Sementis. L.L., T.H.C., K.R.D., P.E. and N.A.P. have had, and/or currently have, salary and/or project support from funds provided, whole or in part, by Sementis. The NHP study was undertaken by Southern Research and was contracted and overseen by the NIAID (NIH, USA) and constitutes an independent assessment of the SCV vaccine vector technology. K.W., R.K., J.G. and F.K. are employees of Southern Research. VACV antibody analyses on the stored serum samples were undertaken at Southern Research under a separate R&D contract paid for by Sementis. CHIKV serology was undertaken at the University of Colorado School of Medicine and was conducted independently of Sementis using serum samples provided by Southern Research. Sementis had no influence on the analysis and interpretation of the data., (© The Author(s) 2020.)

White adipose tissue (WAT) produces interleukin-10 and other immune suppressors in response to pathogen-associated molecular patterns (PAMPs). It also homes a subset of B-cells specialized in the production of IL-10, referred to as regulatory B-cells. We investigated whether viral stimuli, polyinosinic: polycytidylic acid (poly(I:C)) or whole replicative murine cytomegalovirus (MCMV), could stimulate the expression of IL-10 in murine WAT using in vivo and ex vivo approaches. Our results showed that in vivo responses to systemic administration of poly(I:C) resulted in high levels of endogenously-produced IL-10 and IL-21 in WAT. In ex vivo WAT explants, a subset of B-cells increased their endogenous IL-10 expression in response to poly(I:C). Finally, MCMV replication in WAT explants resulted in decreased IL-10 levels, opposite to the effect seen with poly(I:C). Moreover, downregulation of IL-10 correlated with relatively lower number of Bregs. To our knowledge, this is the first report of IL-10 expression by WAT and WAT-associated B-cells in response to viral stimuli.

Chikungunya virus (CHIKV), Ross River virus (RRV), o'nyong nyong virus (ONNV), Mayaro virus (MAYV) and Getah virus (GETV) represent arthritogenic alphaviruses belonging to the Semliki Forest virus antigenic complex. Antibodies raised against one of these viruses can cross-react with other serogroup members, suggesting that, for instance, a CHIKV vaccine (deemed commercially viable) might provide cross-protection against antigenically related alphaviruses. Herein we use human alphavirus isolates (including a new human RRV isolate) and wild-type mice to explore whether infection with one virus leads to cross-protection against viremia after challenge with other members of the antigenic complex. Persistently infected Rag1 -/- mice were also used to assess the cross-protective capacity of convalescent CHIKV serum. We also assessed the ability of a recombinant poxvirus-based CHIKV vaccine and a commercially available formalin-fixed, whole-virus GETV vaccine to induce cross-protective responses. Although cross-protection and/or cross-reactivity were clearly evident, they were not universal and were often suboptimal. Even for the more closely related viruses (e.g., CHIKV and ONNV, or RRV and GETV), vaccine-mediated neutralization and/or protection against the intended homologous target was significantly more effective than cross-neutralization and/or cross-protection against the heterologous virus. Effective vaccine-mediated cross-protection would thus likely require a higher dose and/or more vaccinations, which is likely to be unattractive to regulators and vaccine manufacturers.

Introduction: High Mobility Group Box Protein 1 (HMGB1) is a DNA-binding protein that exerts inflammatory or pro-repair effects upon translocation from the nucleus. We postulate aberrant HMGB1 expression in immune-mediated necrotising myopathy (IMNM)., Methods: Herein, we compare HMGB1 expression (serological and sarcoplasmic) in patients with IMNM with that of other myositis subtypes using immunohistochemistry and ELISA., Results: IMNM ( n = 62) and inclusion body myositis (IBM, n = 14) patients had increased sarcoplasmic HMGB1 compared with other myositis patients ( n = 46). Sarcoplasmic HMGB1 expression correlated with muscle weakness and histological myonecrosis, inflammation, regeneration and autophagy. Serum HMGB1 levels were elevated in patients with IMNM, dermatomyositis and polymositis, and those myositis patients with extramuscular inflammatory features., Discussion: Aberrant HMGB1 expression occurs in myositis patients and correlates with weakness. A unique expression profile of elevated sarcoplasmic and serum HMGB1 was detected in IMNM., (Copyright © 2020 Day, Otto, Cash, Eldi, Hissaria, Proudman, Limaye and Hayball.)

A successful outcome to pregnancy is dependent on the ability of the maternal uterine microenvironment to regulate inflammation processes and establish maternal tolerance. Recently, B cells have been shown to influence pregnancy outcomes as aberrations in their numbers and functions are associated with obstetric complications. In this study, we aimed to comprehensively examine the population frequency and phenotypic profile of B cells over the course of murine pregnancy. Our results demonstrated a significant expansion in B cells within the uterus during the peri-implantation period, accompanied by alterations in B cell phenotype. Functional evaluation of uterine B cells purified from pregnant mice at day 5.5 post-coitus established their regulatory capacity as evidenced by effective suppression of proliferation and activation of syngeneic CD4 + T cells. Flow cytometric analysis revealed that the uterine B cell population has an expanded pool of IL-10-producing B cells bearing upregulated expression of co-stimulatory molecules CD80 and CD86 and activation marker CD27. Our investigations herein demonstrate that during the critical stages surrounding implantation, uterine B cells are amplified and phenotypically modified to act in a regulatory manner that potentially contributes toward the establishment of maternal immunological tolerance in early pregnancy., (Copyright © 2019 Guzman-Genuino, Eldi, Garcia-Valtanen, Hayball and Diener.)

