d-mannose suppresses oxidative response and blocks phagocytosis in experimental neuroinflammation.

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.

Molecular immuno-imaging improves tumor detection in head and neck cancer.

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.

Myeloperoxidase PET Imaging Tracks Intracellular and Extracellular Treatment Changes in Experimental Myocardial Infarction.

Myeloperoxidase (MPO) is a highly oxidative, pro-inflammatory enzyme involved in post-myocardial infarction (MI) injury and is a potential therapeutic target. While multiple MPO inhibitors have been developed, the lack of an imaging reporter to select appropriate patients and assess therapeutic efficacy has hampered clinical development. Thus, a translational imaging method to detect MPO activity non-invasively would help to better understand the role MPO plays in MI and facilitate novel therapy development and clinical validation. Interestingly, many MPO inhibitors affect both intracellular and extracellular MPO, but previous MPO imaging methods can only report extracellular MPO activity. In this study, we found that an MPO-specific PET imaging agent (18F-MAPP) can cross cell membranes to report intracellular MPO activity. We showed that 18F-MAPP can track the treatment effect of an MPO inhibitor (PF-2999) at different doses in experimental MI. The imaging results were corroborated by ex vivo autoradiography and gamma counting data. Furthermore, extracellular and intracellular MPO activity assays revealed that 18F-MAPP imaging can report the changes induced by PF-2999 on both intracellular and extracellular MPO activities. These findings support 18F-MAPP as a translational candidate to noninvasively report MPO activity and accelerate drug development against MPO and other related inflammatory targets.

Angiotensin II drives the production of tumor-promoting macrophages.

Macrophages frequently infiltrate tumors and can enhance cancer growth, yet the origins of the macrophage response are not well understood. Here we address molecular mechanisms of macrophage production in a conditional mouse model of lung adenocarcinoma. We report that overproduction of the peptide hormone Angiotensin II (AngII) in tumor-bearing mice amplifies self-renewing hematopoietic stem cells (HSCs) and macrophage progenitors. The process occurred in the spleen but not the bone marrow, and was independent of hemodynamic changes. The effects of AngII required direct hormone ligation on HSCs, depended on S1P(1) signaling, and allowed the extramedullary tissue to supply new tumor-associated macrophages throughout cancer progression. Conversely, blocking AngII production prevented cancer-induced HSC and macrophage progenitor amplification and thus restrained the macrophage response at its source. These findings indicate that AngII acts upstream of a potent macrophage amplification program and that tumors can remotely exploit the hormone's pathway to stimulate cancer-promoting immunity.

Combined chemotherapy and endoscopic ultrasound-guided intratumoral 32P implantation for locally advanced pancreatic adenocarcinoma: a pilot study.

BACKGROUND: This study evaluated clinical outcomes of combined chemotherapy and endoscopic ultrasound (EUS)-guided intratumoral radioactive phosphorus-32 (32P) implantation in locally advanced pancreatic adenocarcinoma (LAPC). METHODS: Consecutive patients with newly diagnosed LAPC were recruited over 20 months. Baseline computed tomography and 18F-2-fluoro-2-deoxy-D-glucose (18FDG) positron emission tomography-computed tomography were performed and repeated after 12 weeks to assess treatment response. Following two cycles of conventional chemotherapy, patients underwent EUS-guided 32P implantation followed by six chemotherapy cycles. RESULTS: 12 patients with LAPC (median age 69 years [interquartile range 61.5-73.3]; 8 male) completed treatment. Technical success was 100 % with no procedural complications. At 12 weeks, median reduction in tumor volume was 8.2 cm3 (95 % confidence interval 4.95-10.85; P = 0.003), with minimal or no 18FDG uptake in nine patients (75 %). Tumor downstaging was achieved in six patients (50 %), leading to successful resection in five (42 %), including four R0 resections (80 %). CONCLUSIONS: EUS-guided 32P implantation was feasible, well tolerated, and resulted in a 42 % surgical resection rate. Further evaluation in a larger randomized multicenter trial is warranted.

An activatable PET imaging radioprobe is a dynamic reporter of myeloperoxidase activity in vivo.

