PET imaging of TSPO expression in immune cells can assess organ-level pathophysiology in high-consequence viral infections.

Ebola virus (EBOV) disease is characterized by lymphopenia, breach in vascular integrity, cytokine storm, and multiorgan failure. The pathophysiology of organ involvement, however, is incompletely understood. Using [18F]-DPA-714 positron emission tomography (PET) imaging targeting the translocator protein (TSPO), an immune cell marker, we sought to characterize the progression of EBOV-associated organ-level pathophysiology in the EBOV Rhesus macaque model. Dynamic [18F]-DPA-714 PET/computed tomography imaging was performed longitudinally at baseline and at multiple time points after EBOV inoculation, and distribution volumes (Vt) were calculated as a measure of peripheral TSPO binding. Using a mixed-effect linear regression model, spleen and lung Vt decreased, while the bone marrow Vt increased over time after infection. No clear trend was found for liver Vt. Multiple plasma cytokines correlated negatively with lung/spleen Vt and positively with bone marrow Vt. Multiplex immunofluorescence staining in spleen and lung sections confirmed organ-level lymphoid and monocytic loss/apoptosis, thus validating the imaging results. Our findings are consistent with EBOV-induced progressive monocytic and lymphocytic depletion in the spleen, rather than immune activation, as well as depletion of alveolar macrophages in the lungs, with inefficient reactive neutrophilic activation. Increased bone marrow Vt, on the other hand, suggests hematopoietic activation in response to systemic immune cell depletion and leukocytosis and could have prognostic relevance. In vivo PET imaging provided better understanding of organ-level pathophysiology during EBOV infection. A similar approach can be used to delineate the pathophysiology of other systemic infections and to evaluate the effectiveness of newly developed treatment and vaccine strategies.

MRI demonstrates glutamine antagonist-mediated reversal of cerebral malaria pathology in mice.

The deadliest complication of Plasmodium falciparum infection is cerebral malaria (CM), with a case fatality rate of 15 to 25% in African children despite effective antimalarial chemotherapy. No adjunctive treatments are yet available for this devastating disease. We previously reported that the glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) rescued mice from experimental CM (ECM) when administered late in the infection, a time by which mice had already suffered blood-brain barrier (BBB) dysfunction, brain swelling, and hemorrhaging. Herein, we used longitudinal MR imaging to visualize brain pathology in ECM and the impact of a new DON prodrug, JHU-083, on disease progression in mice. We demonstrate in vivo the reversal of disease markers in symptomatic, infected mice following treatment, including the resolution of edema and BBB disruption, findings usually associated with a fatal outcome in children and adults with CM. Our results support the premise that JHU-083 is a potential adjunctive treatment that could rescue children and adults from fatal CM.

In vivo imaging of sterile microglial activation in rat brain after disrupting the blood-brain barrier with pulsed focused ultrasound: [18F]DPA-714 PET study.

