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Aberrant innate immune response drives the pathophysiology of many diseases. Myeloperoxidase (MPO) is a highly oxidative enzyme secreted by activated myeloid pro-inflammatory immune cells such as neutrophils and macrophages, and is a key mediator of the damaging innate immune response. Current technologies for detecting MPO activity in living organisms are sparse and suffer from any combination of low specificity, low tissue penetration, or low spatial resolution. We describe a versatile imaging platform to detect MPO activity using an activatable construct conjugated to a biotin moiety (MPO-activatable biotinylated sensor, MABS) that allows monitoring the innate immune response and its modulation at different scales and settings. Methods: We designed and synthesized MABS that contains MPO-specific and biotin moieties, and validated its specificity and sensitivity combining with streptavidin-labeled fluorescent agent and gold nanoparticles imaging in vitro and in vivo in multiple mouse models of inflammation and infection, including Matrigel implant, dermatitis, cellulitis, cerebritis and complete Fraud's adjuvant (CFA)-induced inflammation. Results: MABS MPO imaging non-invasively detected varying MPO concentrations, MPO inhibition, and MPO deficiency in vivo with high sensitivity and specificity. MABS can be used to obtain not only a fluorescence imaging agent, but also a CT imaging agent, conferring molecular activity information to a structural imaging modality. Importantly, using this method on tissue-sections, we found that MPO enzymatic activity does not always co-localize with MPO protein detected with conventional techniques (e.g., immunohistochemistry), underscoring the importance of monitoring enzymatic activity. Conclusion: By choosing from different available secondary probes, MABS can be used to create systems suitable to investigate and image MPO activity at different scales and settings.

Myeloperoxidase (MPO) is a critical proinflammatory enzyme implicated in cardiovascular, neurological, and rheumatological diseases. Emerging therapies targeting inflammation have raised interest in tracking MPO activity in patients. We describe 18 F-MAPP, an activatable MPO activity radioprobe for positron emission tomography (PET) imaging. The activated radioprobe binds to proteins and accumulates at sites of MPO activity. The radioprobe 18 F-MAPP has a short blood half-life, remains stable in plasma, does not demonstrate cytotoxicity, and crosses the intact blood–brain barrier. The 18 F-MAPP imaging detected sites of elevated MPO activity in living mice embedded with human MPO and in mice induced with chemical inflammation or myocardial infarction. The 18 F-MAPP PET imaging noninvasively differentiated varying amounts of MPO activity, competitive inhibition, and MPO deficiency in living animals, confirming specificity and showing that the radioprobe can quantify changes in in vivo MPO activity. The radiosynthesis has been optimized and automated, an important step in translation. These data indicate that 18 F-MAPP is a promising translational candidate to noninvasively monitor MPO activity and inflammation in patients.

Innate immune cells play a key role in the pathogenesis of multiple sclerosis and experimental autoimmune encephalomyelitis (EAE). Current clinical imaging is restricted to visualizing secondary effects of inflammation, such as gliosis and blood-brain barrier disruption. Advanced molecular imaging, such as iron oxide nanoparticle imaging, can allow direct imaging of cellular and molecular activity, but the exact cell types that phagocytose nanoparticles in vivo and how phagocytic activity relates to disease severity is not well understood. In this study we used MRI to map inflammatory infiltrates using high-field MRI and fluorescently labeled cross-linked iron oxide nanoparticles for cell tracking. We confirmed nanoparticle uptake and MR detectability ex vivo. Using in vivo MRI, we identified extensive nanoparticle signal in the cerebellar white matter and circumscribed cortical gray matter lesions that developed during the disease course (4.6-fold increase of nanoparticle accumulation in EAE compared with healthy controls, P < 0.001). Nanoparticles showed good cellular specificity for innate immune cells in vivo, labeling activated microglia, infiltrating macrophages, and neutrophils, whereas there was only sparse uptake by adaptive immune cells. Importantly, nanoparticle signal correlated better with clinical disease than conventional gadolinium (Gd) imaging (r, 0.83 for nanoparticles vs. 0.71 for Gd-imaging, P < 0.001). We validated our approach using the Food and Drug Administration-approved iron oxide nanoparticle ferumoxytol. Our results show that noninvasive molecular imaging of innate immune responses can serve as an imaging biomarker of disease activity in autoimmune-mediated neuroinflammation with potential clinical applications in a wide range of inflammatory diseases.