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 18F-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 18F-MAPP has a short blood half-life, remains stable in plasma, does not demonstrate cytotoxicity, and crosses the intact blood-brain barrier. The 18F-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 18F-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 18F-MAPP is a promising translational candidate to noninvasively monitor MPO activity and inflammation in patients.

Myeloperoxidase Molecular MRI Reveals Synergistic Combination Therapy in Murine Experimental Autoimmune Neuroinflammation.

Background Despite advances in immunomodulatory agents, most current therapies for multiple sclerosis target lymphocytes or lymphocytic function. However, therapy response may be less than optimal due to demyelination and axonal damage caused by myeloid cells. Purpose To determine if myeloperoxidase (MPO) molecular MRI can evaluate whether combination therapy targeting both lymphoid and myeloid inflammation can improve autoimmune neuroinflammation compared with either drug alone, even at suboptimal doses. Materials and Methods Four groups of 94 female mice (8-10 weeks old) were induced with experimental autoimmune encephalomyelitis (EAE) from August 2, 2016, to March 30, 2018, and divided into saline control (n = 22), 4-aminobenzoic acid hydrazide (ABAH) therapy group (n = 19), glatiramer acetate (GA) therapy group (n = 22), and combination therapy group (n = 31). Mice were administered suboptimal doses of ABAH, an irreversible inhibitor of MPO; GA, a first-line multiple sclerosis drug; both ABAH and GA; or saline (control). Mice were imaged with bis-5-hydroxytryptamide-diethylenetriaminepentaacetate gadolinium (hereafter, MPO-Gd) MRI. One-way analysis of variance, two-way analysis of variance, Kurskal-Wallis, and log-rank tests were used. P < .05 was considered to indicate statistical significance. Results The combination-treated group showed delayed disease onset (day 11.3 vs day 9.8 for ABAH, day 10.4 for GA, day 9.9 for control; P < .05) and reduced disease severity (clinical score during the acute exacerbation period of 1.8 vs 3.8 for ABAH, 3.1 for GA, 3.9 for control; P < .05). The combination-treated group demonstrated fewer MPO-positive lesions (30.2 vs 73.7 for ABAH, 64.8 for GA, 67.2 for control; P < .05), smaller MPO-positive lesion volume (16.7 mm3 vs 65.2 mm3 for ABAH, 69.9 mm3 for GA, 66.0 mm3 for control; P < .05), and lower intensity of MPO-Gd lesion activation ratio (0.7 vs 1.9 for ABAH, 3.2 for GA, 2.3 for control; P < .05). Reduced disease severity in the combination group was confirmed at histopathologic analysis, where MPO expression (1779 vs 2673 for ABAH, 2898 for GA; P < .05) and demyelination (5.3% vs 9.0% for ABAH, 10.6% for GA; P < .05) were ameliorated. Conclusion Myeloperoxidase molecular MRI can track the treatment response from immunomodulatory drugs even if the drug does not directly target myeloperoxidase, and establishes that combination therapy targeting both myeloid and lymphocytic inflammation is effective for murine autoimmune neuroinflammation, even at suboptimal doses. © RSNA, 2019 Online supplemental material is available for this article. See also the editorial by Walczak in this issue.

MRI of Iron Oxide Nanoparticles and Myeloperoxidase Activity Links Inflammation to Brain Edema in Experimental Cerebral Malaria.

