MARCH3 negatively regulates IL-3-triggered inflammatory response by mediating K48-linked polyubiquitination and degradation of IL-3Rα.

Interleukin-3 (IL-3) is a hematopoietic growth factor and critical regulator of inflammatory response such as sepsis. IL-3 binds to IL-3 receptor α (IL-3Rα), which is then associated with IL-3Rß to initiate signaling. How IL-3-triggered physiological and pathological effects are regulated at the receptor level is unclear. Here, we show that the plasma membrane-associated E3 ubiquitin ligase MARCH3 negatively regulates IL-3-triggered signaling. MARCH3 is associated with IL-3Rα, mediates its K48-linked polyubiquitination at K377 and promotes its proteasomal degradation. MARCH3-deficiency promotes IL-3-triggered transcription of downstream effector genes and IL-3-induced expansion of myeloid cells. In the cecal ligation and puncture (CLP) model of sepsis, MARCH3-deficiency aggravates IL-3-ampified expression of inflammatory cytokines, organ damage and inflammatory death. Our findings suggest that regulation of IL-3Rα by MARCH3 plays an important role in IL-3-triggered physiological functions and inflammatory diseases.

Autocrine Regulation of Interleukin-3 in the Activity of Regulatory T Cells and its Effectiveness in the Pathophysiology of Sepsis.

Regulatory T cells (Tregs) play a crucial role in modulating the inflammatory response and participated in sepsis-related immune dysfunctions. However, little is known about the regulatory mechanisms by which Tregs are kept in check during immune responses. Here, we verified the simultaneous expression of interleukin-3 (IL-3) and its receptor (IL-3R) in Tregs. Then, by modulation of IL-3 expression via lentiviral transduction-mediated small interfering RNA, we demonstrated that IL-3 negatively regulated Tregs activity via an autocrine mechanism. Furthermore, we found that anti-IL-3 antibody treatment significantly diminished inflammatory cytokines and organ injury, and improved survival in septic mice, which was associated with enhanced Treg percentage and function. Collectively, these results suggest that IL-3 negatively regulates the activity of Tregs in a previously unrecognized autocrine manner, and plays an important role in the excessive inflammatory response in sepsis, which might be utilized as a therapeutic strategy for the treatment of complications in sepsis.

Interleukin 3-induced GITR promotes the activation of human basophils.

Human basophils regulate allergic reactions by secreting histamine, interleukin 4 (IL-4) and IL-13 through key surface receptors FcεRI as well as IL-3R, which are constitutively expressed on basophils. IL-3/IL-3R signaling axis plays key roles in regulating the development and activation of basophils. We and others have shown that IL-3-induced surface receptors e.g. ST2, IL-17RB and IL-2 receptors regulate the biology of basophils. However, the expression and function of IL-3-induced surface proteins on human basophils remain to be elucidated. We in this study aimed to identify new basophil activation regulators by transcriptomic analysis of IL-3-stimulated basophils. Gene expression microarray analysis of IL-3-treated basophils revealed 2050 differentially expressed genes, of which 323 genes encoded surface proteins including GITR. We identified that GITR was preferentially induced by IL-3 rather than anti-IgE, IL-33, fMLP and C5a. IL-3-induced GITR was suppressed by inhibitors targeting JAK2, PI3K and MEK1/2. Stimulation of IL-3-treated basophils by GITR enhanced the expression of IL-4 and IL-13. Moreover, IgE-mediated degranulation was enhanced by GITRL in the presence of IL-3. This transcriptomic analysis of IL-3-activated basophils helps to identify novel activation regulator. IL-3-induced GITR promoted the activation of basophils, adding new evidence supporting GITR as an important player in Th2-associated immune responses.

Establishment of Humanized Mice from Peripheral Blood Mononuclear Cells or Cord Blood CD34+ Hematopoietic Stem Cells for Immune-Oncology Studies Evaluating New Therapeutic Agents.