Currently, there is no stable and flexible method to label and track cytotoxic T-lymphocytes (CTLs) in vivo in CTL immunotherapy. We aimed to evaluate whether the sulfo-hydroxysuccinimide (NHS)-biotin-streptavidin (SA) platform could chemically modify the cell surface of CTLs for in vivo tracking. CD8+ T lymphocytes were labeled with sulfo-NHS-biotin under different conditions and then incubated with SA-Alexa647. Labeling efficiency was proportional to sulfo-NHS-biotin concentration. CD8+ T lymphocytes could be labeled with higher efficiency with sulfo-NHS-biotin in DPBS than in RPMI (P

Purpose To test whether MPO-Gd, an activatable molecular magnetic resonance (MR) imaging agent specific for myeloperoxidase (MPO) activity, could detect MPO activity in nonalcoholic steatohepatitis (NASH) mouse models and human liver biopsy samples. Materials and Methods In this study, 20 leptin receptor-deficient and three MPO knockout mice were injected with endotoxin (lipopolysaccharide) or fed a methionine and choline-deficient (MCD) diet to induce experimental NASH and underwent MR imaging with MPO-Gd. Saline-injected and control diet-fed leptin receptor-deficient mice were used as respective controls. MPO protein and activity measurements and histologic analyses were performed. Eleven human liver biopsy samples underwent MPO-Gd-enhanced MR imaging ex vivo and subsequent histologic evaluation. Results were compared with Student t test or Mann-Whitney U test. Results With endotoxin, a significantly increased contrast-to-noise ratio (CNR) was found compared with sham (mean CNR, 1.81 [95% confidence interval {CI}: 1.53, 2.10] vs 1.02 [95% CI: 0.89, 1.14]; P = .03) at MPO-Gd MR imaging. In the diet-induced NASH model, an increased CNR was also found compared with sham mice (mean CNR, 1.33 [95% CI: 1.27, 1.40] vs 0.98 [95% CI: 0.83, 1.12]; P = .008). Conversely, CNR remained at baseline in NASH mice imaged with gadopentetate dimeglumine and in MPO knockout NASH mice with MPO-Gd, which proves specificity of MPO-Gd. Ex vivo molecular MR imaging of liver biopsy samples from NASH and control patients confirmed results from animal studies (mean CNR for NASH vs control patients, 2.61 [95% CI: 1.48, 3.74] vs 1.29 [95% CI: 1.06, 1.52]; P = .004). Conclusion MPO-Gd showed elevated MPO activity in NAFLD mouse models and human liver biopsy samples. © RSNA, 2017 Online supplemental material is available for this article. An earlier incorrect version of this article appeared online. This article was corrected on April 6, 2017.

ObjectiveTo develop an endogenous rodent model of postinfectious anti-NMDA receptor (NMDAR) encephalitis.MethodsSix mice were inoculated intranasally with herpes simplex virus (HSV) 1 and subsequently treated with acyclovir for 2 weeks. Serum was collected at 3, 6, and 8 weeks postinoculation and tested for NMDAR antibodies through a cell-based assay. Eight weeks postinoculation, mice were killed and their brains were sectioned and immunostained with antibodies to postsynaptic density (PSD)-95 and NMDARs. Colocalization of hippocampal PSD-95 and NMDAR clusters, representing postsynaptic membrane NMDARs, was quantified via confocal imaging. Hippocampi were additionally analyzed for NMDAR and PSD-95 protein using Western blot analysis.ResultsFour of 6 mice (67%) developed serum antibodies to NMDARs: 1 at 3 weeks, 1 at 6 weeks, and 2 at 8 weeks postinoculation. As compared to inoculated mice that did not develop NMDAR antibodies, immunofluorescence staining revealed decreased hippocampal postsynaptic membrane NMDARs in mice with serum antibodies at 8 weeks postinoculation. Western blot analysis showed that mice that had NMDAR antibodies at 8 weeks had decreased total NMDAR but not PSD-95 protein in hippocampal extracts (p < 0.05).ConclusionsMice inoculated intranasally with HSV-1 developed serum NMDAR antibodies. These antibodies were associated with reduced hippocampal NMDARs, as has been shown in previous models where antibodies from patients with anti-NMDAR encephalitis were infused into mice, paving the way for future studies into the pathophysiology of autoimmune encephalitides.

Gelsolin is the fourth most abundant protein in the body and its depletion in the blood has been found in multiple sclerosis (MS) patients. How gelsolin affects the MS brain has not been studied. We found that while the secreted form of gelsolin (pGSN) decreased in the blood of experimental autoimmune encephalomyelitis (EAE) mice, pGSN concentration increased in the EAE brain. Recombinant human pGSN (rhp-GSN) decreased extracellular actin and myeloperoxidase activity in the brain, resulting in reduced disease activity and less severe clinical disease, suggesting that gelsolin could be a potential therapeutic target for MS.