BACKGROUND: Magnetic resonance imaging (MRI)-guided pulsed focused ultrasound combined with the infusion of microbubbles (pFUS+MB) induces transient blood-brain barrier opening (BBBO) in targeted regions. pFUS+MB, through the facilitation of neurotherapeutics' delivery, has been advocated as an adjuvant treatment for neurodegenerative diseases and malignancies. Sterile neuroinflammation has been recently described following pFUS+MB BBBO. In this study, we used PET imaging with [18F]-DPA714, a biomarker of translocator protein (TSPO), to assess for neuroinflammatory changes following single and multiple pFUS+MB sessions. METHODS: Three groups of Sprague-Dawley female rats received MRI-guided pFUS+MB (Optison™; 5-8 × 107 MB/rat) treatments to the left frontal cortex and right hippocampus. Group A rats were sonicated once. Group B rats were sonicated twice and group C rats were sonicated six times on weekly basis. Passive cavitation detection feedback (PCD) controlled the peak negative pressure during sonication. We performed T1-weighted scans immediately after sonication to assess efficiency of BBBO and T2*-weighted scans to evaluate for hypointense voxels. [18F]DPA-714 PET/CT scans were acquired after the BBB had closed, 24 h after sonication in group A and within an average of 10 days from the last sonication in groups B and C. Ratios of T1 enhancement, T2* values, and [18F]DPA-714 percent injected dose/cc (%ID/cc) values in the targeted areas to the contralateral brain were calculated. Histological assessment for microglial activation/astrocytosis was performed. RESULTS: In all groups, [18F]DPA-714 binding was increased at the sonicated compared to non-sonicated brain (%ID/cc ratios > 1). Immunohistopathology showed increased staining for microglial and astrocytic markers in the sonicated frontal cortex compared to contralateral brain and to a lesser extent in the sonicated hippocampus. Using MRI, we documented BBB disruption immediately after sonication with resolution of BBBO 24 h later. We found more T2* hypointense voxels with increasing number of sonications. In a longitudinal group of animals imaged after two and after six sonications, there was no cumulative increase of neuroinflammation on PET. CONCLUSION: Using [18F]DPA-714 PET, we documented in vivo neuroinflammatory changes in association with pFUS+MB. Our protocol (utilizing PCD feedback to minimize damage) resulted in neuroinflammation visualized 24 h post one sonication. Our findings were supported by immunohistochemistry showing microglial activation and astrocytosis. Experimental sonication parameters intended for BBB disruption should be evaluated for neuroinflammatory sequelae prior to implementation in clinical trials.

Brain 18F-FDG PET of SIV-infected macaques after treatment interruption or initiation.

BACKGROUND: Although rates of severe HIV-associated neurocognitive disorders have declined in the post-antiretroviral treatment (ART) era, subtle deficits persist, possibly exacerbated by treatment non-adherence. The actual effects of ART interruption/initiation on brain glucose metabolism as a reflection of viral replication and neuroinflammation remain unclear. Our study investigates how treatment initiation and interruption alter brain glucose metabolism in SIV-infected macaques, using 18F-FDG PET in correlation with plasma and CSF viral loads (VL) and cytokine levels. METHODS: SIV-infected macaques (n = 7) underwent ART initiation only, ART interruption only, or both. Five uninfected animals served as controls. 18F-FDG PET imaging was performed at baseline and 1, 3, and 6 months after treatment modification. Mean and maximum standardized uptake values (SUV) for the whole-brain and subregions were calculated. Plasma and CSF VL and cytokine levels were measured. Paired t tests evaluated acute changes in whole-brain SUV from baseline to 1 month, while mixed-effect linear regression models evaluated changes over multiple timepoints and correlated SUV values with disease markers. RESULTS: ART interruption was associated with increased SUVmean and SUVmax acutely, after 1 month (SUVmean 95% CI [0.044-0.786 g/ml], p = 0.037; SUVmax 95% CI [0.122-3.167 g/ml], p = 0.041). The correlation between SUV and time, however, was not significant when evaluated across all timepoints. Increased SUVmean and SUVmax correlated with decreased CD4+ and CD8+ T-cell counts and increased plasma VL. SUVmax was positively associated with increases in CSF VL, and there were borderline positive associations between SUVmax and IL-2, and between SUVmean and IL-15. The treatment initiation group showed no associations between imaging and disease biomarkers despite viral suppression, reduced cytokine levels, and increased CD4+ and CD8+ T-cell counts. CONCLUSIONS: ART interruption is associated with increased brain glucose metabolism within 1 month of treatment cessation, which, in concert with increased levels of pro-inflammatory cytokines in the CSF, may reflect neuroinflammation in the setting of viral rebound. Although we cannot assert neurologic damage in association with cerebral hypermetabolism, it is a concerning outcome of ART non-adherence. Treatment initiation, meanwhile, did not result in significant changes in brain metabolism. HIV-induced neuroinflammation may require a longer period to abate than our follow-up period allowed.

