MCYTOMAG-70K MilliporeMILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel - Immunology Multiplex Assay

Autism spectrum disorder (ASD) is a cluster of neurodevelopmental disorders characterized by impairments in communication, social interest and stereotypical behaviour. Dysfunction of the intestinal tract is reported in patients with ASD and implicated in the development and severity of ASD symptoms. However, more research is required to investigate the association of intestinal problems with ASD and the potential underlying mechanisms. The purpose of this study was to investigate comorbid symptoms of intestinal inflammation in a murine model of ASD induced by prenatal exposure to valproic acid (VPA). Pregnant BALB/c females were treated subcutaneously with 600 mg/kg VPA or phosphate buffered saline on gestational day 11. Offspring were housed with their mother until weaning on postnatal day 21 (P21). All pups were exposed to a social behaviour test on P28. Inflammatory correlates and activity of the serotonergic system were measured in brain and intestinal tissue. Here we demonstrate, in addition to reduced social behaviour and increased expression of neuroinflammatory markers in the brain, that VPA in utero- exposed male offspring showed epithelial cell loss and neutrophil infiltration in the intestinal tract. Furthermore, reduced levels of serotonin were not only observed the prefrontal cortex and amygdala of VPA in utero- exposed males, but also in the small intestine. Overall, we demonstrate that gender-specific inflammatory conditions are present in the small intestines of VPA in utero- exposed mice and are accompanied by a disturbed serotonergic system in the brain as well as in the intestinal tract.

Combinatorial use of iron oxide nanoparticles (IONPs) and an alternating magnetic field (AMF) can induce local hyperthermia in tumors in a controlled and uniform manner. Heating B16 primary tumors at 43°C for 30min activated dendritic cells (DCs) and subsequently CD8(+) T cells in the draining lymph node (dLN) and conferred resistance against rechallenge with B16 (but not unrelated Lewis Lung carcinoma) given 7days post hyperthermia on both the primary tumor side and the contralateral side in a CD8(+) T cell-dependent manner. Mice with heated primary tumors also resisted rechallenge given 30days post hyperthermia. Mice with larger heated primary tumors had greater resistance to secondary tumors. No rechallenge resistance occurred when tumors were heated at 45°C. Our results demonstrate the promising potential of local hyperthermia treatment applied to identified tumors in inducing anti-tumor immune responses that reduce the risk of recurrence and metastasis.Local heating of tumors via iron oxide NPs and an alternating magnetic field led to activation of anti-cancer CD8 T cells, which resulted in resistance against re-challenge and greater resistance to secondary tumors. Similar local heating-based strategies may become an important weapon in enhancing tumor elimination via a naturally existing but attenuated immune response.

Dendritic cells (DCs) are immune cells found in the peripheral tissues where they sample the organism for infections or malignancies. There they take up antigens and migrate towards immunological organs to contact and activate T lymphocytes that specifically recognize the antigen presented by these antigen presenting cells. In the steady state there are several types of resident DCs present in various different organs. For example, in the mouse, splenic DC populations characterized by the co-expression of CD11c and CD8 surface markers are specialized in cross-presentation to CD8 T cells, while CD11c/SIRP-1α DCs seem to be dedicated to activating CD4 T cells. On the other hand, DCs have also been associated with the development of various diseases such as cancer, atherosclerosis, or inflammatory conditions. In such disease, DCs can participate by inducing angiogenesis or immunosuppression (tumors), promoting autoimmune responses, or exacerbating inflammation (atherosclerosis). This change in DC biology can be prompted by signals in the microenvironment. We have previously shown that the interaction of DCs with various extracellular matrix components modifies the immune properties and angiogenic potential of these cells. Building on those studies, herewith we analyzed the angiogenic profile of murine myeloid DCs upon interaction with 2D and 3D type-I collagen environments. As determined by PCR array technology and quantitative PCR analysis we observed that interaction with these collagen environments induced the expression of particular angiogenic molecules. In addition, DCs cultured on collagen environments specifically upregulated the expression of CXCL-1 and -2 chemokines. We were also able to establish DC cultures on type-IV collagen environments, a collagen type expressed in pathological conditions such as atherosclerosis. When we examined DC populations in atherosclerotic veins of Apolipoprotein E deficient mice we observed that they expressed adhesion molecules capable of interacting with collagen. Finally, to further investigate the interaction of DCs with collagen in other pathological conditions, we determined that both murine ovarian and breast cancer cells express several collagen molecules that can contribute to shape their particular tumor microenvironment. Consistently, tumor-associated DCs were shown to express adhesion molecules capable of interacting with collagen molecules as determined by flow cytometry analysis. Of particular relevance, tumor-associated DCs expressed high levels of CD305/LAIR-1, an immunosuppressive receptor. This suggests that signaling through this molecule upon interaction with collagen produced by tumor cells might help define the poorly immunogenic status of these cells in the tumor microenvironment. Overall, these studies demonstrate that through interaction with collagen proteins, DCs can be capable of modifying the microenvironments of inflammatory disease such as cancer or atherosclerosis.