Purpose To investigate the association of inflammation and brain edema in a cerebral malaria (CM) mouse model with a combination of bis-5-hydroxy-tryptamide-diethylenetriaminepentaacetate gadolinium, referred to as MPO-Gd, and cross-linked iron oxide nanoparticle (CLIO-NP) imaging. Materials and Methods Female wild-type (n = 23) and myeloperoxidase (MPO) knock-out (n = 5) mice were infected with the Plasmodium berghei ANKA strain from May 2016 to July 2018. Seven healthy mice served as control animals. At a Rapid Murine Coma and Behavioral Scale (RMCBS) score of less than 15, mice underwent MRI at 9.4 T and received gadodiamide, MPO-Gd, or CLIO-NPs. T1-weighted MRI was used to assess MPO activity, and T2*-weighted MRI was used to track CLIO-NPs. Immunofluorescent staining and flow cytometric analyses characterized CLIO-NPs, MPO, endothelial cells, and leukocytes. An unpaired, two-tailed Student t test was used to compare groups; Spearman correlation analysis was used to determine the relationship of imaging parameters to clinical severity. Results MPO-Gd enhancement occurred in inflammatory CM hotspots (olfactory bulb > rostral migratory stream > brainstem > cortex, P < .05 for all regions compared with control mice; mean olfactory bulb signal intensity ratio: 1.40 ± 0.07 vs 0.96 ± 0.01, P < .01). The enhancement was reduced in MPO knockout mice (mean signal intensity ratio at 60 minutes: 1.13 ± 0.04 vs 1.40 ± 0.07 in CM, P < .05). Blood-brain barrier compromise was suggested by parenchymal gadolinium enhancement, leukocyte recruitment, and endothelial activation. CLIO-NPs accumulated mainly intravascularly and at the vascular endothelium. CLIO-NPs were also found in the choroid plexus, indicating inflammation of the ventricular system. Blood-cerebrospinal fluid barrier breakdown showed correlation with brain swelling (r2: 0.55, P < .01) and RMCBS score (r2: 0.75, P < .001). Conclusion Iron oxide nanoparticle imaging showed strong inflammatory involvement of the microvasculature in a murine model of cerebral malaria. Furthermore, bis-5-hydroxy-tryptamide-diethylenetriaminepentaacetate gadolinium imaging depicted parenchymal and intraventricular inflammation. This combined molecular imaging approach links vascular inflammation to breakdown of the blood-brain barrier and blood-cerebrospinal fluid barrier that correlate with global brain edema and disease severity. © RSNA, 2018 Online supplemental material is available for this article. See also the editorial by Kiessling in this issue.

High rates of indeterminate interferon-gamma release assays for the diagnosis of latent tuberculosis infection in liver transplantation candidates.

BACKGROUND AND AIMS: Screening for latent tuberculosis infection (LTBI) is recommended prior to solid organ transplantation. Interferon-gamma release assays (IGRAs) are the most widely used test for LTBI screening; however, assessment of IGRA performance in patients with end-stage liver disease is limited. The purpose of this study was to evaluate the prevalence and predictors of indeterminate (INDT) IGRA results in liver transplantation candidates. METHODS: Between March 2011 and May 2018, we retrospectively analyzed 155 patients undergoing liver transplantation assessment, who underwent IGRA testing (Quantiferon-TB Gold, QFT-G) to exclude LTBI. Characteristics of patients, including age, gender, etiology of liver disease, MELD score, and absolute lymphocyte counts, were compared by QFT-G result (determinate vs INDT). RESULTS: Of the 155 patients screened, the rate of positive, negative, and INDT results were 5.2%, 69.8%, and 25%, respectively. The only variable independently associated with an indeterminate test on multivariate analysis was MELD score (odds ratio = 1.07, 95% CI = 1.01-1.14 per unit increase; P = 0.014). In 95% of INDT tests, both TB antigen tube and the positive control tube were negative and repeat testing gave the same indeterminate result, suggestive of anergy rather than laboratory error. CONCLUSIONS: Our study suggests a high rate of INDT IGRA results during screening of liver transplant candidates for LTBI, associated with severity of liver disease and anergy. Because of the high rate of INDT QFT-G testing in this setting, individualized risk assessment is required including a thorough assessment of clinical risk factors and knowledge of local TB prevalence.

Exploring pre-surgery donor-specific antibodies in the context of organ shortage in liver transplant.