Problem: A successful outcome to pregnancy is critically dependent on the initiation of maternal immune tolerance before embryo implantation. Cells of embryonic origin that come in contact with the uterine microenvironment can exert influence over the phenotype and function of immune cells to facilitate robust implantation; however, what influence they may have on B cells remains unknown. In this study, we investigate the effect of human trophoblast cells on B-cell phenotype and the subsequent effect on peri-implantation events., Method of Study: We cultured purified human B cells with the first-trimester human trophoblast cell line Swan 71 to investigate trophoblast-B-cell interactions and utilized trophoblast spheroids in an in vitro implantation model of migration and invasion., Results: Trophoblast-educated B cells or TE-B cells were found to consist of B cells in committed lineages such as plasmablasts and memory B cells, as well as increased proportions in subsets of CD24 hi CD27 + regulatory B cells and CD19 + IL-10 + B cells. Conditioned media from the TE-B cells showed reduced production of pro-inflammatory cytokines that influenced the T-cell proliferation and cytokine production. Using trophoblast spheroids, we assessed the role of TE-B cells in trophoblast invasion and migration. Our results demonstrate a protective effect of TE-B-conditioned media against deleterious inflammation as evidenced by survival of the trophoblast spheroid in the presence of an immune assault and promotion of a migratory phenotype., Conclusion: We posit that trophoblast-mediated education of B cells leads to their acquisition of properties capable of modulating inflammation in the uterine environment during the peri-implantation period., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Delta inulin, also known as microparticulate inulin (MPI), was modified by covalently attaching doxorubicin to its nanostructured surface for use as a targeted drug delivery vehicle. MPI is readily endocytosed by monocytes, macrophages, and dendritic cells and in this study, we sought to utilize this property to develop a system to target anti-cancer drugs to lymphoid organs. We investigated, therefore, whether MPI could be used as a vehicle to deliver doxorubicin selectively, thereby reducing the toxicity of this antibiotic anthracycline drug. Doxorubicin was covalently attached to the surface of MPI using an acid-labile linkage to enable pH-controlled release. The MPI-doxorubicin conjugate was characterized using FTIR and SEM, confirming covalent attachment and indicating doxorubicin coupling had no obvious impact on the physical nanostructure, integrity, and cellular uptake of the MPI particles. To simulate the stability of the MPI-doxorubicin in vivo, it was stored in artificial lysosomal fluid (ALF, pH 4.5). Although the MPI-doxorubicin particles were still visible after 165 days in ALF, 53% of glycosidic bonds in the inulin particles were hydrolyzed within 12 days in ALF, reflected by the release of free glucose into solution. By contrast, the fructosidic bonds were much more stable. Drug release studies of the MPI-doxorubicin in vitro, demonstrated a successful pH-dependent controlled release effect. Confocal laser scanning microscopy studies and flow cytometric analysis confirmed that when incubated with live cells, MPI-doxorubicin was efficiently internalized by immune cells. An assay of cell metabolic activity demonstrated that the MPI carrier alone had no toxic effects on RAW 264.7 murine monocyte/macrophage-like cells, but exhibited anti-cancer effects against HCT116 human colon cancer cells. MPI-doxorubicin had a greater anti-cancer cell effect than free doxorubicin, particularly when at lower concentrations, suggesting a drug-sparing effect. This study establishes that MPI can be successfully modified with doxorubicin for chemotherapeutic drug delivery.

Sepsis is a highly complex and often fatal syndrome which varies widely in its clinical manifestations, and therapies that target the underlying uncontrolled immune status in sepsis are needed. The failure of preclinical approaches to provide significant sepsis survival benefit in the clinic is often attributed to inappropriate animal disease models. It has been demonstrated that high mobility group box protein 1 (HMGB1) blockade can reduce inflammation, mortality and morbidity in experimental sepsis without promoting immunosuppression. Within this study, we explored the use of ovine anti-HMGB1 antibodies in a model of ovine septic shock incorporating intensive care supports (OSSICS). Results: Septic sheep exhibited elevated levels of HMGB1 within 12 h after the induction of sepsis. In this study, sepsis was induced in six anaesthetized adult Border Leicester × Merino ewes via intravenous instillation of E. coli and sheep monitored according to intensive care unit standard protocols for 26 h, with the requirement for noradrenaline as the primary endpoint. Septic sheep exhibited a hyperdynamic circulation, renal dysfunction, deranged coagulation profile and severe metabolic acidosis. Sheep were assigned a severity of illness score, which increased over time. While a therapeutic effect of intravenous anti-HMGB1 antibody could not be observed in this model due to limited animal numbers, a reduced bacterial dose induced a septic syndrome of much lower severity. With modifications including a reduced bacterial dose, a longer timeframe and broad spectrum antibiotics, the OSSICS model may become a robust tool for preclinical assessment of sepsis therapeutics., (Copyright: © Stevens et al.)

The use of particles for monocyte-mediated delivery could be a more efficient strategy and approach to achieve intracellular targeting and delivery of antitubercular drugs to host macrophages. In this study, the potential of inulin microparticles to serve as a drug vehicle in the treatment of chronic tuberculosis using a monocytes-mediated drug targeting approach was evaluated. Isoniazid (INH) was conjugated to inulin via hydrazone linkage in order to obtain a pH-sensitive inulin-INH conjugate. The conjugate was then characterized using proton nuclear magnetic resonance ( 1 HNMR), Fourier transform infrared spectroscopy (FTIR) as well as in vitro, cellular uptake and intracellular Mycobacterium tuberculosis (Mtb) antibacterial efficacy. The acid-labile hydrazone linkage conferred pH sensitivity to the inulin-INH conjugate with ~95, 77 and 65% of the drug released after 5 h at pH 4.5, 5.2, and 6.0 respectively. Cellular uptake studies confirm that RAW 264.7 monocytic cells efficiently internalized the inulin conjugates into endocytic compartments through endocytosis. The intracellular efficacy studies demonstrate that the inulin conjugates possess a dose-dependent targeting effect against Mtb-infected monocytes. This was through efficient internalization and cleavage of the hydrazone bond by the acidic environment of the lysosome, which subsequently released the isoniazid intracellularly to the Mtb reservoir. These results clearly suggest that inulin conjugates can serve as a pH-sensitive intracellular drug delivery system for TB treatment.