Treatment Outcomes in American Football Players After Intervertebral Disk Herniation: Systematic Review and Meta-Analysis.

BACKGROUND: American football players are at increased risk for many forms of spinal injury. Intervertebral disk herniations are particularly concerning as they are the leading cause of days lost to injury and can have long-term effects on player careers. Disk herniation management plays a major role in the likelihood and success of return-to-play (RTP). OBJECTIVE: To assess the incidence, demographic variables, treatment approaches, outcomes, and RTP rates of disk herniations in American football players. METHODS: A systematic review of the literature investigating disk herniations in American football players using PubMed, Cochrane Library, and Embase was performed. RTP estimates were calculated by pooling study-specific data using a random-effects model. RESULTS: Four hundred twenty-two studies were identified, with 18 meeting inclusion criteria. Offensive and defensive linemen were the 2 most commonly injured positions. Players undergoing operations were on average younger, with higher body mass indexes, fewer seasons played, and longer post-treatment careers than nonsurgical counterparts. Postsurgical recovery periods lasted an average 106 d, with a mean RTP duration of 33 games over 2.7 yr and an 8.45% reoperation rate. Operative treatment offered a nonsignificant increase in the likelihood of return-to-play compared with nonoperative treatment (odds ratio = 2.81, 95% CI 0.83-9.51). CONCLUSION: Disk herniations are a common injury, with surgery potentially improving post-treatment outcomes. The literature suffers from heterogeneous definitions of RTP and varying performance metrics, making it difficult to draw clear conclusions. To better understand the impact of disk herniation and treatment on player health and performance, more studies should be performed prospectively and with standardized metrics.

Assessing organ-level immunoreactivity in a rat model of sepsis using TSPO PET imaging.

There is current need for new approaches to assess/measure organ-level immunoreactivity and ensuing dysfunction in systemic inflammatory response syndrome (SIRS) and sepsis, in order to protect or recover organ function. Using a rat model of systemic sterile inflammatory shock (intravenous LPS administration), we performed PET imaging with a translocator protein (TSPO) tracer, [18F]DPA-714, as a biomarker for reactive immunoreactive changes in the brain and peripheral organs. In vivo dynamic PET/CT scans showed increased [18F]DPA-714 binding in the brain, lungs, liver and bone marrow, 4 hours after LPS injection. Post-LPS mean standard uptake values (SUVmean) at equilibrium were significantly higher in those organs compared to baseline. Changes in spleen [18F]DPA-714 binding were variable but generally decreased after LPS. SUVmean values in all organs, except the spleen, positively correlated with several serum cytokines/chemokines. In vitro measures of TSPO expression and immunofluorescent staining validated the imaging results. Noninvasive molecular imaging with [18F]DPA-714 PET in a rat model of systemic sterile inflammatory shock, along with in vitro measures of TSPO expression, showed brain, liver and lung inflammation, spleen monocytic efflux/lymphocytic activation and suggested increased bone marrow hematopoiesis. TSPO PET imaging can potentially be used to quantify SIRS and sepsis-associated organ-level immunoreactivity and assess the effectiveness of therapeutic and preventative approaches for associated organ failures, in vivo.

Symptomatic Epidural Hematoma After Elective Cervical Spine Surgery: Incidence, Timing, Risk Factors, and Associated Complications.