The clinical success of immune checkpoint modulators and the development of next-generation immune-oncology (IO) agents underscore the need for robust preclinical models to evaluate novel IO therapeutics. Human immune system (HIS) mouse models enable in vivo studies in the context of the HIS via a human tumor. The immunodeficient mouse strains NOG (Prkdcscid Il2rgtm1Sug ) and triple-transgenic NOG-EXL [Prkdcscid Il2rgtm1Sug Tg (SV40/HTLV-IL3, CSF2)], which expresses human IL-3 and GM-CSF, allow for human CD34+ hematopoietic stem cell (huCD34+ HSC) engraftment and multilineage immune cell development by 12 to 16 weeks post-transplant and facilitate studies of immunomodulatory agents. A more rapid model of human immune engraftment utilizes peripheral blood mononuclear cells (PBMCs) transplanted into immunodeficient murine hosts, permitting T-cell engraftment within 2 to 3 weeks without outgrowth of other human immune cells. The PBMC-HIS model can be limited due to onset of xenogeneic graft-versus-host disease (xGVHD) within 3 to 5 weeks post-implantation. Host deficiency in MHC class I, as occurs in beta-2 microglobulin knockout in either NOG or NSG mice, results in resistance to xGVHD, which permits a longer therapeutic window. In this article, detailed processes for generating humanized mice by transplantation of HSCs from cord blood-derived huCD34+ HSCs or PBMCs into immunodeficient mouse strains to respectively generate HSC-HIS and PBMC-HIS mouse models are provided. In addition, the co-engraftment and growth kinetics of patient-derived and cell line-derived xenograft tumors in humanized mice and recovery of tumor-infiltrating lymphocytes from growing tumors to evaluate immune cell subsets by flow cytometry are described. © 2020 The Authors. Basic Protocol 1: Establishment of patient-derived xenograft tumors in CD34+ hematopoietic stem cell-humanized mice Basic Protocol 2: Establishment of patient-derived xenograft tumors in peripheral blood mononuclear cell-humanized mice Support Protocol 1: Flow cytometry assessment of humanization in mice Support Protocol 2: Flow cytometry assessment of tumor-infiltrating lymphocytes in tumor-bearing humanized mouse models.

IL-3 Receptor Expression on Activated Human Th Cells Is Regulated by IL-4, and IL-3 Synergizes with IL-4 to Enhance Th2 Cell Differentiation.

IL-3, a cytokine secreted by activated T lymphocytes, is known to regulate the proliferation, survival, and differentiation of hematopoietic cells. However, the role of IL-3 in regulation of T cell functions is not fully delineated. Previously, we have reported that IL-3 plays an important role in development of regulatory T cells in mice. In this study, we investigated the regulation of IL-3R expression on human Th cells and also examined the role of IL-3 in effector functions of these cells. We found that human peripheral blood Th cells in resting state do not show surface expression of IL-3R; however, its expression was observed at transcript and intracellular protein levels. The functional IL-3R expression on the surface was seen only after antigenic stimulation. When naive Th cells were activated in the presence of various cytokines, we found that IL-4 significantly increases the surface expression of IL-3R and also increases the number of IL-3R+ Th cells. Interestingly, IL-3R+ cells exhibit a Th2 cell-like phenotype and show high GATA-3 expression. Moreover, Th2 cells in presence of IL-3 show increased expression of type 2 effector cytokines, such as IL-4, IL-5, and IL-13. Furthermore, IL-3R expressing and IL-3-secreting Th cells were high in house dust mite-allergic patients. Thus, to our knowledge, we provide the first evidence that the expression of IL-3R on activated human Th cells is modulated by IL-4, and IL-3 regulates the effector functions of Th2 cells. Our results suggest that IL-3 may play an important role in regulating allergic immune responses.

Eosinophil cytolysis on Immunoglobulin G is associated with microtubule formation and suppression of rho-associated protein kinase signalling.

