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Detection and accurate delineation of tumor is important for the management of head and neck squamous cell carcinoma (HNSCC) but is challenging with current imaging techniques. In this study, we evaluated whether molecular immuno-imaging targeting myeloperoxidase (MPO) activity, an oxidative enzyme secreted by many myeloid innate immune cells, would be superior in detecting tumor extent compared to conventional contrast agent (DTPA-Gd) in a carcinogen-induced immunocompetent HNSCC murine model and corroborated in human surgical specimens. In C57BL/6 mice given 4-nitroquinoline-N-oxide (4-NQO), there was increased MPO activity in the head and neck region as detected by luminol bioluminescence compared to that of the control group. On magnetic resonance imaging, the mean enhancing volume detected by the MPO-targeting agent (MPO-Gd) was higher than that by the conventional agent DTPA-Gd. The tumor volume detected by MPO-Gd strongly correlated with tumor size on histology, and higher MPO-Gd signal corresponded to larger tumor size found by imaging and histology. On the contrary, the tumor volume detected by DTPA-Gd did not correlate as well with tumor size on histology. Importantly, MPO-Gd imaging detected areas not visualized with DTPA-Gd imaging that were confirmed histopathologically to represent early tumor. In human specimens, MPO was similarly associated with tumors, especially at the tumor margins. Thus, molecular immuno-imaging targeting MPO not only detects oxidative immune response in HNSCC, but can better detect and delineate tumor extent than nonselective imaging agents. Thus, our findings revealed that MPO imaging could improve tumor resection as well as be a useful imaging biomarker for tumor progression, and potentially improve clinical management of HNSCC once translated.

Simple Summary Inflammation and oxidative stress are important host defense responses. However, while the host response can be cytotoxic and kill tumor cells, tumor cells can also alter and exploit the host immune environment to further their survival. Thus, the host response can impact both tumor suppression and progression. Modulating the tumor–host response interaction to favor tumor suppression would be highly desirable. D-mannose has been found to have anti-inflammatory properties and can block signaling related to myeloperoxidase (MPO), a highly oxidizing pro-inflammatory enzyme secreted in host defense. However, the effect of D-mannose on host immune response in the glioma microenvironment has not been explored. We found that D-mannose slowed glioma growth by increasing MPO activity and oxidative stress in the glioma microenvironment. Our findings revealed that D-mannose may be able to shift the host immune response toward tumor suppression and could be a potential new therapeutic direction for these difficult-to-treat tumors. Abstract Host immune response in the tumor microenvironment plays key roles in tumorigenesis. We hypothesized that D-mannose, a simple sugar with anti-inflammatory properties, could decrease oxidative stress and slow glioma progression. Using a glioma stem cell model in immunocompetent mice, we induced gliomas in the brain and tracked MPO activity in vivo with and without D-mannose treatment. As expected, we found that D-mannose treatment decreased the number of MPO+ cells and slowed glioma progression compared to PBS-treated control animals with gliomas. Unexpectedly, instead of decreasing MPO activity, D-mannose increased MPO activity in vivo, revealing that D-mannose boosted the MPO activity per MPO+ cell. On the other hand, D-glucose had no effect on MPO activity. To better understand this effect, we examined the effect of D-mannose on bone marrow-derived myeloid cells. We found that D-mannose modulated MPO activity via two mechanisms: directly via N-glycosylation of MPO, which boosted the MPO activity of each molecule, and indirectly by increasing H2O2 production, the main substrate for MPO. This increased host immune response acted to reduce tumor size, suggesting that increasing MPO activity such as through D-mannose administration may be a potential new therapeutic direction for glioma treatment.

