In Vivo 19F MR Imaging Cell Tracking of Inflammatory Macrophages and Site-specific Development of Colitis-associated Dysplasia.

Purpose To investigate whether the magnitude of in vivo fluorine 19 (19F) magnetic resonance (MR) imaging signal is associated with subsequent development of colitis-associated dysplasia after in situ fluorination of inflammatory macrophages in a mouse model of inflammatory bowel disease (IBD). Materials and Methods Experiments were approved by the institutional animal care and use committee. Mice in the experimental group (n = 10) were administered azoxymethane and dextran sulfate sodium to induce colitis-associated dysplasia. Five mice were in the noninduced control group. Animals were injected with a commercially available perfluorocarbon and were examined in vivo with an 11.7-T MR imager for up to 110 days. Colons were then harvested followed by high-spatial-resolution ex vivo MR imaging. Multiple colon segments with or without 19F signal were histologically graded and were correlated with 19F signal intensity by using a Spearman correlation test. The signal intensity in mice with colitis-associated dysplasia was compared with that in control mice with a two-tailed Mann-Whitney U test. Results Patchy distributions of 19F signal intensity in the colon wall were seen on in vivo and ex vivo images, representing chronic inflammation as shown by immunohistochemistry. Histologic scores of inflammation and site-specific development of colitis-associated dysplasia in the descending colon showed good correlation with normalized 19F signal intensity (r = 0.88, P = .033 for the ascending colon; r = 0.82, P = .006 for the descending colon). A significantly (P = .002) higher normalized 19F signal-to-noise ratio was found at sites that developed dysplasia (mean, 0.58 ± 0.09 [standard deviation]) as compared with sites that did not (mean, 0.17 ± 0.22). Conclusion 19F MR imaging cell tracking of macrophages can be used to assess local inflammation in a mouse model of IBD. The resulting local 19F signal intensity, representing the magnitude of inflammation, has a positive correlation with the development of colitis-associated dysplasia. © RSNA, 2016 Online supplemental material is available for this article.

Assaying macrophage activity in a murine model of inflammatory bowel disease using fluorine-19 MRI.

Macrophages have an important role in the pathogenesis of most chronic inflammatory diseases. A means of non-invasively quantifying macrophage migration would contribute significantly towards our understanding of chronic inflammatory processes and aid the evaluation of novel therapeutic strategies. We describe the use of a perfluorocarbon tracer reagent and in vivo (19)F magnetic resonance imaging (MRI) to quantify macrophage burden longitudinally. We apply these methods to evaluate the severity and three-dimensional distribution of macrophages in a murine model of inflammatory bowel disease (IBD). MRI results were validated by histological analysis, immunofluorescence and quantitative real-time polymerase chain reaction. Selective depletion of macrophages in vivo was also performed, further validating that macrophage accumulation of perfluorocarbon tracers was the basis of (19)F MRI signals observed in the bowel. We tested the effects of two common clinical drugs, dexamethasone and cyclosporine A, on IBD progression. Whereas cyclosporine A provided mild therapeutic effect, unexpectedly dexamethasone enhanced colon inflammation, especially in the descending colon. Overall, (19)F MRI can be used to evaluate early-stage inflammation in IBD and is suitable for evaluating putative therapeutics. Due to its high macrophage specificity and quantitative ability, we envisage (19)F MRI having an important role in evaluating a wide range of chronic inflammatory conditions mediated by macrophages.

In vivo observation of intracellular oximetry in perfluorocarbon-labeled glioma cells and chemotherapeutic response in the CNS using fluorine-19 MRI.