The nature of the protein corona forming on biomaterial surfaces can affect the performance of implanted devices. This study investigated the role of surface chemistry and wettability on human serum-derived protein corona formation on biomaterial surfaces and the subsequent effects on the cellular innate immune response. Plasma polymerization, a substrate-independent technique, was employed to create nanothin coatings with four specific chemical functionalities and a spectrum of surface charges and wettability. The amount and type of protein adsorbed was strongly influenced by surface chemistry and wettability but did not show any dependence on surface charge. An enhanced adsorption of the dysopsonin albumin was observed on hydrophilic carboxyl surfaces while high opsonin IgG2 adsorption was seen on hydrophobic hydrocarbon surfaces. This in turn led to a distinct immune response from macrophages; hydrophilic surfaces drove greater expression of anti-inflammatory cytokines by macrophages, whilst surface hydrophobicity caused increased production of proinflammatory signaling molecules. These findings map out a unique relationship between surface chemistry, hydrophobicity, protein corona formation, and subsequent cellular innate immune responses; the potential outcomes of these studies may be employed to tailor biomaterial surface modifications, to modulate serum protein adsorption and to achieve the desirable innate immune response to implanted biomaterials and devices.

Regulatory T cells (Tregs) are essential for maternal tolerance in allogeneic pregnancy. In preeclampsia, Tregs are fewer and display aberrant phenotypes, particularly in the thymic Treg (tTreg) compartment, potentially because of insufficient priming to male partner alloantigens before conception. To investigate how tTregs as well as peripheral Tregs (pTregs) respond to male partner seminal fluid, Foxp3 + CD4 + Tregs were examined in the uterus and uterus-draining lymph nodes in virgin estrus mice and 3.5 d postcoitum. Mating elicited 5-fold increases in uterine Tregs accompanied by extensive Treg proliferation in the uterus-draining lymph nodes, comprising 70% neuropilin 1 + tTregs and 30% neuropilin 1 - pTregs. Proliferation marker Ki67 and suppressive competence markers Foxp3 and CTLA4 were induced after mating in both subsets, and Ki67, CTLA4, CD25, and GITR were higher in tTregs than in pTregs. Analysis by t -stochastic neighbor embedding confirmed phenotypically distinct tTreg and pTreg clusters, with the proportion of tTregs but not pTregs among CD4 + T cells expanding in response to seminal fluid. Bisulphite sequencing revealed increased demethylation of the Treg-specific demethylation region in the Foxp3 locus in tTregs but not pTregs after mating. These data show that tTregs and pTregs with distinct phenotypes both respond to seminal fluid priming, but the Foxp3 epigenetic signature is uniquely increased in tTregs. We conclude that reproductive tract tTregs as well as pTregs are sensitive to local regulation by seminal fluid, providing a candidate mechanism warranting evaluation for the potential to influence preeclampsia susceptibility in women., (Copyright © 2019 by The American Association of Immunologists, Inc.)

The propensity of monocytes to migrate into sites of mycobacterium tuberculosis (TB) infection and then become infected themselves makes them potential targets for delivery of drugs intracellularly to the tubercle bacilli reservoir. Conventional TB drugs are less effective because of poor intracellular delivery to this bacterial sanctuary. This study highlights the potential of using semicrystalline delta inulin particles that are readily internalised by monocytes for a monocyte-based drug delivery system. Pyrazinoic acid was successfully attached covalently to the delta inulin particles via a labile linker. The formation of new conjugate and amide bond was confirmed using zeta potential, Proton Nuclear Magnetic Resonance ( 1 HNMR) and Fourier transform infrared spectroscopy (FTIR). Scanning electron microscopy (SEM) confirmed that no significant change in size after conjugation which is an important parameter for monocyte targeting. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were used to establish the change in thermal properties. The analysis of in-vitro release demonstrated pH-triggered drug cleavage off the delta inulin particles that followed a first-order kinetic process. The efficient targeting ability of the conjugate for RAW 264.7 monocytic cells was supported by cellular uptake studies. Overall, our finding confirmed that semicrystalline delta inulin particles (MPI) can be modified covalently with drugs and such conjugates allow intracellular drug delivery and uptake into monocytes, making this system potentially useful for the treatment of TB.

Introduction: With the increasing number of vaccines and vaccine-preventable diseases, the pressure to generate multi-valent and multi-pathogen vaccines grows. Combining individual established vaccines to generate single-shot formulations represents an established path, with significant ensuing public health and cost benefits. Poxvirus-based vector systems have the capacity for large recombinant payloads and have been widely used as platforms for the development of recombinant vaccines encoding multiple antigens, with considerable clinical trials activity and a number of registered and licensed products., Areas Covered: Herein we discuss design strategies, production processes, safety issues, regulatory hurdles and clinical trial activities, as well as pertinent new technologies such as systems vaccinology and needle-free delivery. Literature searches used PubMed, Google Scholar and clinical trials registries, with a focus on the recombinant vaccinia-based systems, Modified Vaccinia Ankara and the recently developed Sementis Copenhagen Vector., Expert Commentary: Vaccinia-based platforms show considerable promise for the development of multi-valent and multi-pathogen vaccines, especially with recent developments in vector technologies and manufacturing processes. New methodologies for defining immune correlates and human challenge models may also facilitate bringing such vaccines to market.

Zika and chikungunya viruses have caused major epidemics and are transmitted by Aedes aegypti and/or Aedes albopictus mosquitoes. The "Sementis Copenhagen Vector" (SCV) system is a recently developed vaccinia-based, multiplication-defective, vaccine vector technology that allows manufacture in modified CHO cells. Herein we describe a single-vector construct SCV vaccine that encodes the structural polyprotein cassettes of both Zika and chikungunya viruses from different loci. A single vaccination of mice induces neutralizing antibodies to both viruses in wild-type and IFNAR -/- mice and protects against (i) chikungunya virus viremia and arthritis in wild-type mice, (ii) Zika virus viremia and fetal/placental infection in female IFNAR -/- mice, and (iii) Zika virus viremia and testes infection and pathology in male IFNAR -/- mice. To our knowledge this represents the first single-vector construct, multi-pathogen vaccine encoding large polyproteins, and offers both simplified manufacturing and formulation, and reduced "shot burden" for these often co-circulating arboviruses.

Murine models of Salmonella enterica serovar Typhimurium infection are one of the commonest tools to study host-pathogen interactions during bacterial infections. Critically, the outcome of S . Typhimurium infection is impacted by the genetic background of the mouse strain used, with macrophages from C57BL/6 and BALB/c mice lacking the capacity to control intracellular bacterial replication. For this reason, the use of congenic strains, which mix the genetic backgrounds of naturally protected mouse strains with those of susceptible strains, has the capacity to significantly alter results and interpretation of S . Typhimurium infection studies. Here, we describe how macrophage knockout cell lines generated by CRISPR/Cas9 gene editing can help determine the contribution of background contaminations in the phenotypes of primary macrophages from congenic mice, on the outcome of S . Typhimurium infection studies. Our own experience illustrates how the CRISPR/Cas9 technology can be used to complement pre-existing knockout models, and shows that there is great merit in performing concurrent studies with both genetic models, to exclude unanticipated side-effects on host-pathogen interactions.