To determine whether cellular miRNAs play a role in West Nile virus (WNV) neuropathogenesis, we evaluated WNV-infected mice brain for the expression profile of miRNAs, their potential functions and their correlation with genes involved in inflammatory pathways. A total of 528 miRNAs and 168 mRNA genes were examined. One hundred thirty-nine miRNAs were significantly differentially expressed in WNV-infected mice brain. Ingenuity pathway analysis demonstrated that these miRNAs and their target genes are involved in pathways related to inflammatory response, immune-cell trafficking and cell death. Moreover, we demonstrate an inverse correlation between WNV-modulated miRNAs and their target neuroinflammatory genes in the same mice brain. We demonstrate that miR-196a, miR-202-3p, miR-449c, and miR-125a-3p target multiple genes involving cytokines, chemokines, and apoptotic genes, which belong to different signaling pathways that play critical role in WNV neuropathogenesis. Functional studies targeting specific miRNA are warranted to develop therapeutics for the management of WNV disease.

Although peripheral low-grade inflammation has been associated with a high incidence of mood symptoms in patients with metabolic syndrome (MetS), much less is known about the potential involvement of brain activation of cytokines in that context. Recently we showed in a mouse model of MetS, namely the db/db mice, an enhanced hippocampal inflammation associated with increased anxiety-like behavior (Dinel et al., 2011). However, depressive-like behavior was not affected in db/db mice. Based on the strong association between depressive-like behavior and cytokine-induced brain activation of indoleamine 2,3-dioxygenase (IDO), the enzyme that metabolizes tryptophan along the kynurenine pathway, these results may suggest an impairment of brain IDO activation in db/db mice. To test this hypothesis, we measured the ability of db/db mice and their healthy db/+ littermates to enhance brain IDO activity and depressive-like behavior after a systemic immune challenge with lipopolysaccharide (LPS). Here we show that LPS (5 μg/mouse) significantly increased depressive-like behavior (increased immobility time in a forced-swim test, FST) 24h after treatment in db/+ mice, but not in db/db mice. Interestingly, db/db mice also displayed after LPS treatment blunted increase of brain kynurenine/tryptophan ratio compared to their db/+ counterparts, despite enhanced induction of hippocampal cytokine expression (interleukin-1β, tumor necrosis factor-α). Moreover, this was associated with an impaired effect of LPS on hippocampal expression of the brain-derived neurotrophic factor (BDNF) that contributes to mood regulation, including under inflammatory conditions. Collectively, these data indicate that the rise in brain tryptophan catabolism and depressive-like behavior induced by innate immune system activation is impaired in db/db mice. These findings could have relevance in improving the management and treatment of inflammation-related complications in MetS.

This study explored the novel use of iron oxide (IO) nanoparticles (<20 nm) as a vaccine delivery platform without additional adjuvants. A recombinant malaria vaccine antigen, the merozoite surface protein 1 (rMSP1), was conjugated to IO nanoparticles (rMSP1-IO). Immunizations in outbred mice with rMSP1-IO achieved 100% responsiveness with antibody titers comparable to those obtained with rMSP1 formulated with a clinically acceptable adjuvant, Montanide ISA51 (2.7×10 vs. 1.6×10; respectively). Only rMSP1-1O could induce significant levels (80%) of parasite inhibitory antibodies. The rMSP1-IO was highly stable at 4°C and was amenable to lyophilization, maintaining its antigenicity, immunogenicity, and ability to induce inhibitory antibodies. Further testing in nonhuman primates, Aotus monkeys, also elicited 100% immune responsiveness and high levels of parasite inhibitory antibodies (55-100% inhibition). No apparent local or systemic toxicity was associated with IO immunizations. Murine macrophages and dendritic cells efficiently (>90%) internalized IO nanoparticles, but only the latter were significantly activated, with elevated expression/secretion of CD86, cytokines (IL-6, TNF-α, IL1-b, IFN-γ, and IL-12), and chemokines (CXCL1, CXCL2, CCL2, CCL3, CCL4, and CXCL10). Thus, the IO nanoparticles is a novel, safe, and effective vaccine platform, with built-in adjuvancy, that is highly stable and field deployable for cost-effective vaccine delivery.