Preclinical development of therapeutic agents against cancer could greatly benefit from noninvasive markers of tumor killing. Potentially, the intracellular partial pressure of oxygen (pO(2) ) can be used as an early marker of antitumor efficacy. Here, the feasibility of measuring intracellular pO(2) of central nervous system glioma cells in vivo using (19) F magnetic resonance techniques is examined. Rat 9L glioma cells were labeled with perfluoro-15-crown-5-ether ex vivo and then implanted into the rat striatum. (19) F MRI was used to visualize tumor location in vivo. The mean (19) F T(1) of the implanted cells was measured using localized, single-voxel spectroscopy. The intracellular pO(2) in tumor cells was determined from an in vitro calibration curve. The basal pO(2) of 9L cells (day 3) was determined to be 45.3 ± 5 mmHg (n = 6). Rats were then treated with a 1 × LD10 dose of bischloroethylnitrosourea intravenously and changes in intracellular pO(2) were monitored. The pO(2) increased significantly (P = 0.042, paired T-test) to 141.8 ± 3 mmHg within 18 h after bischloroethylnitrosourea treatment (day 4) and remained elevated (165 ± 24 mmHg) for at least 72 h (day 6). Intracellular localization of the perfluoro-15-crown-5-ether emulsion in 9L cells before and after bischloroethylnitrosourea treatment was confirmed by histological examination and fluorescence microscopy. Overall, noninvasive (19) F magnetic resonance techniques may provide a valuable preclinical tool for monitoring therapeutic response against central nervous system or other deep-seated tumors.

Self-delivering nanoemulsions for dual fluorine-19 MRI and fluorescence detection.

We report the design, synthesis, and biological testing of highly stable, nontoxic perfluoropolyether (PFPE) nanoemulsions for dual 19F MRI-fluorescence detection. A linear PFPE polymer was covalently conjugated to common fluorescent dyes (FITC, Alexa647 and BODIPy-TR), mixed with pluronic F68 and linear polyethyleneimine (PEI), and emulsified by microfluidization. Prepared nanoemulsions (<200 nm) were readily taken up by both phagocytic and non-phagocytic cells in vitro after a short (approximately 3 h) co-incubation. Following cell administration in vivo, 19F MRI selectively visualizes cell migration. Exemplary in vivo MRI images are presented of T cells labeled with a dual-mode nanoemulsion in a BALB/c mouse. Fluorescence detection enables fluorescent microscopy and FACS analysis of labeled cells, as demonstrated in several immune cell types including Jurkat cells, primary T cells and dendritic cells. The intracellular fluorescence signal is directly proportional to the 19F NMR signal and can be used to calibrate cell loading in vitro.

Reducing severe hypoglycaemia in hospitalised patients with diabetes: Early outcomes of standardised reporting and management.

BACKGROUND: Severe hypoglycaemic events (HGEs) in hospitalised patients are associated with poor outcomes and prolonged hospitalization. Systematic, coordinated care is required for acute management and prevention of HGEs; however, studies evaluating quality control efforts are scarce. OBJECTIVE: To investigate the effectiveness of system-based interventions to improve management response to HGEs. METHODS: System-based interventions were designed and implemented following a root cause analysis of HGE in adult patients with diabetes from two general medical wards with the highest incidence of HGE. Interventions included electronic medical record programming for a standardised order set for basal-bolus insulin regimen and hypoglycemia protocol, automated dextrose order, automated MD notification, and recommendation for endocrine consultation after two critical HGEs. The Pyxis MedStation was programmed to alert nurses to recheck blood glucose 15 min after the treatment. A card with the HGE management protocol was attached to each provider's ID badge and educational seminars were given to all providers. MAIN OUTCOMES AND MEASURES: Primary outcomes were to evaluate median time from HGE (glucose <50 mg/dL) to euglycemia (>100 mg/dL), and time from HGE to follow-up finger-stick (FS) testing preintervention and postintervention. Secondary outcomes were cumulative incidence of HGEs, recurrent hypoglycemia, rate of physician notification and use of standardised treatments among adults with diabetes on the two general medical wards. RESULTS: Among hospitalised adults with diabetes and HGE, median time from HGE to euglycemia declined from 225±46 min preintervention to 87±26 min postintervention (p=0.03). Median time from HGE to next FS testing also declined (76±14 min to 28±10 min, p<0.001). Standardised treatment administration for HGE improved significantly from 34% (12/35) to 97% (36/37); physician notification rate improved significantly from 51% (18/35) to 78% (29/37).Among hospitalised adults with diabetes, incidence of HGE decreased from 12% (35/295) over 3 months (preintervention period) to 6% (37/610) over 6 months (postintervention period) (p<0.001), while recurrent HGE did not show significant differences (37% (13/35) to 24% (9/37), p=0.09). CONCLUSIONS: System-based interventions had a clinically important impact on decreasing time from HGE to euglycemia and to next FS testing. This hypoglycemia bundle of care may be applied and tested in other community hospitals to improve patient safety.