BACKGROUND: The presence of eosinophils in the airway is associated with asthma severity and risk of exacerbations. Cell-free eosinophil granules are found in tissues in eosinophilic diseases, including asthma. This suggests that eosinophils have lysed and released cellular content, likely harming tissues. OBJECTIVE: The present study explores the mechanism of CD32- and αMß2 integrin-dependent eosinophil cytolysis of IL3-primed blood eosinophils seeded on heat-aggregated immunoglobulin G (HA-IgG). METHODS: Cytoskeletal events and signalling pathways potentially involved in cytolysis were assessed using inhibitors. The level of activation of the identified events and pathways involved in cytolysis was measured. In addition, the links between these identified pathways and changes in degranulation (exocytosis) and adhesion were analysed. RESULTS: Cytolysis of IL3-primed eosinophils was dependent on the production of reactive oxygen species (ROS) and downstream phosphorylation of p-38 MAPK. In addition, formation of microtubule (MT) arrays was necessary for cytolysis and was accompanied by changes in MT dynamics as measured by phosphorylation status of stathmin and microtubule-associated protein 4 (MAP4), the latter of which was regulated by ROS production. Reduced ROCK signalling preceded cytolysis, which was associated with eosinophil adhesion and reduced migration. CONCLUSION AND CLINICAL RELEVANCE: In this CD32- and αMß2 integrin-dependent adhesion model, lysing eosinophils exhibit reduced migration and ROCK signalling, as well as both MT dynamic changes and p-38 phosphorylation downstream of ROS production. We propose that interfering with these pathways would modulate eosinophil cytolysis and subsequent eosinophil-driven tissue damage.

Seroepidemiology Study of Helicobacter pylori Infection and Its Effect on Cardiovascular Diseases in Karaj, Iran.

Helicobacter pylori infection occurs throughout the world between countries and races. A seroprevalence study of H. pylori infection in the occurrence of atherosclerosis (ASc) has been considered to be the main cause of cardiovascular diseases (CVDs) in part of Iran. From 600 volunteers, 97 serum samples were selected and tested for the presence of H. pylori infection. Positive and negative H. pylori infection samples were then selected for Chlamydia pneumonia and cytomegalovirus infection. Their serum levels for high-sensitive C-reactive protein (hs-CRP) and IL-6 titer were measured using an ELISA test. Prevalence of H. pylori infection was ∼68.04% in Karaj. Among the 22 samples of cytokine titration, 4 individuals were HP- and 18 were HP+. It was also noticed that 17 samples had normal titration (77.27%) of IL-6 and 3 had a high level of IL-6 (13.63%), 20 people had high hs-CRP titration (90.90%), and 2 were low level hs-CRP (9.1%). Obtained results showed that there is no significant difference between HP+ and HP- infection and IL-6 and hs-CRP titration. Results showed that H. pylori could not lead to ASc by increasing the levels of proinflammatory cytokines such as IL-6 and hs-CRP. Further studies are necessary for the detection of mechanism of H. pylori on ASc.

Human Eosinophils Express a Distinct Gene Expression Program in Response to IL-3 Compared with Common ß-Chain Cytokines IL-5 and GM-CSF.

Despite recent advances in asthma management with anti-IL-5 therapies, many patients have eosinophilic asthma that remains poorly controlled. IL-3 shares a common ß subunit receptor with both IL-5 and GM-CSF but, through α-subunit-specific properties, uniquely influences eosinophil biology and may serve as a potential therapeutic target. We aimed to globally characterize the transcriptomic profiles of GM-CSF, IL-3, and IL-5 stimulation on human circulating eosinophils and identify differences in gene expression using advanced statistical modeling. Human eosinophils were isolated from the peripheral blood of healthy volunteers and stimulated with either GM-CSF, IL-3, or IL-5 for 48 h. RNA was then extracted and bulk sequencing performed. DESeq analysis identified differentially expressed genes and weighted gene coexpression network analysis independently defined modules of genes that are highly coexpressed. GM-CSF, IL-3, and IL-5 commonly upregulated 252 genes and downregulated 553 genes, producing a proinflammatory and survival phenotype that was predominantly mediated through TWEAK signaling. IL-3 stimulation yielded the most numbers of differentially expressed genes that were also highly coexpressed (n = 119). These genes were enriched in pathways involving JAK/STAT signaling. GM-CSF and IL-5 stimulation demonstrated redundancy in eosinophil gene expression. In conclusion, IL-3 produces a distinct eosinophil gene expression program among the ß-chain receptor cytokines. IL-3-upregulated genes may provide a foundation for research into therapeutics for patients with eosinophilic asthma who do not respond to anti-IL-5 therapies.