Prostate cancer cells frequently overexpress the gastrin-releasing peptide receptor, and various strategies have been applied in preclinical settings to target this receptor for the specific delivery of anticancer compounds. Recently, elastin-like polypeptide (ELP)-based self-assembling micelles with tethered GRP on the surface have been suggested to actively target prostate cancer cells. Poorly soluble chemotherapeutics such as docetaxel (DTX) can be loaded into the hydrophobic cores of ELP micelles, but only limited drug retention times have been achieved. Herein, we report the generation of hybrid ELP/liposome nanoparticles which self-assembled rapidly in response to temperature change, encapsulated DTX at high concentrations with slow release, displayed the GRP ligand on the surface, and specifically bound to GRP receptor expressing PC-3 cells as demonstrated by flow cytometry. This novel type of drug nanocarrier was successfully used to reduce cell viability of prostate cancer cells in vitro through the specific delivery of DTX., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

The Australian plant Acacia ligulata has a number of traditional food and medicinal uses by Australian Aboriginal people, although no bioactive compounds have previously been isolated from this species. Bioassay-guided fractionation of an ethanolic extract of the mature pods of A. ligulata led to the isolation of the two new echinocystic acid triterpenoid saponins, ligulatasides A (1) and B (2), which differ in the fine structure of their glycan substituents. Their structures were elucidated on the basis of 1D and 2D NMR, GC-MS, LC-MS/MS, and saccharide linkage analysis. These are the first isolated compounds from A. ligulata and the first fully elucidated structures of triterpenoid saponins from Acacia sensu stricto having echinocystic acid reported as the aglycone. Compounds 1 and 2 were evaluated for cytotoxic activity against a human melanoma cancer cell line (SK-MEL28) and a diploid fibroblast cell line (HFF), but showed only weak activity.

Vaccinia-based systems have been extensively explored for the development of recombinant vaccines. Herein we describe an innovative vaccinia virus (VACV)-derived vaccine platform technology termed Sementis Copenhagen Vector (SCV), which was rendered multiplication-defective by targeted deletion of the essential viral assembly gene D13L. A SCV cell substrate line was developed for SCV vaccine production by engineering CHO cells to express D13 and the VACV host-range factor CP77, because CHO cells are routinely used for manufacture of biologics. To illustrate the utility of the platform technology, a SCV vaccine against chikungunya virus (SCV-CHIK) was developed and shown to be multiplication-defective in a range of human cell lines and in immunocompromised mice. A single vaccination of mice with SCV-CHIK induced antibody responses specific for chikungunya virus (CHIKV) that were similar to those raised following vaccination with a replication-competent VACV-CHIK and able to neutralize CHIKV. Vaccination also provided protection against CHIKV challenge, preventing both viremia and arthritis. Moreover, SCV retained capacity as an effective mouse smallpox vaccine. In summary, SCV represents a new and safe vaccine platform technology that can be manufactured in modified CHO cells, with pre-clinical evaluation illustrating utility for CHIKV vaccine design and construction., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Central to pregnancy success is a state of T cell tolerance to paternal antigens, which is initiated at conception. The role and regulation of specific phenotypes of CD8 + T cells in mediating pregnancy tolerance is not clear. This study aimed to investigate the impact on pregnancy outcome of altering the cytokine environment during maternal CD8 + T cell priming in early pregnancy. Transgenic Act-mOVA male mice were mated to C57BL/6 (B6) females to generate fetuses expressing ovalbumin (OVA) as a model paternal antigen. OVA-reactive CD8 + OT-I T cells were activated in vitro with OVA in the presence of either transforming growth factor-β1 (TGFB1) plus interleukin-10 (IL10), or IL2, to mimic normal or dysregulated uterine conditions, respectively, and transferred into pregnant mice on gestational day 3.5. OT-I T cells activated with TGFB1 and IL10, like naive OT-I T cells, did not alter embryo implantation or fetal viability. In contrast, OT-I T cells activated with IL2 caused extensive fetal loss manifesting in mid-gestation. IL2-activated OT-I T cells expressed less FOXP3 and higher interferon-γ (IFNG) than cells activated with TGFB1 and IL10. Fetal loss did not occur in females mated with B6 males, demonstrating the antigen specificity of fetal loss, and was not abrogated by maternal genetic C1q deficiency indicating a mechanism independent of antibody-mediated cytotoxicity. These data indicate that alternative phenotypes generated in maternal CD8 + T cells at the time of priming with paternal antigens can impact pregnancy outcome, such that inappropriate activation of CD8 + T cells before implantation is capable of causing antigen-specific fetal loss later in pregnancy.

The use of cost-effective vaccines capable of inducing robust CD8 + T cell immunity will contribute significantly towards the elimination of persistent viral infections and cancers worldwide. We have previously reported that a cytolytic DNA vaccine encoding an immunogen and a truncated mouse perforin (PRF) protein significantly augments anti-viral T cell (including CD8 + T cell) immunity. Thus, the current study investigated whether this vaccine enhances activation of dendritic cells (DCs) resulting in greater priming of CD8 + T cell immunity. In vitro data showed that transfection of HEK293T cells with the cytolytic DNA resulted in the release of lactate dehydrogenase, indicative of necrotic/lytic cell death. In vitro exposure of this lytic cell debris to purified DCs from naïve C57BL/6 mice resulted in maturation of DCs as determined by up-regulation of CD80/CD86. Using activation/proliferation of adoptively transferred OT-I CD8 + T cells to measure antigen presentation by DCs in vivo, it was determined that cytolytic DNA immunisation resulted in a time-dependent increase in the proliferation of OT-I CD8 + T cells compared to canonical DNA immunisation. Overall, the data suggest that the cytolytic DNA vaccine increases the activity of DCs which has important implications for the design of DNA vaccines to improve their translational prospects.

