Molecular Imaging Of Inflammation By Targeting Myeloperoxidase And Fluorescent Tracking Of Mouse T Lymphocytes

Molecular Imaging is in vivo characterization and measurement of biologic processes at the cellular and molecular level by imaging. Molecular imaging must meet the following conditions:1. clinically meaningful targets; 2. suitable molecular probes for targets and appropriate signal amplification mechanism; 3. imaging device with high sensitivity and resolution, or mixed imaging device.Targets for molecular imaging needs to be substances that express a lot under certain physiological and pathological conditions. Since neuroinflammation is involved in almost all of the central nervous system diseases, it has been popular in research. In the central nervous system, when there is infection, trauma, poisoning or autoimmune startup, etc., the BBB (blood-brain barrier, BBB) becomes weak, circulating immune cells may cross the BBB, and induce the release of a variety of enzymes involved in neuroinflammation. Myeloperoxidase (MPO) is a more crucial one, and is powerful inflammatory mediators of many activated leukocytes. With hydrogen peroxide and chloride ions as substrates, MPO catalyzed a lot of reactions to produce hypochlorous acid and a variety of radicals. If the physical body can not effectively clear the free radicals and oxidants, they may cause a variety of pathological processes and tissue damage. Thus, MPO activity can be a target of reactive neuroinflammation.A molecular probe is a compound that is a composed of a substrate material linked to a signal producer. At present, most of the molecular probes are the targeting probes. The targeting probe works by specifically binding to the target, mainly used in direct imaging of the distribution of interest molecules in the body. Activable probes, which are also known as smart probes, can be activated by target molecules or involved in the specific molecular events, with a very good specificity.For detection of the probe, there are advantages and disadvantages for various devices. MRI has a very high spatial resolution and is suitable for the central nervous system; but much lower detection sensitivity, which needs large amounts of contrast agent accumulation and strong signal amplification system in the target tissue. Optical molecular imaging has a lot of advantages, like non-invasiveness, non radiation hazards, low price, high sensitivity, real-time imaging and so on. But molecular probes still need to be used in conjunction with high resolution devices to give a full play of its advantages. Multi-mode imaging probe and device is imperative.In this study, we aim to target MPO, a key enzyme in neuroinflammation. MPO can specifically activate the magnetic resonance and fluorescence smart probe. We plan to study the application of the probes in neuroinflammation-related diseases. We may also explore the the fluorescent tracking of T lymphocytes targeting glioma in vitro preliminarily.Part ⅠMolecular MRI of Myeloperoxidase Detects Effective Combination Treatment in a Mouse Model of Multiple SclerosisPurpose:Glatiramer acetate (GA), a first-line drug for multiple sclerosis (MS), primarily increased Th2 anti-inflammatory lymphocytes while 4-aminobenzoic acid hydrazide (ABAH) is an irreversible inhibitor for myeloperoxidase (MPO), a major product of pro-inflammatory myeloid cells. The aim of this study was to investigate whether the combination of these two agents could be more beneficial than each treatment alone, and whether this benefit can be evaluated and tracked by molecular imaging targeting MPO as an imaging biomarker.Materials and methods:Experimental autoimmune encephalomyelitis (EAE) was induced using proteolipid protein (PLP) in 110 SJL female mice divided into 4 groups given the following at half doses:1) ABAH IP 20mg/kg bid,2) GA IP 75ug/qd,3) both ABAH IP 20 mg/kg bid and GA 75 ug (combination) and (4) saline as control. As an additional control, five mice were induced with PBS instead of PLP. Mice were imaged with the agent bis-5HT-DTPA-Gd that targets MPO in T1WI pre-contrast and post-contrast sequences (n=3 in ABAH group, n=3 in GA group, n=4 in combination group) when they first become symptomatic. Analysis of the lesion number, lesion size and CNR from the pre-contrast and 60min post-contrast images was conducted. Histologic staining with HE, anti-MPO and luxol fast blue was used to analyze the inflammatory infiltration, MPO expression and demyelination. Statistical analysis was performed using ANOVA for multiple groups with p<0.05 as significant.Results:The combination group showed delayed disease onset (day 9) and reduced disease severity compared to the ABAH (day8; P<0.001) and GA group (day8; P<0.05). At remission, the disease burden was also less severe in the combination group compared to the ABAH group (P<0.05) and GA group (P<0.05). The combined treatment group also tended to have improved survival compared to the ABAH group (P=0.11) and GA group (P=0.1671). On imaging, the combination group showed fewer lesions (ABAH P<0.01; GA P<0.05), smaller lesion size (ABAH P<0.05; GA P<0.05) and lower image intensity (ABAH P<0.01; GA P<0.05). On histopathology, the combination group showed attenuation of inflammatory cells infiltration, less MPO expression and less demyelination.Conclusion:When used at sub-optimal doses, neither medication affected EAE but combination at those doses led to the significant alleviation of EAE disease severity that can be tracked by molecular imaging targeting MPO. Therefore, MPO-Gd is a potential key imaging