BACKGROUND: There is a growing disparity between the number of liver transplant (LT) candidates and availability of suitable liver allografts. Antibody-mediated rejection (AMR), secondary to positive donor-specific antibodies (DSA), remains a concern in liver transplantation. This study aimed to correlate expression of DSA on pre-transplant screening and outcomes of LT, specifically development of AMR in liver allografts and liver function profile in the post-operative period. METHODS: Data of consecutive patients undergoing orthotopic LT (OLT) at the South Australian Liver Transplant Unit was analysed. All patients underwent DSA testing pre-transplant. RESULTS: Within a cohort of 96 patients, over a post-OLT median follow-up of 849 days, only 2 patients (2%) developed AMR. While both patients had a positive DSA test preoperatively, overall DSA positivity was noted in 31% patients, with a specificity for prediction of AMR of 0.708. No significant association was noted between AMR (p = 0.092), T cell-mediated rejection/TCMR (p = 0.797) or late hepatic artery thrombosis/LHAT (p = 0.521). There was no significant interaction effect between DSA positivity and serum bilirubin or transaminases over a period of 100 days. CONCLUSION: AMR following LT is uncommon. A positive DSA pre-transplant does not imply a definite risk of AMR. Also, there does not exist a significant interaction in time between DSA expression and serum bilirubin or transaminase levels. Until there emerges evidence to the contrary, it appears reasonable to consider DSA-positive donors within the broad context of marginal donors in the context of a worldwide shortage of LT donor allografts.

In vivo nanoparticle imaging of innate immune cells can serve as a marker of disease severity in a model of multiple sclerosis.

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.

Ultrasmall Superparamagnetic Iron Oxide Imaging Identifies Tissue and Nerve Inflammation in Pain Conditions.

Objective: Correlation between radiologic structural abnormalities and clinical symptoms in low back pain patients is poor. There is an unmet clinical need to image inflammation in pain conditions to aid diagnosis and guide treatment. Ferumoxytol, an ultrasmall superparamagnetic iron oxide (USPIO) nanoparticle, is clinically used to treat iron deficiency anemia and showed promise in imaging tissue inflammation in human. We explored whether ferumoxytol can be used to identify tissue and nerve inflammation in pain conditions in animals and humans. Methods: Complete Freud's adjuvant (CFA) or saline was injected into mice hind paws to establish an inflammatory pain model. Ferumoxytol (20 mg/kg) was injected intravenously. Magnetic resonance imaging (MRI) was performed prior to injection and 72 hours postinjection. The changes in the transverse relaxation time (T2) before and after ferumoxytol injection were compared between mice that received CFA vs saline injection. In the human study, we administered ferumoxytol (4 mg/kg) to a human subject with clinical symptoms of lumbar radiculopathy and compared the patient with a healthy subject. Results: Mice that received CFA exhibited tissue inflammation and pain behaviors. The changes in T2 before and after ferumoxytol injection were significantly higher in mice that received CFA vs saline (20.8 ± 3.6 vs 2.2 ± 2.5, P = 0.005). In the human study, ferumoxytol-enhanced MRI identified the nerve root corresponding to the patient's symptoms, but the nerve root was not impinged by structural abnormalities, suggesting the potential superiority of this approach over conventional structural imaging techniques. Conclusions: Ferumoxytol-enhanced MRI can identify tissue and nerve inflammation and may provide a promising diagnostic tool in assessing pain conditions in humans.

Spinal Cord Inflammation: Molecular Imaging after Thoracic Aortic Ischemia Reperfusion Injury.