The β-1, 3 (d)-glucan (β-glucan) present in the cell wall of Candida albicans induces epigenetic changes in human monocytes resulting in primed macrophages exhibiting increased cytokine responsiveness to reinfection. This phenomenon is referred to as trained immunity or innate immune memory. However, whether β-glucan can reprogramme murine monocytes in vitro or induce lasting effects in vivo has yet to be elucidated. Thus, purified murine spleen-derived monocytes were primed with β-glucan in vitro and assessed for markers of differentiation and survival. Important macrophage cell markers during monocyte-to-macrophage differentiation were downregulated and survival enhanced due to partial inhibition of apoptosis. Increased survival and not the β-glucan training effect explained the elevated production of tumour necrosis factor-α (TNFα) and interleukin-6 (IL-6) induced by subsequent lipopolysaccharide (LPS) challenge. In vivo, 4 days after systemic administration of β-glucan, mice were more responsive to LPS challenge as shown by the increased serum levels of TNFα, IL-6 and IL-10, an effect shown to be short lived as enhanced cytokine production was lost by day 20. Here, we have characterised murine macrophages derived from β-glucan-primed monocytes based on their surface marker expression and for the first time provide evidence that the training effect of β-glucan in vivo declines within a 3-week period.

Sepsis remains a significant health burden and a major clinical need exists for therapeutics to dampen the excessive and uncontrolled immune activation. Nuclear protein high mobility group box protein 1 (HMGB1) is released following cell death and is a late mediator in sepsis pathogenesis. While approaches targeting HMGB1 have demonstrated reduced mortality in pre-clinical models of sepsis, the impact of HMGB1 blockade on the complex septic inflammatory milieu and the development of subsequent immunosuppression remain enigmatic. Analysis of plasma samples obtained from septic shock patients established an association between increased HMGB1 and non-survival, higher APACHE II scores, and increased pro-inflammatory cytokine responses. Pre-clinically, administration of neutralising ovine anti-HMGB1 polyclonal antibodies improved survival in murine endotoxaemia and caecal ligation and puncture-induced sepsis models, and altered early cytokine profiles to one which corresponded to patterns observed in the surviving patient cohort. Additionally, anti-HMGB1 treated murine sepsis survivors were significantly more resistant to secondary bacterial infection and exhibited altered innate immune cell phenotypes and cytokine responses. These findings demonstrate that anti-HMGB1 antibodies alter inflammation in murine sepsis models and reduce sepsis mortality without potentiating immunosuppression.

Immune checkpoint molecules function as a system of checks and balances that enhance or inhibit immune responses to infectious agents, foreign tissues, and cancerous cells. Immunotherapies that target immune checkpoint molecules, particularly the inhibitory molecules programmed cell death 1 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), have revolutionized human oncology in recent years, yet little is known about these key immune signaling molecules in species other than primates and rodents. The Tasmanian devil facial tumor disease is caused by transmissible cancers that have resulted in a massive decline in the wild Tasmanian devil population. We have recently demonstrated that the inhibitory checkpoint molecule PD-L1 is upregulated on Tasmanian devil ( Sarcophilus harrisii ) facial tumor cells in response to the interferon-gamma cytokine. As this could play a role in immune evasion by tumor cells, we performed a thorough comparative analysis of checkpoint molecule protein sequences among Tasmanian devils and eight other species. We report that many of the key signaling motifs and ligand-binding sites in the checkpoint molecules are highly conserved across the estimated 162 million years of evolution since the last common ancestor of placental and non-placental mammals. Specifically, we discovered that the CTLA-4 (MYPPPY) ligand-binding motif and the CTLA-4 (GVYVKM) inhibitory domain are completely conserved across all nine species used in our comparative analysis, suggesting that the function of CTLA-4 is likely conserved in these species. We also found that cysteine residues for intra- and intermolecular disulfide bonds were also highly conserved. For instance, all 20 cysteine residues involved in disulfide bonds in the human 4-1BB molecule were also present in devil 4-1BB. Although many key sequences were conserved, we have also identified immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and immunoreceptor tyrosine-based switch motifs (ITSMs) in genes and protein domains that have not been previously reported in any species. This checkpoint molecule analysis and review of salient features for each of the molecules presented here can serve as road map for the development of a Tasmanian devil facial tumor disease immunotherapy. Finally, the strategies can be used as a guide for veterinarians, ecologists, and other researchers willing to venture into the nascent field of wild immunology.

Recombinant vaccinia viruses (rVACVs) are promising antigen-delivery systems for vaccine development that are also useful as research tools. Two common methods for selection during construction of rVACV clones are (i) co-insertion of drug resistance or reporter protein genes, which requires the use of additional selection drugs or detection methods, and (ii) dominant host-range selection. The latter uses VACV variants rendered replication-incompetent in host cell lines by the deletion of host-range genes. Replicative ability is restored by co-insertion of the host-range genes, providing for dominant selection of the recombinant viruses. Here, we describe a new method for the construction of rVACVs using the cowpox CP77 protein and unmodified VACV as the starting material. Our selection system will expand the range of tools available for positive selection of rVACV during vector construction, and it is substantially more high-fidelity than approaches based on selection for drug resistance.