Anti-CTLA-4 synergizes with dendritic cell-targeted vaccine to promote IL-3-dependent CD4+ effector T cell infiltration into murine pancreatic tumors.

One successful class of cancer immunotherapies, immune checkpoint inhibitory antibodies, disrupts key pathways that regulate immune checkpoints, such as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4). These agents unleash the potency of antigen-experienced T cells that have already been induced as a consequence of the existing tumor. But only 20% of cancers naturally induce T cells. For most cancers, vaccines are require to induce and mobilize T effector cells (Teffs ) to traffick into tumors. We evaluated the effects of anti-CTLA-4 given in combination with an antigen-specific dendritic cell vaccine on intratumoral Teffs in a murine pancreatic cancer model. The dendritic cell-targeted tumor antigen plus anti-CTLA-4 significantly increased the number of vaccine-induced CD4+ Teffs within the tumor. This increase was accompanied by a reduction in the size of the peripheral CD4+ Teff pool. We also found that IL-3 production by activated CD4+ T cells was significantly increased with this combination. Importantly, the CD4+ Teff response was attenuated in Il3-/- mice, suggesting mediation of the effect by IL-3. Finally, the induced T cell infiltration was associated with activation of the tumor endothelium by T cell-derived IL-3. Our findings collectively provide a new insight into the mechanism driving Teff infiltration and vascular activation in a murine pancreatic cancer model, specifically identifying a new role for IL-3 in the anticancer immune response.

Stepping out of the shadow: STIM2 promotes IL-3-induced cytokine release.

Basophils are a small population of innate immune cells, but their release of the cytokine interleukin-4 (IL-4) is important for mounting an efficient immune response against distinct parasites. Yoshikawa et al (in the 9 April 2019 issue) showed that whereas STIM1 is essential for IL-4 release after stimulation of FcεRI, STIM2 mediates a delayed IL-3/IL-33-induced IL-4 release independent of STIM1.

Pivotal role of STIM2, but not STIM1, in IL-4 production by IL-3-stimulated murine basophils.

Basophils have nonredundant roles in various immune responses that require Ca2+ influx. Here, we examined the role of two Ca2+ sensors, stromal interaction molecule 1 and 2 (STIM1 and STIM2), in basophil activation. We found that loss of STIM1, but not STIM2, impaired basophil IL-4 production after stimulation with immunoglobulin E (IgE)-containing immune complexes. In contrast, when basophils were stimulated with IL-3, loss of STIM2, but not STIM1, reduced basophil IL-4 production. This difference in STIM proteins was associated with distinct time courses of Ca2+ influx and transcription of the Il4 gene that were elicited by each stimulus. Similarly, basophil-specific STIM1 expression was required for IgE-driven chronic allergic inflammation in vivo, whereas STIM2 was required for IL-4 production after combined IL-3 and IL-33 treatment in mice. These data indicate that STIM1 and STIM2 have differential roles in the production of IL-4, which are stimulus dependent. Furthermore, these results illustrate the vital role of STIM2 in basophils, which is often considered to be less important than STIM1.

GM-CSF, IL-3, and IL-5 Family of Cytokines: Regulators of Inflammation.

The ß common chain cytokines GM-CSF, IL-3, and IL-5 regulate varied inflammatory responses that promote the rapid clearance of pathogens but also contribute to pathology in chronic inflammation. Therapeutic interventions manipulating these cytokines are approved for use in some cancers as well as allergic and autoimmune disease, and others show promising early clinical activity. These approaches are based on our understanding of the inflammatory roles of these cytokines; however, GM-CSF also participates in the resolution of inflammation, and IL-3 and IL-5 may also have such properties. Here, we review the functions of the ß common cytokines in health and disease. We discuss preclinical and clinical data, highlighting the potential inherent in targeting these cytokine pathways, the limitations, and the important gaps in understanding of the basic biology of this cytokine family.