Purpose To evaluate whether noninvasive molecular imaging technologies targeting myeloperoxidase (MPO) can reveal early inflammation associated with spinal cord injury after thoracic aortic ischemia-reperfusion (TAR) in mice. Materials and Methods The study was approved by the institutional animal care and use committee. C57BL6 mice that were 8-10 weeks old underwent TAR (n = 55) or sham (n = 26) surgery. Magnetic resonance (MR) imaging (n = 6) or single photon emission computed tomography (SPECT)/computed tomography (CT) (n = 15) studies targeting MPO activity were performed after intravenous injection of MPO sensors (bis-5-hydroxytryptamide-tetraazacyclododecane [HT]-diethyneletriaminepentaacetic acid [DTPA]-gadolinium or indium 111-bis-5-HT-DTPA, respectively). Immunohistochemistry and flow cytometry were used to identify myeloid cells and neuronal loss. Proinflammatory cytokines, keratinocyte chemoattractant (KC), and interleukin 6 (IL-6) were measured with enzyme-linked immunosorbent assay. Statistical analyses were performed by using nonparametric tests and the Pearson correlation coefficient. P < .05 was considered to indicate a significant difference. Results Myeloid cells infiltrated into the injured cord at 6 and 24 hours after TAR. MR imaging confirmed the presence of ischemic lesions associated with mild MPO-mediated enhancement in the thoracolumbar spine at 24 hours compared with the sham procedure. SPECT/CT imaging of MPO activity showed marked MPO-sensor retention at 6 hours (P = .003) that continued to increase at 24 hours after TAR (P = .0001). The number of motor neurons decreased substantially at 24 hours after TAR (P < .01), which correlated inversely with in vivo inflammatory changes detected at molecular imaging (r = 0.64, P = .0099). MPO was primarily secreted by neutrophils, followed by lymphocyte antigen 6 complexhigh monocytes and/or macrophages. There were corresponding increased levels of proinflammatory cytokines KC (P = .0001) and IL-6 (P = .0001) that mirrored changes in MPO activity. Conclusion MPO is a suitable imaging biomarker for identifying and tracking inflammatory damage in the spinal cord after TAR in a mouse model. © RSNA, 2016 Online supplemental material is available for this article.

Molecular MR Imaging of Myeloperoxidase Distinguishes Steatosis from Steatohepatitis in Nonalcoholic Fatty Liver Disease.

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.

Outcomes and predictors of harmful relapse following liver transplantation for alcoholic liver disease in an Australian population.

BACKGROUND: Limited studies of patient survival and alcoholic relapse following transplantation for alcoholic liver disease have been described in Australian populations. AIM: To describe these outcomes in an Australian population, to determine the association between harmful alcoholic relapse and patient survival, and to examine pre-transplant variables associated with harmful relapse. METHODS: Single centre, retrospective review of consecutive patients transplanted at the South Australian Liver Transplant Unit. Relapse was identified by an independent investigator using case note review and confidential patient questionnaire. RESULTS: A total of 87 patients (median age 52 years, 84% male, median Model for End-Stage Liver Disease (MELD) score 18) was included in the study with a median follow-up time of 50 months. The 1-, 3- and 5-year survival of patients was 93.1, 87.4 and 82.0% respectively. Two deaths were directly attributable to graft failure due to alcohol. Fourteen (16%) patients fulfilled criteria for harmful relapse and 18 (21%) patients experienced any form of relapse to alcohol. Harmful relapse was associated with increased mortality (hazard ratio (HR) 3.2, 95% confidence interval (CI) 1.1-9.7, P = 0.041). Only two factors were independently associated with harmful relapse on multivariate analysis; prior alcohol rehabilitation (HR 8.4, 95% CI 2.5-28.4, P = 0.001) and single versus married status (HR 0.09, 95% CI 0.02-1.2, P = 0.019). CONCLUSION: Good patient survival outcomes were seen for this South Australian population. Harmful alcohol relapse occurs in a minority of patients and rarely results in direct graft loss. Modifiable pre-transplant factors that predict harmful relapse were not identified.

Surface biotinylation of cytotoxic T lymphocytes for in vivo tracking of tumor immunotherapy in murine models.

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 < 0.05). Incubation temperature was not a key factor. CTLs maintained sufficient labeling for at least 72 h (P < 0.05), without altering cell viability. After co-culturing labeled CTLs with mouse glioma stem cells (GSCs) engineered to present biotin on their surface, targeting CTLs could specifically target biotin-presenting GSCs and inhibited cell proliferation (P < 0.01) and tumor spheres formation. In a biotin-presenting GSC brain tumor model, targeting CTLs could be detected in biotin-presenting gliomas in mouse brains but not in the non-tumor-bearing contralateral hemispheres (P < 0.05). In vivo fluorescent molecular tomography imaging in a subcutaneous U87 mouse model confirmed that targeting CTLs homed in on the biotin-presenting U87 tumors but not the control U87 tumors. PET imaging with 89Zr-deferoxamine-biotin and SA showed a rapid clearance of the PET signal over 24 h in the control tumor, while only minimally decreased in the targeted tumor. Thus, sulfo-NHS-biotin-SA labeling is an efficient method to noninvasively track the migration of adoptive transferred CTLs and does not alter CTL viability or interfere with CTL-mediated cytotoxic activity.