The idiopathic inflammatory myopathies (IIMs) are a heterogeneous group of systemic muscle conditions that are believed to be autoimmune in nature. They have distinct pathological features, but the aetiopathogenesis of each subtype remains largely unknown. Recently, there has been increased interest in the complex role the innate immune system plays in initiating and perpetuating these conditions, and how this may differ between subtypes. This article summarises the traditional paradigms of IIM pathogenesis and reviews the accumulating evidence for disturbances in innate immune processes in these rare, but debilitating chronic conditions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The devil facial tumor disease (DFTD) is caused by clonal transmissible cancers that have led to a catastrophic decline in the wild Tasmanian devil ( Sarcophilus harrisii ) population. The first transmissible tumor, now termed devil facial tumor 1 (DFT1), was first discovered in 1996 and has been continually transmitted to new hosts for at least 20 years. In 2015, a second transmissible cancer [devil facial tumor 2 (DFT2)] was discovered in wild devils, and the DFT2 is genetically distinct and independent from the DFT1. Despite the estimated 136,559 base pair substitutions and 14,647 insertions/deletions in the DFT1 genome as compared to two normal devil reference genomes, the allograft tumors are not rejected by the host immune system. Additionally, genome sequencing of two sub-strains of DFT1 detected greater than 15,000 single-base substitutions that were found in only one of the DFT1 sub-strains, demonstrating the transmissible tumors are evolving and that generation of neoantigens is likely ongoing. Recent evidence in human clinical trials suggests that blocking PD-1:PD-L1 interactions promotes antitumor immune responses and is most effective in cancers with a high number of mutations. We hypothesized that DFTD cells could exploit the PD-1:PD-L1 inhibitory pathway to evade antitumor immune responses. We developed recombinant proteins and monoclonal antibodies (mAbs) to provide the first demonstration that PD-1 binds to both PD-L1 and PD-L2 in a non-placental mammal and show that PD-L1 is upregulated in DFTD cells in response to IFN-γ. Immunohistochemistry showed that PD-L1 is rarely expressed in primary tumor masses, but low numbers of PD-L1 + non-tumor cells were detected in the microenvironment of several metastatic tumors. Importantly, in vitro testing suggests that PD-1 binding to PD-L1 and PD-L2 can be blocked by mAbs, which could be critical to understanding how the DFT allografts evade the immune system.

Adjuvants are used to enhance the immune response against specific antigens for the production of antibodies, with the choice of adjuvant most critical for poorly immunogenic and self-antigens. This study quantitatively and qualitatively evaluated CoVaccine HT™ and Freund's adjuvants for eliciting therapeutic ovine polyclonal antibodies targeting the endogenous alarmin, high mobility group box-1 (HMGB1). Sheep were immunised with HMGB1 protein in CoVaccine HT™ or Freund's adjuvants, with injection site reactions and antibody titres periodically assessed. The binding affinity of antibodies for HMGB1 and their neutralisation activity was determined in-vitro, with in vivo activity confirmed using a murine model of endotoxemia. Results indicated that CoVaccine HT™ elicited significantly higher antibody tires with stronger affinity and more functional potency than antibodies induced with Freund's adjuvants. These studies provide evidence that CoVaccine HT™ is superior to Freund's adjuvants for the production of antibodies to antigens with low immunogenicity and supports the use of this alternative adjuvant for clinical and experimental use antibodies., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Passive immunotherapy may have particular benefits for the treatment of severe influenza infection in at-risk populations, however little is known of the impact of passive immunotherapy on the formation of memory responses to the virus. Ideally, passive immunotherapy should attenuate the severity of infection while still allowing the formation of adaptive responses to confer protection from future exposure. In this study, we sought to determine if administration of influenza-specific ovine polyclonal antibodies could inhibit adaptive immune responses in a murine model of lethal influenza infection. Ovine polyclonal antibodies generated against recombinant PR8 (H1N1) hemagglutinin exhibited potent prophylactic capacity and reduced lethality in an established influenza infection, particularly when administered intranasally. Surviving mice were also protected against reinfection and generated normal antibody and cytotoxic T lymphocyte responses to the virus. The longevity of ovine polyclonal antibodies was explored with a half-life of over two weeks following a single antibody administration. These findings support the development of an ovine passive polyclonal antibody therapy for treatment of severe influenza infection which does not affect the formation of subsequent acquired immunity to the virus.

Chimeric antigen receptor (CAR) T cells have shown great promise in the treatment of hematologic malignancies but more variable results in the treatment of solid tumors and the persistence and expansion of CAR T cells within patients has been identified as a key correlate of antitumor efficacy. Lack of immunological "space", functional exhaustion, and deletion have all been proposed as mechanisms that hamper CAR T-cell persistence. Here we describe the events following activation of third-generation CAR T cells specific for GD2. CAR T cells had highly potent immediate effector functions without evidence of functional exhaustion in vitro, although reduced cytokine production reversible by PD-1 blockade was observed after longer-term culture. Significant activation-induced cell death (AICD) of CAR T cells was observed after repeated antigen stimulation, and PD-1 blockade enhanced both CAR T-cell survival and promoted killing of PD-L1(+) tumor cell lines. Finally, we assessed CAR T-cell persistence in patients enrolled in the CARPETS phase 1 clinical trial of GD2-specific CAR T cells in the treatment of metastatic melanoma. Together, these data suggest that deletion also occurs in vivo and that PD-1-targeted combination therapy approaches may be useful to augment CAR T-cell efficacy and persistence in patients.

Implantable devices have become an established part of medical practice. However, often a negative inflammatory host response can impede the integration and functionality of the device. In this paper, we interrogate the role of surface nanotopography and chemistry on the potential molecular role of the inflammasome in controlling macrophage responses. To achieve this goal we engineered model substrata having precisely controlled nanotopography of predetermined height and tailored outermost surface chemistry. Bone marrow derived macrophages (BMDM) were harvested from genetically engineered mice deficient in the inflammasome components ASC, NLRP3 and AIM2. These cells were then cultured on these nanoengineered substrata and assessed for their capacity to attach and express pro-inflammatory cytokines. Our data provide evidence that the inflammasome components ASC, NLRP3 and AIM2 play a role in regulating macrophage adhesion and activation in response to surface nanotopography and chemistry. The findings of this paper are important for understanding the inflammatory consequences caused by biomaterials and pave the way to the rational design of future implantable devices having controlled and predictable inflammatory outcomes.