CD8+ T cells expand stem and progenitor cells in favorable but not adverse risk acute myeloid leukemia.

CD8+ T cell immunosurveillance is crucial in solid tumors and T cell dysfunction leads to tumor progression. In contrast, the role of CD8+ T cells in the control of leukemia is less clear. We characterized the molecular signature of leukemia stem/progenitor cells (LSPCs) and paired CD8+ T cells in patients with acute myeloid leukemia (AML). Epigenetic alterations via histone deacetylation reduced the expression of immune-related genes in bone marrow (BM)-infiltrating CD8+ T cells. Surprisingly, a silenced gene expression pattern in CD8+ T cells significantly correlated with an improved prognosis. To define interactions between CD8+ T cells and LSPCs, we performed comprehensive correlative network modeling. This analysis indicated that CD8+ T cells contribute to the maintenance/expansion of LSPCs, particularly in favorable risk AML. Functionally, CD8+ T cells in favorable AML induced the expansion of LSPCs by stimulating the autocrine production of important hematopoietic cytokines such as interleukin (IL)-3. In contrast, LSPCs in aggressive AML were characterized by a higher activation of stemness/proliferation-related pathways and develop independent of BM CD8+ T cells. Overall, our study indicates that CD8+ T cells support and expand LSPCs in favorable risk AML whereas intermediate and adverse risk AML possess the intrinsic molecular abnormalities to develop independently.

Self-reactive CD4+ IL-3+ T cells amplify autoimmune inflammation in myocarditis by inciting monocyte chemotaxis.

Acquisition of self-reactive effector CD4+ T cells is a major component of the autoimmune response that can occur during myocarditis, an inflammatory form of cardiomyopathy. Although the processes by which self-reactive T cells gain effector function have received considerable attention, how these T cells contribute to effector organ inflammation and damage is less clear. Here, we identified an IL-3-dependent amplification loop that exacerbates autoimmune inflammation. In experimental myocarditis, we show that effector organ-accumulating autoreactive IL-3+ CD4+ T cells stimulate IL-3R+ tissue macrophages to produce monocyte-attracting chemokines. The newly recruited monocytes differentiate into antigen-presenting cells that stimulate local IL-3+ CD4+ T cell proliferation, thereby amplifying organ inflammation. Consequently, Il3 -/- mice resist developing robust autoimmune inflammation and myocardial dysfunction, whereas therapeutic IL-3 targeting ameliorates disease. This study defines a mechanism that orchestrates inflammation in myocarditis, describes a previously unknown function for IL-3, and identifies IL-3 as a potential therapeutic target in patients with myocarditis.

Expression of IL-3 receptors and impact of IL-3 on human T and B cells.

A large number of animal models revealed that IL-3 plays an important role for the development of T and B cell-mediated autoimmune diseases. However, little is known about the expression and regulation of IL-3 receptors in human T and B cells and how IL-3 modulates the activation and survival of these cells. We show that the IL-3 receptor CD123 is substantially upregulated on proliferating CD4+ and CD8+ T as well as B cells. Upregulation of CD123 differs between various activators and can be further modulated by cytokines. Exposure of human T and B cells to IL-3 enhances proliferation and survival. IL-3 also induces a shift towards secretion of proinflammatory cytokines in T and B cells and reduces the expression of IL-10 in B cells. Thus IL-3 may have proinflammatory and immunostimulatory properties also in human autoimmune diseases.

Control of cytokine-driven eosinophil migratory behavior by TGF-beta-induced protein (TGFBI) and periostin.