Herpes Simplex Virus-2 Esophagitis in a Young Immunocompetent Adult.

Herpes simplex esophagitis (HSE) is commonly identified in immunosuppressed patients. It is rare among immunocompetent patients and almost all of the reported cases are due to HSV-1 infection. HSV-2 esophagitis is extremely rare. We report the case of a young immunocompetent male who presented with dysphagia, odynophagia, and epigastric pain. Endoscopy showed multitudes of white nummular lesions in the distal esophagus initially suspected to be candida esophagitis. However, classic histopathological findings of multinucleated giant cells with eosinophilic intranuclear inclusions and positive HSV-2 IgM confirmed the diagnosis of HSV-2 esophagitis. The patient rapidly responded to acyclovir treatment. Although HSV-2 is predominantly associated with genital herpes, it can cause infections in other parts of the body previously attributed to only HSV-1 infection.

Pulmonary Nocardiosis in a Multiple Myeloma Patient Treated with Proteasome Inhibitors.

BACKGROUND: The use of proteasome inhibitors like Bortezomib to treat multiple myeloma has been associated with increased rates of opportunistic infections, including Nocardia, especially when lymphopenia is present. The prevalence or association of such infections with newer agents like Carfilzomib is not known. CASE REPORT: A 71-year-old man with multiple myeloma presented with a 6-week history of respiratory symptoms and cyclic fevers. He was undergoing chemotherapy with Carfilzomib. Work-up revealed severe lymphopenia and a CT chest showed multiple lung nodules and a mass-like consolidation. He underwent a bronchoscopy, and respiratory cultures grew Nocardia species. He responded well to intravenous antibiotics with resolution of symptoms and CT findings. CONCLUSIONS: With the introduction of newer agents like Carfilzomib for the treatment of multiple myeloma, clinicians must maintain a high degree of suspicion for opportunistic infections to achieve early diagnosis and treatment.

Paradoxical decrease in the capture and lymph node delivery of cancer vaccine antigen induced by a TLR4 agonist as visualized by dual-mode imaging.

Traditionally, cell-mediated immune responses to vaccination in animal models are evaluated by invasive techniques such as biopsy and organ extraction. We show here that by combining two noninvasive imaging technologies, MRI and bioluminescence imaging (BLI), we can visualize both the afferent and efferent arms of cellular events following vaccination longitudinally. To this end, we evaluated the immune response elicited by a novel Toll-like receptor 4 agonist vaccine adjuvant, glucopyranosyl lipid A (GLA), using a whole-cell tumor vaccine. After magnetovaccination, MRI was used to visualize antigen-presenting cell-mediated antigen capture and subsequent migration to draining lymph nodes (DLN). Paradoxically, we observed that the incorporation of GLA in the vaccine reduced these critical parameters of the afferent immune response. For the efferent arm, the magnitude of the ensuing antigen-specific T-cell response in DLN visualized using BLI correlated with antigen delivery to the DLN as measured by MRI. These findings were confirmed using flow cytometry. In spite of the GLA-associated reduction in antigen delivery to the DLN, however, the use of GLA as a vaccine adjuvant led to a massive proliferation of vaccine primed antigen-specific T cells in the spleen. This was accompanied by an enhanced tumor therapeutic effect of the vaccine. These findings suggest that GLA adjuvant changes the temporal and anatomical features of both the afferent and efferent arms of the vaccine response and illustrates the utility of quantitative noninvasive imaging as a tool for evaluating these parameters during vaccine optimization.

Advances in using MRI probes and sensors for in vivo cell tracking as applied to regenerative medicine.