Synthetic materials employed for enhancing, replacing, or restoring biological functionality may be compromised by the host immune responses that they evoke. Surface modification has attracted substantial attention as a tool to modulate the host response to synthetic materials; however, how surface nanotopography combined with chemistry affects immune effector cell responses is still poorly understood. To address this open question, a unique set of model surfaces with controlled surface nanotopography in the range of 16, 38, and 68 nm has been generated. Tailored outermost surface chemistry that was amine, carboxyl, or methyl group rich has been provided. The combinations of these properties yield 12 surface types that are subject to functional assays assessing key immune effector cells, namely, primary neutrophil and macrophage responses in vitro. The data demonstrate that surface nanotopography leads to enhanced matrix metalloproteinase-9 production from primary neutrophils, and a decrease in pro-inflammatory cytokine secretion from primary macrophages. Together, these results are the first to directly compare the immunomodulatory effects of the cooperative interplay between surface nanotopography and chemistry., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Detailing the inflammatory mechanisms of biomaterial-implant induced foreign body responses (FBR) has implications for revealing targetable pathways that may reduce leukocyte activation and fibrotic encapsulation of the implant. We have adapted a model of poly(methylmethacrylate) (PMMA) bead injection to perform an assessment of the mechanistic role of the ASC-dependent inflammasome in this process. We first demonstrate that ASC(-/-) mice subjected to PMMA bead injections had reduced cell infiltration and altered collagen deposition, suggesting a role for the inflammasome in the FBR. We next investigated the NLRP3 and AIM2 sensors because of their known contributions in recognising damaged and apoptotic cells. We found that NLRP3 was dispensable for the fibrotic encapsulation; however AIM2 expression influenced leukocyte infiltration and controlled collagen deposition, suggesting a previously unexplored link between AIM2 and biomaterial-induced FBR.

Investigating immune cell populations within various reproductive tissues commonly utilises flow cytometric methods. With advances in fluorophore technology and equipment capabilities, multiple cell types from a single tissue sample can be identified by using different combinations of cell surface markers to distinguish specific cell populations. Here a protocol optimized for mouse uterine tissue was used to show the proportional changes in dendritic cells, monocyte/macrophages, T and B cells, NK and NK T cells, and the granulocytes, neutrophils and eosinophils at each of the four stages of the estrous cycle. Importantly, we demonstrate that use of anti-SiglecF or assessment of FSC/SSC plots could be used to differentiate monocyte/macrophage and eosinophil populations that otherwise cannot be distinguished by use of the common combination of antibodies against F4/80 and CD11b. Our results clearly indicate that within the uterus a dynamic population of immune cells resides, with many cell types reaching peak abundance at estrus and metestrus phases of the cycle, consistent with their importance in the response to paternal antigens and/or pathogens encountered after insemination., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The purpose of this study was to assess the biofilm-removing efficacy and inflammatory activity of a serrulatane diterpenoid, 8-hydroxyserrulat-14-en-19-oic acid (1), isolated from the Australian medicinal plant Eremophila neglecta. Biofilm breakup activity of compound 1 on established Staphylococcus epidermidis and Staphylococcus aureus biofilms was compared to the antiseptic chlorhexidine and antibiotic levofloxacin. In a time-course study, 1 was deposited onto polypropylene mesh to mimic a wound dressing and tested for biofilm removal. The ex-vivo cytotoxicity and effect on lipopolysaccharide-induced pro-inflammatory cytokine release were studied in mouse primary bone-marrow-derived macrophage (BMDM) cells. Compound 1 was effective in dispersing 12 h pre-established biofilms with a 7 log10 reduction of viable bacterial cell counts, but was less active against 24 h biofilms (approximately 2 log10 reduction). Compound-loaded mesh showed dosage-dependent biofilm-removing capability. In addition, compound 1 displayed a significant inhibitory effect on tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) secretion from BMDM cells, but interleukin-1 beta (IL-1β) secretion was not significant. The compound was not cytotoxic to BMDM cells at concentrations effective in removing biofilm and lowering cytokine release. These findings highlight the potential of this serrulatane diterpenoid to be further developed for applications in wound management.

Medical device associated infections are a persistent medical problem which has not found a comprehensive solution yet. Over the last decades, there have been intense research efforts toward developing antibacterial coatings that could potentially improve medical outcomes. Silver nanoparticles have attracted a great deal of attention as a potent alternative to conventional antibiotics. Herein, we present a biologically inspired approach to synthesize phospholipid encapsulated silver nanoparticles and their surface immobilization to a functional plasma polymer interlayer to generate antibacterial coatings. The antibacterial efficacy of the coatings was evaluated against three medically relevant pathogens including the Gram-positive Staphylococcus aureus and Staphylococcus epidermidis , and the Gram-negative Pseudomonas aeruginosa . The innate immune response to the coatings was assessed in vitro using primary bone marrow derived macrophages (BMDM). Any potential cytotoxicity was studied with primary human dermal fibroblasts (HDFs). Overall, the coatings had excellent inhibition of bacterial growth. We also observed reduced expression of pro-inflammatory cytokines from BMDM which suggests a reduced inflammatory response. The combined properties of coatings developed in this study may make them a good candidate for application on medical devices such as catheters and wound dressings.

Hybrid micro and nanoparticles have become a topic of intense research in recent years. This is due to the special properties of these materials that open new avenues in advanced applications. Herein, we report a novel method for the generation of hybrid particles utilising plasma polymerization. Poly (methyl methacrylate) (PMMA) beads were first coated with a thin allylamine based plasma polymer layer. Gold nanoparticles of engineered size and surface structure were then attached in a controlled manner to the plasma polymer coated beads. To generate uniform chemistry on the outermost surface and to preserve the nanotopography, we deposited a 5-10 nm thin layer of Acpp. We demonstrated that these particles can be utilized in in vivo models to interrogate important biological phenomena. Specifically, we used them in mice to study the inflammatory and foreign body responses to surface nanotopography. The data strongly indicates that surface nanotopography and chemistry can modulate collagen production and the number of adhering immune cells. The method for generating hybrid particles reported here is solvent free and can open new opportunities in fields such as tissue engineering, drug delivery, biosensors, and regenerative medicine., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The tyrosine kinase receptor, EphB4, mediates cross-talk between stromal and hematopoietic populations during bone remodeling, fracture repair and arthritis, through its interactions with the ligand, ephrin-B2. This study demonstrated that transgenic EphB4 mice (EphB4 Tg), over-expressing EphB4 under the control of collagen type-1 promoter, exhibited higher frequencies of osteogenic cells and hematopoietic stem/progenitor cells (HSC), correlating with a higher frequency of long-term culture-initiating cells (LTC-IC), compared with wild type (WT) mice. EphB4 Tg stromal feeder layers displayed a greater capacity to support LTC-IC in vitro, where blocking EphB4/ephrin-B2 interactions decreased LTC-IC output. Similarly, short hairpin RNA-mediated EphB4 knockdown in human bone marrow stromal cells reduced their ability to support high ephrin-B2 expressing CD34(+) HSC in LTC-IC cultures. Notably, irradiated EphB4 Tg mouse recipients displayed enhanced bone marrow reconstitution capacity and enhanced homing efficiency of transplanted donor hematopoietic stem/progenitor cells relative to WT controls. Studies examining the expression of hematopoietic supportive factors produced by stromal cells indicated that CXCL12, Angiopoietin-1, IL-6, FLT-3 ligand, and osteopontin expression were more highly expressed in EphB4 Tg stromal cells compared with WT controls. These findings indicate that EphB4 facilitates stromal-mediated support of hematopoiesis, and constitute a novel component of the HSC niche., (© 2015 AlphaMed Press.)