BACKGROUND: As the rate of elective cervical spine surgery increases, studies of complications may improve quality of care. Symptomatic postoperative cervical epidural hematomas (PCEH) are rare but result in significant morbidity. Because of their low incidence, the risk factors and complications associated with symptomatic PCEH remain unclear. OBJECTIVE: To clarify the prevalence, timing, variables, and complications associated with PCEH following elective cervical spine surgery. METHODS: Using the American College of Surgeons National Surgical Quality Improvement Program database, cervical spine surgeries performed between 2012 and 2016 were identified using Current Procedural Terminology codes. Symptomatic PCEH was defined as readmission or reoperation events specifically associated with International Classification of Diseases code diagnoses of postoperative hematoma within 30 d of index surgery. Multivariate models were created to assess the independent association of symptomatic PCEH with other postoperative complications. RESULTS: There were 53233 patients included for analysis. The overall incidence of symptomatic PCEH was 0.4% (n = 198). Reoperation occurred in 158 cases (78.8%), of which 2 required a second reoperation (1.3%). The majority (91.8%) of hematomas occurred within 15 d of surgery. Multivariate analysis identified male gender, American Society of Anesthesiologists classes 3 to 5, bleeding disorder, increasing number of operative levels, revision surgery, dural repair, and perioperative transfusion as independent factors associated with PCEH. Upon controlling for those confounders, PCEH was independently associated with cardiac arrest, stroke, deep vein thrombosis, surgical site infection, and pneumonia. CONCLUSION: Postoperative epidural hematomas requiring readmission or reoperation following elective cervical spine surgery occurred at an incidence of 0.4%. Symptomatic PCEHs are associated with increased rates of numerous major morbidities.

Postoperative Prophylactic Antibiotics in Spine Surgery: A Propensity-Matched Analysis.

BACKGROUND: Surgical site infections are common and costly complications after spine surgery. Prophylactic antibiotics are the standard of care; however, the appropriate duration of antibiotics has yet to be adequately addressed. We sought to determine whether the duration of antibiotic administration (preoperatively only versus preoperatively and for 24 hours postoperatively) impacts postoperative infection rates. METHODS: All patients undergoing inpatient spinal procedures at a single institution from 2011 to 2018 were evaluated for inclusion. A minimum of 1 year of follow-up was used to adequately capture postoperative infections. The 1:1 nearest-neighbor propensity score matching technique was used between patients who did and did not receive postoperative antibiotics, and multivariable logistic regression analysis was conducted to control for confounding. RESULTS: A total of 4,454 patients were evaluated and, of those, 2,672 (60%) received 24 hours of postoperative antibiotics and 1,782 (40%) received no postoperative antibiotics. After propensity-matched analysis, there was no difference between patients who received postoperative antibiotics and those who did not in terms of the infection rate (1.8% compared with 1.5%). No significant decrease in the odds of postoperative infection was noted in association with the use of postoperative antibiotics (odds ratio = 1.17; 95% confidence interval, 0.620 to 2.23; p = 0.628). Additionally, there was no observed increase in the risk of Clostridium difficile infection or in the short-term rate of infection with multidrug-resistant organisms. CONCLUSIONS: There was no difference in the rate of surgical site infections between patients who received 24 hours of postoperative antibiotics and those who did not. Additionally, we found no observable risks, such as more antibiotic-resistant infections and C. difficile infections, with prolonged antibiotic use. LEVEL OF EVIDENCE: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

Validation of rule-based algorithms to determine colorectal, breast, and cervical cancer screening status using electronic health record data from an urban healthcare system in New York City.

Although cancer screening has greatly reduced colorectal cancer, breast cancer, and cervical cancer morbidity and mortality over the last few decades, adherence to cancer screening guidelines remains inconsistent, particularly among certain demographic groups. This study aims to validate a rule-based algorithm to determine adherence to cancer screening. A novel screening algorithm was applied to electronic health record (EHR) from an urban healthcare system in New York City to automatically determine adherence to national cancer screening guidelines for patients deemed eligible for screening. First, a subset of patients was randomly selected from the EHR and their data were exported in a de-identified manner for manual review of screening adherence by two teams of human reviewers. Interrater reliability for manual review was calculated using Cohen's Kappa and found to be high in all instances. The sensitivity and specificity of the algorithm was calculated by comparing the algorithm to the final manual dataset. When assessing cancer screening adherence, the algorithm performed with a high sensitivity (79%, 70%, 80%) and specificity (92%, 99%, 97%) for colorectal cancer, breast cancer, and cervical cancer screenings, respectively. This study validates an algorithm that can effectively determine patient adherence to colorectal cancer, breast cancer, and cervical cancer screening guidelines. This design improves upon previous methods of algorithm validation by using computerized extraction of essential components of patients' EHRs and by using de-identified data for manual review. Use of the described algorithm could allow for more precise and efficient allocation of public health resources to improve cancer screening rates.