The field of molecular and cellular imaging allows molecules and cells to be visualized in vivo non-invasively. It has uses not only as a research tool but in clinical settings as well, for example in monitoring cell-based regenerative therapies, in which cells are transplanted to replace degenerating or damaged tissues, or to restore a physiological function. The success of such cell-based therapies depends on several critical issues, including the route and accuracy of cell transplantation, the fate of cells after transplantation, and the interaction of engrafted cells with the host microenvironment. To assess these issues, it is necessary to monitor transplanted cells non-invasively in real-time. Magnetic resonance imaging (MRI) is a tool uniquely suited to this task, given its ability to image deep inside tissue with high temporal resolution and sensitivity. Extraordinary efforts have recently been made to improve cellular MRI as applied to regenerative medicine, by developing more advanced contrast agents for use as probes and sensors. These advances enable the non-invasive monitoring of cell fate and, more recently, that of the different cellular functions of living cells, such as their enzymatic activity and gene expression, as well as their time point of cell death. We present here a review of recent advancements in the development of these probes and sensors, and of their functioning, applications and limitations.

Fluorine-19 Labeling of Stromal Vascular Fraction Cells for Clinical Imaging Applications.

UNLABELLED: Stromal vascular fraction (SVF) cells are used clinically for various therapeutic targets. The location and persistence of engrafted SVF cells are important parameters for determining treatment failure versus success. We used the GID SVF-1 platform and a clinical protocol to harvest and label SVF cells with the fluorinated ((19)F) agent CS-1000 as part of a first-in-human phase I trial (clinicaltrials.gov identifier NCT02035085) to track SVF cells with magnetic resonance imaging during treatment of radiation-induced fibrosis in breast cancer patients. Flow cytometry revealed that SVF cells consisted of 25.0% ± 15.8% CD45+, 24.6% ± 12.5% CD34+, and 7.5% ± 3.3% CD31+ cells, with 2.1 ± 0.7 × 105 cells per cubic centimeter of adipose tissue obtained. Fluorescent CS-1000 (CS-ATM DM Green) labeled 87.0% ± 13.5% of CD34+ progenitor cells compared with 47.8% ± 18.5% of hematopoietic CD45+ cells, with an average of 2.8 ± 2.0 × 10¹² ¹9F atoms per cell, determined using nuclear magnetic resonance spectroscopy. The vast majority (92.7% ± 5.0%) of CD31+ cells were also labeled, although most coexpressed CD34. Only 16% ± 22.3% of CD45-/CD31-/CD34- (triple-negative) cells were labeled with CS-ATM DM Green. After induction of cell death by either apoptosis or necrosis, >95% of ¹9F was released from the cells, indicating that fluorine retention can be used as a surrogate marker for cell survival. Labeled-SVF cells engrafted in a silicone breast phantom could be visualized with a clinical 3-Tesla magnetic resonance imaging scanner at a sensitivity of approximately 2 × 106 cells at a depth of 5 mm. The current protocol can be used to image transplanted SVF cells at clinically relevant cell concentrations in patients. SIGNIFICANCE: Stromal vascular fraction (SVF) cells harvested from adipose tissue offer great promise in regenerative medicine, but methods to track such cell therapies are needed to ensure correct administration and monitor survival. A clinical protocol was developed to harvest and label SVF cells with the fluorinated (¹9F) agent CS-1000, allowing cells to be tracked with (19)F magnetic resonance imaging (MRI). Flow cytometry evaluation revealed heterogeneous ¹9F uptake in SVF cells, confirming the need for careful characterization. The proposed protocol resulted in sufficient ¹9F uptake to allow imaging using a clinical MRI scanner with point-of-care processing.

Label-free in vivo molecular imaging of underglycosylated mucin-1 expression in tumour cells.

Alterations in mucin expression and glycosylation are associated with cancer development. Underglycosylated mucin-1 (uMUC1) is overexpressed in most malignant adenocarcinomas of epithelial origin (for example, colon, breast and ovarian cancer). Its counterpart MUC1 is a large polymer rich in glycans containing multiple exchangeable OH protons, which is readily detectable by chemical exchange saturation transfer (CEST) MRI. We show here that deglycosylation of MUC1 results in >75% reduction in CEST signal. Three uMUC1(+) human malignant cancer cell lines overexpressing uMUC1 (BT20, HT29 and LS174T) show a significantly lower CEST signal compared with the benign human epithelial cell line MCF10A and the uMUC1(-) tumour cell line U87. Furthermore, we demonstrate that in vivo CEST MRI is able to make a distinction between LS174T and U87 tumour cells implanted in the mouse brain. These results suggest that the mucCEST MRI signal can be used as a label-free surrogate marker to non-invasively assess mucin glycosylation and tumour malignancy.