biomarker for MS and could be used to guide treatment decisions.Part ⅡMyeloperoxidase Sensitive Fluorescent Probe, Characteristics and ImagingPurpose:Based the mechianism of MPO-Gd, we aim to compound a new MPO activable fluorescent probe, and some preliminary studies about its imaging properties.Materials and methods:5-HT was used as a substrate for this probe (which can be activated by MPO), and a biotin group was added to this substrate to avoid self-quenching. Probe can then be tracked by fluorescent streptavidin. In vitro, matrigel was mixed with human MPO or no human MPO and plated in 96-well plate. Probe and SA-AF-647 was added and washed separately. Plate was then imaged by FRI. Matrigel with different concentrations of human MPO was injected subcutaneously into mice thigh, followed by tail vein injection of MPO probe and SA-AF-647. Streptococcus pneumoniae was subcutaneously injected into a mouse thigh, with the other side injected with PBS as self-control. Sytox Green was also subcutaneously injected and then tail vein injection of MPO probe and SA-AF-647. Salmonella was intrathetically injected to make a meningitis mouse model. Flow cytometry and MPO activity was done to test this model.24 hours later, followed by tail vein injection of MPO probe and SA-AF-647. Fluorescent imaging was taken at 0,15,30,45,60min after tail vein injection of SA-AF-647.Results:The MPO specific fluorescent probe was successfully compounded. The in vitro matrigel experiments showed that fluorescence signal only came from matrigel with human MPO, but not matrigel without human MPO. This means the probe could be activated by human MPO and oligomerize in matrigel; the imaging based on the biotin-streptavidin system worked well. After subcutaneously injection of matrigel with different concentrations of MPO, we can see great increase of fluorescence coming from the matrigel with MPO. There is a linear correlation between the concentration of MPO and fluorescence intensity (r2=0.9572). After subcutaneous injection of streptococcus pneumonia, in both the GFP and RFP channel, the fluorescence imaging showed pieces of positive areas and they colocalized with each other. No signal came from the control side. In a central nervous system disease model of meningitis,60min after tail vein injection of SA-AF-647, ex vitro fluorescent imaging showed clear meninges contrast, while no contrast from the control brain. Huge amounts of myeloid cell infiltrated into the meningitis brain and the MPO activity was also very high.Conclusion:In this study, a new MPO activable and specific smart fluorescent probe was successfully compounded. In vitro and in vivo experiments proved that this probe could be activated by humanial and mouse MPO and imaged with fluorescent streptavidin.Part Ⅲ In Vitro Fluorescent Tracking of Cytotoxic T Lymphocytes to Glioma Stem CellsPurpose:Since there is high sensitivity with fluorescent imaging and based on the probe synthesis from the first and second part, we aim to biotinylate the cell surface of T lymphocytes and label T cells with fluorescence conjugated streptavidin. Then plan to verify if streptavidin would help track T lymphocytes targeting biotinylated glioma stem cells.Materials and methods:CD8+ T lymphocytes were isolated from mouse spleen by positive isolation, then activated and stimulated for 1-2 days. Both lentivirus infection and Sulfo-NHS-biotin were used to biotinylate T lymphocytes. Labeling efficiency was detected by flow cytometry. The labeling efficiency and T lymphocytes viability of covalent biotinylation was analysed to 72 hours after labeling. Glioma stem cells were infected by lentivirus to express biotin on cells surface and to secret bioluminescence. Flow cytometry and bioluminence test was used to detect the infection efficiency. Biotinylated T lymphocytes and glioma stem cells were co-cultured to detect the specific recognition and killing of glioma stem cells by the T lymphocytes.Results:The biotinylation of T lymphocytes by lentivirus infection is very low, with an efficiency around 15% and very severe cell damage. While the covalent biotinylation method could get a labeling efficiency of more than 90%. Flow cytometry results showed that until 72 hours, the cells kept the labeling and very good survivability. Glioma stem cells could be easily biotinylated by lentivirus infection. Flow cytometry results showed successful surface biotin expression and secretion of bioluminescence. Glioma stem cells grew well as spheres. The co-culture of T lymphocytes and glioma stem cells showed that growth of glioma stem cells from the targeted group were greatly inhibited and cells lost the normal shape as sphere. Bioluminescence results also showed decreased growth of the targeted glioma stem cells, compared to the untargeted and control groups.Conclusion:T lymphocytes could be easily and very efficiently biotinylated with covalent bonding of Sulfo-NHS-biotin on cell surface protein. Glioma stem cells could be easiy biotinylated by lentivirus infection. Streptavidin fluorescent labeling could help track T lymphocytes targeting biotinylated glioma stem cells in vitro.In all, in this study, we compounded the MPO activable specific MPO-Gd and tracked the synthetical treatment efficiency of GA and ABAH in a mouse model of EAE. We compounded a fluorescent probe and detected the imaging of human and mouse MPO, both in vitro and in vivo. By biotinylating both the lymphocytes and glioma stem cells, streptavidin fluorescent labeling could help track T lymphocytes targeting biotinylated glioma stem cells in vitro.