Myeloperoxidase Nuclear Imaging for Epileptogenesis.

PURPOSE: To determine if myeloperoxidase (MPO) is involved in epileptogenesis and if molecular nuclear imaging can be used to noninvasively map inflammatory changes in epileptogenesis. MATERIALS AND METHODS: The animal and human studies were approved by the institutional review boards. Pilocarpine-induced epileptic mice were treated with 4-aminobenzoic acid hydrazide (n = 46), a specific irreversible MPO inhibitor, or saline (n = 42). Indium-111-bis-5-hydroxytryptamide-diethylenetriaminepentaacetate was used to image brain MPO activity (n = 6 in the 4-aminobenzoic acid hydrazide and saline groups; n = 5 in the sham group) by using single photon emission computed tomography/computed tomography. The role of MPO in the development of spontaneous recurrent seizures was assessed by means of clinical symptoms and biochemical and histopathologic data. Human brain specimens from a patient with epilepsy and a patient without epilepsy were stained for MPO. The Student t test, one-way analysis of variance, and Mann-Whitney and Kruskal-Wallis tests were used. Differences were regarded as significant if P was less than .05. RESULTS: MPO and leukocytes increased in the brain during epileptogenesis (P < .05). Blocking MPO delayed spontaneous recurrent seizures (99.6 vs 142 hours, P = .016), ameliorated the severity of spontaneous recurrent seizures (P < .05), and inhibited mossy fiber sprouting (Timm index, 0.31 vs 0.03; P = .003). Matrix metalloproteinase activity was upregulated during epileptogenesis in an MPO-dependent manner (1.44 vs 0.94 U/mg, P = .049), suggesting that MPO acts upstream of matrix metalloproteinases. MPO activity was mapped during epileptogenesis in vivo in the hippocampal regions. Resected temporal lobe tissue from a human patient with refractory epilepsy but not the temporal lobe tissue from a patient without seizures demonstrated positive MPO immunostaining, suggesting high translational potential for this imaging technology. CONCLUSION: The findings of this study highlight an important role for MPO in epileptogenesis and show MPO to be a potential therapeutic target and imaging biomarker for epilepsy.

Myeloperoxidase Inhibition Increases Neurogenesis after Ischemic Stroke.

The relationship between inflammation and neurogenesis in stroke is currently not well understood. Focal ischemia enhances cell proliferation and neurogenesis in the neurogenic regions, including the subventricular zone (SVZ), dentate gyrus, as well as the non-neurogenic striatum, and cortex in the ischemic hemisphere. Myeloperoxidase (MPO) is a potent oxidizing enzyme secreted during inflammation by activated leukocytes, and its enzymatic activity is highly elevated after stroke. In this study, we investigated whether the inhibition of MPO activity by a specific irreversible inhibitor, 4-aminobenzoic acid hydrazide (ABAH) (MPO-/- mice) can increase neurogenesis after transient middle cerebral artery occlusion in mice. ABAH administration increased the number of proliferating bromodeoxyuridine (BrdU)-positive cells expressing markers for neural stems cells, astrocytes, neuroprogenitor cells (Nestin), and neuroblasts (doublecortin) in the ischemic SVZ, anterior SVZ, striatum, and cortex. MPO inhibition also increased levels of brain-derived neurotrophic factor, phosphorylation of cAMP response element-binding protein (Ser133), acetylated H3, and NeuN to promote neurogenesis in the ischemic SVZ. ABAH treatment also increased chemokine CXC receptor 4 expression in the ischemic SVZ. MPO-deficient mice treated with vehicle or ABAH both showed similar effects on the number of BrdU+ cells in the ischemic hemisphere, demonstrating that ABAH is specific to MPO. Taken together, our results underscore a detrimental role of MPO activity to postischemia neurogenesis and that a strategy to inhibit MPO activity can increase cell proliferation and improve neurogenesis after ischemic stroke.