Periostin, which is induced by interleukin (IL)-13, is an extracellular matrix (ECM) protein that supports αMß2 integrin-mediated adhesion and migration of IL-5-stimulated eosinophils. Transforming growth factor (TGF)-ß-induced protein (TGFBI) is a widely expressed periostin paralog known to support monocyte adhesion. Our objective was to compare eosinophil adhesion and migration on TGFBI and periostin in the presence of IL-5-family cytokines. Eosinophil adhesion after 1 h and random motility over 20 h in the presence of various concentrations of IL-5, IL-3, or granulocyte macrophage-colony stimulating factor (GM-CSF) were quantified in wells coated with various concentrations of TGFBI or periostin. Results were compared to video microscopy of eosinophils. Cytokine-stimulated eosinophils adhered equivalently well to TGFBI or periostin in a coating concentration-dependent manner. Adhesion was blocked by anti-αMß2 and stimulated at the lowest concentration by GM-CSF. In the motility assay, periostin was more potent than TGFBI, the coating-concentration effect was bimodal, and IL-3 was the most potent cytokine. Video microscopy revealed that under the optimal coating condition of 5 µg/ml periostin, most eosinophils migrated persistently and were polarized and acorn-shaped with a ruffling forward edge and granules gathered together, in front of the nucleus. On 10 µg/ml periostin or TGFBI, more eosinophils adopted a flattened pancake morphology with dispersed granules and nuclear lobes, and slower migration. Conversion between acorn and pancake morphologies were observed. We conclude that TGFBI or periostin supports two modes of migration by IL-5 family cytokine-activated eosinophils. The rapid mode is favored by intermediate protein coatings and the slower by higher coating concentrations. We speculate that eosinophils move by haptotaxis up a gradient of adhesive ECM protein and then slow down to surveil the tissue.

IL-3-producing basophils are required to exacerbate airway hyperresponsiveness in a murine inflammatory model.

BACKGROUND: Basophils are commonly associated with allergic responses because of their ability to produce large amounts of pro-Th2 cytokines and histamine. However, the mechanisms through which bone marrow-resident basophils (BMRB) become fully competent cytokine and histamine producers in response to IgE crosslinking are poorly understood. Here, we sought to determine the role of IL-3 in promoting pro-Th2 basophils. METHODS: BMRB and basophils exposed to IL-3 in vitro and in vivo were evaluated for their production of Th2 cytokines and histamine in response to FcεRI crosslinking on both protein and gene expression levels. In vivo relevance of our findings was assessed in a model of ovalbumin-induced allergic asthma using IL-3-deficient and wild-type mice in a protocol of adoptive basophil transfer. RESULTS: We show that BMRB and basophils previously exposed to IL-3 differ in their ability to generate cytokines (IL-4, IL-6, IL-13, and GM-CSF) and histamine in response to FcεRI crosslinking, reflecting two stages of maturation. Exposure to IL-3 initiated an autocrine loop of endogenous IL-3 production that enhanced histamine and cytokine production upon FcεRI crosslinking. This increased responsiveness required calcium flux and was dependent on calcineurin and store-operated calcium channels. Our findings are of pathophysiological relevance, as assessed by the failure of IL-3-deficient mice to develop airway hyperreactivity, which could be restored by adoptive transfer of IL-3-derived basophils recovered from wild-type mice. CONCLUSION: IL-3-dependent basophils promote Th2 allergic AHR, which designates the IL-3/basophil axis as a promising therapeutic target for the treatment of basophil-dependent asthma.

IL-3 but not monomeric IgE regulates FcεRI levels and cell survival in primary human basophils.

Binding of allergen-specific IgE to its primary receptor FcεRI on basophils and mast cells represents a central event in the development of allergic diseases. The high-affinity interaction between IgE and FcεRI results in permanent sensitization of these allergic effector cells and critically regulates their release of pro-inflammatory mediators upon IgE cross-linking by allergens. In addition, binding of monomeric IgE has been reported to actively regulate FcεRI surface levels and promote survival of mast cells in the absence of allergen through the induction of autocrine cytokine secretion including interleukin-3 (IL-3). As basophils and mast cells share many biological commonalities we sought to assess the role of monomeric IgE binding and IL-3 signaling in FcεRI regulation and cell survival of primary human basophils. FcεRI cell surface levels and survival of isolated blood basophils were assessed upon addition of monomeric IgE or physiologic removal of endogenous cell-bound IgE with a disruptive IgE inhibitor by flow cytometry. We further determined basophil cell numbers in both low and high serum IgE blood donors and mice that are either sufficient or deficient for FcεRI. Ultimately, we investigated the effect of IL-3 on basophil surface FcεRI levels by protein and gene expression analysis. Surface levels of FcεRI were passively stabilized but not actively upregulated in the presence of monomeric IgE. In contrast to previous observations with mast cells, monomeric IgE binding did not enhance basophil survival. Interestingly, we found that IL-3 transcriptionally regulates surface levels of FcεRI in human primary basophils. Our data suggest that IL-3 but not monomeric IgE regulates FcεRI expression and cell survival in primary human basophils. Thus, blocking of IL-3 signaling in allergic effector cells might represent an interesting approach to diminish surface FcεRI levels and to prevent prolonged cell survival in allergic inflammation.