The role of intracellular calcium ion oscillations in T-cell physiology is being increasingly appreciated by studies that describe how unique temporal and spatial calcium ion signatures can control different signalling pathways. Within this review, we provide detailed mechanisms of calcium ion oscillations, and emphasise the pivotal role that calcium signalling plays in directing crucial events pertaining to T-cell functionality. We also describe methods of calcium ion quantification, and take the opportunity to discuss how a deeper understanding of calcium signalling combined with new detection and quantification methodologies can be used to better design immunotherapies targeting T-cell responses.

Increasing evidence demonstrates induction of proinflammatory Toll-like receptor (TLR) 2 and TLR4 signaling by morphine and, TLR4 signaling by alcohol; thus indicating a common site of drug action and a potential novel innate immune-dependent hypothesis for opioid and alcohol drug interactions. Hence, the current study aimed to assess the role of TLR2, TLR4, MyD88 (as a critical TLR-signaling participant), NF-κB, Interleukin-1β (IL-1β; as a downstream proinflammatory effector molecule) and the μ opioid receptor (MOR; as a classical site for morphine action) in acute alcohol-induced sedation (4.5g/kg) and alcohol (2.5g/kg) interaction with morphine (5mg/kg) by assessing the loss of righting reflex (LORR) as a measure of sedation. Wild-type male Balb/c mice and matched genetically-deficient TLR2, TLR4, and MyD88 strains were utilized, together with pharmacological manipulation of MOR, NF-κB, TLR4 and Interleukin-1β. Alcohol induced significant LORR in wild-type mice; this was halved by MyD88 and TLR4 deficiency, and surprisingly nearly completely eliminated by TLR2 deficiency. In contrast, the interaction between morphine and alcohol was found to be MOR-, NF-κB-, TLR2- and MyD88-dependent, but did not involve TLR4 or Interleukin-1β. Morphine-alcohol interactions caused acute elevations in microglial cell counts and NF-κB-p65 positive cells in the motor cortex in concordance with wild-type and TLR2 deficient mouse behavioral data, implicating neuroimmunopharmacological signaling as a pivotal mechanism in this clinically problematic drug-drug interaction., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The capture and activation of individual T cells on functionalised surfaces enables real-time analyses of the magnitude and rhythm of intracellular calcium release. Application of Haarlet transformations generate a calcium flux 'threshold', with the frequency of the 'threshold crossings' correlating with the strength of the original T cell stimulus. These findings represent a new method to evaluate graduations in T cell activation in real time, and at a single-cell level.

Biomaterial implants are an established part of medical practice, encompassing a broad range of devices that widely differ in function and structural composition. However, one common property amongst biomaterials is the induction of the foreign body response: an acute sterile inflammatory reaction which overlaps with tissue vascularisation and remodelling and ultimately fibrotic encapsulation of the biomaterial to prevent further interaction with host tissue. Severity and clinical manifestation of the biomaterial-induced foreign body response are different for each biomaterial, with cases of incompatibility often associated with loss of function. However, unravelling the mechanisms that progress to the formation of the fibrotic capsule highlights the tightly intertwined nature of immunological responses to a seemingly noncanonical "antigen." In this review, we detail the pathways associated with the foreign body response and describe possible mechanisms of immune involvement that can be targeted. We also discuss methods of modulating the immune response by altering the physiochemical surface properties of the biomaterial prior to implantation. Developments in these areas are reliant on reproducible and effective animal models and may allow a "combined" immunomodulatory approach of adapting surface properties of biomaterials, as well as treating key immune pathways to ultimately reduce the negative consequences of biomaterial implantation.

Vaccinia virus (VACV) has achieved unprecedented success as a live viral vaccine for smallpox which mitigated eradication of the disease. Vaccinia virus has a complex virion morphology and recent advances have been made to answer some of the key outstanding questions, in particular, the origin and biogenesis of the virion membrane, the transformation from immature virion (IV) to mature virus (MV), and the role of several novel genes, which were previously uncharacterized, but have now been shown to be essential for VACV virion formation. This new knowledge will undoubtedly contribute to the rational design of safe, immunogenic vaccine candidates, or effective antivirals in the future. This review endeavors to provide an update on our current knowledge of the VACV maturation processes with a specific focus on the initiation of VACV replication through to the formation of mature virions.

Infections arising from bacterial adhesion and colonization on medical device surfaces are a significant healthcare problem. Silver based antibacterial coatings have attracted a great deal of attention as a potential solution. This paper reports on the development of a silver nanoparticles based antibacterial surface that can be applied to any type of material surface. The silver nanoparticles were surface engineered with a monolayer of 2-mercaptosuccinic acid, which facilitates the immobilization of the nanoparticles to the solid surface, and also reduces the rate of oxidation of the nanoparticles, extending the lifetime of the coatings. The coatings had excellent antibacterial efficacy against three clinically significant pathogenic bacteria i.e. Staphylococcus epidermidis, Staphylococcus aureus and Pseudomonas aeruginosa. Studies with primary human fibroblast cells showed that the coatings had no cytotoxicity in vitro. Innate immune studies in cultures of primary macrophages demonstrated that the coatings do not significantly alter the level of expression of pro-inflammatory cytokines or the adhesion and viability of these cells. Collectively, these coatings have an optimal combination of properties that make them attractive for deposition on medical device surfaces such as wound dressings, catheters and implants., (Copyright © 2014 Elsevier Ltd. All rights reserved.)