Decompressive craniectomy is often required after head trauma, stroke, or cranial bleeding to control subsequent brain swelling and prevent death. The infection rate after cranial bone flap replacement ranges from 0.8% to 15%, with an alarming frequency caused by methicillin-resistant Staphylococcus aureus, which is problematic because of recalcitrance to antibiotic therapy. Herein we report the establishment of a novel mouse model of S. aureus cranial bone flap infection that mimics several aspects of human disease. Bacteria colonized bone flaps for up to 4 months after infection, as revealed by scanning electron microscopy and quantitative culture, demonstrating the chronicity of the model. Analysis of a human cranial bone flap with confirmed S. aureus infection by scanning electron microscopy revealed similar structural attributes as the mouse model, demonstrating that it closely parallels structural facets of human disease. Inflammatory indices were most pronounced within the subcutaneous galeal compartment compared with the underlying brain parenchyma. Specifically, neutrophil influx and chemokine expression (CXCL2 and CCL5) were markedly elevated in the galea, which demonstrated substantial edema on magnetic resonance images, whereas the underlying brain parenchyma exhibited minimal involvement. Evaluation of immune mechanisms required for bacterial containment and inflammation revealed critical roles for MyD88-dependent signaling and neutrophils. This novel mouse model of cranial bone flap infection can be used to identify key immunologic and therapeutic mechanisms relevant to persistent bone flap infection in humans.

By inhibiting target gene expression, microRNAs (miRNAs) play major roles in various physiological and pathological processes. miR-146a, a miRNA induced upon lipopolysaccharide (LPS) stimulation and virus infection, is also highly expressed in patients with immune disorders such as rheumatoid arthritis, Sjögren's syndrome, and psoriasis. Whether the high level of miR-146a contributes to any of these pathogenesis-related processes remains unknown. To elucidate the function of miR-146a in vivo, we generated a transgenic (TG) mouse line overexpressing miR-146a. Starting at an early age, these TG mice developed spontaneous immune disorders that mimicked human autoimmune lymphoproliferative syndrome (ALPS) with distinct manifestations, including enlarged spleens and lymph nodes, inflammatory infiltration in the livers and lungs, increased levels of double-negative T cells in peripheral blood, and increased serum immunoglobulin G levels. Moreover, with the adoptive transfer approach, we found that the B-cell population was the major etiological factor and that the expression of Fas, a direct target of miR-146a, was significantly dampened in TG germinal center B cells. These results indicate that miR-146a may be involved in the pathogenesis of ALPS by targeting Fas and may therefore serve as a novel therapeutic target.

Sulfur mustard (SM) is a highly reactive vesicating agent that can induce severe ocular injury. The clinical features of this injury have been well documented, but the molecular basis for this pathology is not well understood. Identification and validation of specific targets is necessary in the effort to develop effective therapeutics for this injury. Currently used rabbit models are not well suited for many molecular studies because the necessary reagents are not widely available. However, these reagents are widely available for the mouse model.Our objective is to develop a mouse model of SM-induced ocular injury suitable for the study of the molecular mechanisms of injury and the evaluation of therapeutics.Ocular exposure to sulfur mustard vapor was accomplished by using a vapor cup method. Dose response studies were conducted in female BALB/c mice. An exposure dose which produced moderate injury was selected for further study as moderate injury was determined to be amenable to studying the beneficial effects of potential therapeutics. Histopathology and inflammatory markers were evaluated for up to 28 days after exposure, while clinical injury progression was evaluated for 1 year post-exposure.A biphasic ocular injury was observed in mice exposed to SM. Acute phase SM ocular injury in mice was characterized by significant corneal epithelium loss, corneal edema, limbal engorgement, and ocular inflammation. This was followed by a brief recovery phase. A delayed injury phase then ensued in the following weeks to months and was characterized by keratitis, stromal edema, infiltrates, neovascularization, and eventual corneal scarring.SM-induced ocular injury in mice is consistent with observations of SM-induced ocular injury in humans and rabbit models. However, in the mouse model, the SM ocular injury, a more rapid onset of the delayed injury phase was observed. We have developed an animal model of SM injury that is suitable for studies to elucidate molecular mechanisms of injury and identify potential therapeutic targets.