ScFv-modified graphene-coated IDE-arrays for 'label-free' screening of cardiovascular disease biomarkers in physiological saline.

Fatty-acid binding proteins (FABP) and myeloperoxidases (MPO) are associated with many chronic conditions in humans and considered to be important biomarkers for diagnosis of cardiac diseases. Here we assemble a new electrical biosensor platform based on graphene-coated interdigitated electrode arrays (IDE-arrays) towards ultrafast, label-free screening of heart type-FABP and MPO. Arrays of nanoscale (nanoIDE) and microscale (microIDE) electrode-arrays were fabricated on wafer-scale by combining nanoimprint and photolithography processes. Chemical vapor deposition grown multilayer graphene was transferred onto nano/microIDE-arrays and used as a high surface-to-volume ratio electrical transducer. Novel biofunctional layers of specially engineered anti-h-FABP and anti-MPO single-chain fragment variables (scFv) were immobilized onto graphene-coated IDE-array sensor platform for electrical detection of h-FABP and MPO in physiological saline. scFv fragments show increased sensitivity in comparison to the state-of-the-art competitive ELISA for their higher affinity towards target analytes. Deploying FABP and MPO specific scFvs as receptor molecules onto our high-sensitivity graphene-coated IDE-arrays with identical sensor characteristics and assays covering clinically relevant concentrations in physiological saline, we demonstrate realization of a simple and versatile biosensor platform capable of high performance cardiac-bioassays for point-of-care applications.

Characterisation of the porcine cytokines which activate the CD131ßc common sub-unit, for potential immune-augmentation.

Early acting cytokines and growth factors such as those of the CD131 ßc subunit, may offer an alternative method to the current use of antibiotics and chemicals such as anthelmintics in maintaining Porcine (Po) health. Thus far, the recombinant Po (rPo) Granulocyte-macrophage colony-stimulating factor (GM-CSF), rPo interleukin-3 (IL-3) and rPo interleukin-5 (IL-5) proteins have been identified and cloned and the biological activity of each cytokine has been confirmed in vitro, however, in vivo immune system regulation and hematopoietic stem cell (HSC) augmentation are regulated by numerous cytokines and cellular signals within the bone marrow (BM) niche. In order to quantify the use of recombinant cytokines in augmenting the immune response, it is necessary to determine the stages of hematopoiesis induced by each cytokine and possible areas of synergy requiring further investigation. Here we used the chemotherapeutic agent 5-fluorouracil (5-FU), to chemically induce a state of myelosuppression in young pigs. This allowed for the monitoring of both the autologous BM reconstitution and recombinant cytokine induced BM repopulation, precursor cell proliferation and cellular differentiation. The recombinant cytokines PoGM-CSF, PoIL-3 and PoIL-5 were administered by intramuscular injections (i.m.) following confirmation of 5-FU induced leukocytopenia. Blood and BM samples were collected and then analysed for cell composition. Statistically significant results were observed in several blood cell populations including eosinophils for animals treated with rPoIL-5, rPoGM-CSF and basophils for animals treated with rPoIL-3. BM analysis of CD90+ and CD172a+ cells confirmed myelosuppression in week one with significant results observed between rPoIL-3 and the 5-FU control group in week two and for the rPoGM-CSF group in week three. These results have demonstrated the effects of each of these rPo cytokines within the hematopoietic processes of the pig and may demonstrate similar outcomes in other mammalian models including human.