(19)F spin-lattice relaxation of perfluoropolyethers: Dependence on temperature and magnetic field strength (7.0-14.1T).

Fluorine ((19)F) MRI of perfluorocarbon-labeled cells has become a powerful technique to track the migration and accumulation of cells in living organisms. It is common to label cells for (19)F MRI with nanoemulsions of perfluoropolyethers that contain a large number of chemically equivalent fluorine atoms. Understanding the mechanisms of (19)F nuclear relaxation, and in particular the spin-lattice relaxation of these molecules, is critical to improving experimental sensitivity. To date, the temperature and magnetic field strength dependence of spin-lattice relaxation rate constant (R1) for perfluoropolyethers has not been described in detail. In this study, we evaluated the R1 of linear perfluoropolyether (PFPE) and cyclic perfluoro-15-crown-5 ether (PCE) at three magnetic field strengths (7.0, 9.4, and 14.1T) and at temperatures ranging from 256-323K. Our results show that R1 of perfluoropolyethers is dominated by dipole-dipole interactions and chemical shift anisotropy. R1 increased with magnetic field strength for both PCE and PFPE. In the temperature range studied, PCE was in the fast motion regime (ωτc<1) at all field strengths, but for PFPE, R1 passed through a maximum, from which the rotational correlation time was estimated. The importance of these measurements for the rational design of new (19)F MRI agents and methods is discussed.

Preclinical evidence that PD1 blockade cooperates with cancer vaccine TEGVAX to elicit regression of established tumors.

Biomarker studies have shown that expression of the T-cell coregulatory ligand PDL1 on tumor cells correlates with clinical responsiveness to the PD1 antibody nivolumab. Here, we report the findings of a preclinical cancer vaccine study demonstrating vaccine-dependent PDL1 upregulation in the tumor microenvironment. We formulated an IFNγ-inducing cancer vaccine called TEGVAX that combined GM-CSF and multiple Toll-like receptor agonists to increase the number of activated dendritic cells. Treatment of established tumors with TEGVAX retarded tumor growth in a manner associated with enhanced systemic antitumor immunity. Unexpectedly, TEGVAX also upregulated PDL1 expression in the tumor microenvironment, possibly explaining why tumors were not eliminated completely. In support of this likelihood, PDL1 upregulation in this setting relied upon IFNγ-expressing tumor-infiltrating CD4(+) and CD8(+) T cells and administration of a PD1-blocking antibody with TEGVAX elicited complete regression of established tumors. Taken together, our findings provide a mechanistic rationale to combine IFNγ-inducing cancer vaccines with immune checkpoint blockade.

Inflammation driven by overexpression of the hypoglycosylated abnormal mucin 1 (MUC1) links inflammatory bowel disease and pancreatitis.

OBJECTIVE: Pancreatitis occurs as an extraintestinal complication of inflammatory bowel disease (IBD), but the cause is poorly understood. Mucin 1 (MUC1) is overexpressed in an abnormal, hypoglycosylated form on the colonic epithelium in human IBD where it contributes to inflammation. MUC1 is also expressed on pancreatic ductal epithelia. We tested the possibility that in IBD, MUC1 expression on pancreatic ducts is also abnormal leading to inflammation and pancreatitis. METHODS: We used MUC1/interleukin-10 mice that develop IBD. We imaged abnormal MUC1 expression in these mice by adoptively transferring T cells from T cell receptor transgenic mice specific for abnormal MUC1. Cells were labeled with a novel perfluorocarbon tracer reagent and quantified and visualized in vivo using high-throughput F nuclear magnetic resonance spectroscopy and magnetic resonance imaging. RESULTS: MUC1-specific T cells migrated to the colon in mice with IBD and also to the pancreas. Immunohistochemistry confirmed increased expression on the pancreatic ducts of the abnormal MUC1 seen in the colon and the presence of cellular infiltrate. CONCLUSIONS: Migration of MUC1-specific T cells to the colon and the pancreas in diseased mice suggests that pancreatitis is an extraintestinal site of IBD, characterized by proinflammatory abnormal expression of MUC1. Therapies directed against abnormal MUC1 have the potential of targeting the disease in both sites.