Inflammation drives the pathology of many neurological diseases. d-mannose has been found to exert an antiinflammatory effect in peripheral diseases, but its effects on neuroinflammation and inflammatory cells in the central nervous system have not been studied. We aimed to determine the effects of d-mannose on key macrophage/microglial functions—oxidative stress and phagocytosis. In murine experimental autoimmune encephalomyelitis (EAE), we found d-mannose improved EAE symptoms compared to phosphate-buffered saline (PBS)-control mice, while other monosaccharides did not. Multiagent molecular MRI performed to assess oxidative stress (targeting myeloperoxidase [MPO] using MPO-bis-5-hydroxytryptamide diethylenetriaminepentaacetate gadolinium [Gd]) and phagocytosis (using cross-linked iron oxide [CLIO] nanoparticles) in vivo revealed that d-mannose–treated mice had smaller total MPO-Gd(+) areas than those of PBS-control mice, consistent with decreased MPO-mediated oxidative stress. Interestingly, d-mannose–treated mice exhibited markedly smaller CLIO(+) areas and much less T2 shortening effect in the CLIO(+) lesions compared to PBS-control mice, revealing that d-mannose partially blocked phagocytosis. In vitro experiments with different monosaccharides further confirmed that only d-mannose treatment blocked macrophage phagocytosis in a dose-dependent manner. As phagocytosis of myelin debris has been known to increase inflammation, decreasing phagocytosis could result in decreased activation of proinflammatory macrophages. Indeed, compared to PBS-control EAE mice, d-mannose–treated EAE mice exhibited significantly fewer infiltrating macrophages/activated microglia, among which proinflammatory macrophages/microglia were greatly reduced while antiinflammatory macrophages/microglia increased. By uncovering that d-mannose diminishes the proinflammatory response and boosts the antiinflammatory response, our findings suggest that d-mannose, an over-the-counter supplement with a high safety profile, may be a low-cost treatment option for neuroinflammatory diseases such as multiple sclerosis.

ObjectiveTo identify and develop the optimal active immunization induction method for NMDAR encephalitis in rodents.BackgroundEncephalitis is a devastating neurologic disorder with high morbidity and mortality. Many cases are autoimmune. N-Methyl-D-aspartic acid receptor (NMDAR) encephalitis (NMDARE), characterized by antibodies against the NMDAR in the blood and spinal fluid of patients, is the most common form of autoimmune encephalitis (AE). A translational rodent model of NMDARE would allow for in-depth studies into AE pathophysiology, leading to advances in the diagnosis and treatment of this debilitating neuropsychiatric disorder.Design/Methods7-week-old female C57BL/6J mice were injected subcutaneously with an emulsion of complete Freund's adjuvant, attenuated Mycobacterium tuberculosis (TB), and a 30 amino acid peptide flanking the NMDAR NR1 subunit N368/G369 residue targeted by antibodies in NMDARE patients. Three different induction methods were tested by varying the amount and injection method of pertussis toxin, subcutaneous injection sites, reimmunization, and amounts of TB. Mice were bled biweekly and sacrificed at 2, 4, 6, 8, and 14 weeks. Serum and CSF NMDAR antibody titer; mouse behavior; hippocampal NMDAR protein and cluster density; and brain immune cell entry and cytokine content were examined.ResultsImmunized mice had serum and CSF NMDAR antibodies. Mice exhibited behavioral changes, altered hippocampal NMDAR protein, brain immune cell entry, and elevated cytokines in their brains. Titers were higher and changes were sustained in reimmunized mice.ConclusionsActive immunization against the portion of the NMDAR targeted in patients with NMDARE resulted in robust production of NMDAR antibodies in the blood and spinal fluid, changes in hippocampal NMDAR protein, elevations in brain immune cells and cytokines, and behavioral changes in mice. Reimmunization was needed to sustain the responses. Active immunization therefore holds potential as a translational model of NMDARE, allowing for the creation of a novel generation of diagnostics and therapeutics.

Myeloperoxidase (MPO) is a key component of innate immunity but can damage tissues when secreted abnormally. We developed a new generation of a highly efficient MPO-activatable MRI probe (heMAMP) to report MPO activity. heMAMP has improved Gd stability compared to bis-5-HT-Gd-DTPA (MPO-Gd) and demonstrates no significant cytotoxicity. Importantly, heMAMP is more efficiently activated by MPO compared to MPO-Gd, 5HT-DOTA(Gd), and 5HT-DOTAGA-Gd. Molecular docking simulations revealed that heMAMP has increased rigidity via hydrogen bonding intramolecularly and improved binding affinity to the active site of MPO. In animals with subcutaneous inflammation, activated heMAMP showed a 2-3-fold increased contrast-to-noise ratio (CNR) compared to activated MPO-Gd and 4-10 times higher CNR compared to conventional DOTA-Gd. This increased efficacy was further confirmed in a model of unstable atherosclerotic plaque where heMAMP demonstrated a comparable signal increase and responsiveness to MPO inhibition at a 3-fold lower dosage compared to MPO-Gd, further underscoring heMAMP as a potential translational candidate.

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.