Monitoring Immune Activation with Whole-Body Fluorodeoxyglucose-Positron-Emission Tomography in Simian Immunodeficiency Virus-Infected Rhesus Macaques.

This study aimed to assess immune activation in tissues by measuring glucose metabolism with 18F-fluorodeoxyglucose (FDG) and investigate the associations of various peripheral markers of disease progression with initiation and interruption of combination antiretroviral therapy in SIV-infected rhesus macaques (Macaca mulatta). Mixed-effect linear models revealed a significant inverse association of peripheral blood CD4+ T cell counts (p < 0.01) and a direct association of plasma viral load (p < 0.01) with the FDG uptake in the spleen, bone marrow, and most clusters of lymph nodes. In contrast, no significant associations were found for the liver and the bowel FDG uptake. We also found no association of the fraction of proliferating peripheral blood T and B lymphocytes with FDG uptake in any analyzed tissues. The bowel FDG uptake of uninfected animals was heterogeneous and reached levels as high as those seen in the bowel or the clusters of lymph nodes or the spleen of high viremic SIV-infected animals, suggesting that factors beyond SIV-induced immune activation dominate the gut FDG uptake.

Longitudinal in vivo imaging of acute neuropathology in a monkey model of Ebola virus infection.

Ebola virus (EBOV) causes neurological symptoms yet its effects on the central nervous system (CNS) are not well-described. Here, we longitudinally assess the acute effects of EBOV on the brain, using quantitative MR-relaxometry, 18F-Fluorodeoxyglucose PET and immunohistochemistry in a monkey model. We report blood-brain barrier disruption, likely related to high cytokine levels and endothelial viral infection, with extravasation of fluid, Gadolinium-based contrast material and albumin into the extracellular space. Increased glucose metabolism is also present compared to the baseline, especially in the deep gray matter and brainstem. This regional hypermetabolism corresponds with mild neuroinflammation, sporadic neuronal infection and apoptosis, as well as increased GLUT3 expression, consistent with increased neuronal metabolic demands. Neuroimaging changes are associated with markers of disease progression including viral load and cytokine/chemokine levels. Our results provide insight into the pathophysiology of CNS involvement with EBOV and may help assess vaccine/treatment efficacy in real time.

Potential Mechanism for HIV-Associated Depression: Upregulation of Serotonin Transporters in SIV-Infected Macaques Detected by 11C-DASB PET.

Purpose: Increased incidence of depression in HIV+ patients is associated with lower adherence to treatment and increased morbidity/mortality. One possible underlying pathophysiology is serotonergic dysfunction. In this study, we used an animal model of HIV, the SIV-infected macaque, to longitudinally image serotonin transporter (SERT) expression before and after inoculation, using 11C-DASB (SERT ligand) PET imaging. Methods: We infected seven rhesus macaques with a neurovirulent SIV strain and imaged them at baseline and multiple time points after inoculation (group A). Pyrosequencing methylation analysis of the SERT promoter region was performed. We also measured SERT mRNA/protein in brain single-cell suspensions from another group (group B) of SIV-infected animals (n = 13). Results: Despite some animals showing early fluctuations, 86% of our group A animals eventually showed a net increase in midbrain/thalamus binding potential (BPND) over the course of their disease (mean increased binding between last time point and baseline = 30.2% and 32.2%, respectively). Repeated-measures mixed-model analysis showed infection duration to be predictive of midbrain BPND (p = 0.039). Thalamic BPND was statistically significantly associated with multiple CSF cytokines (P < 0.05). There was higher SERT protein levels in the second group (group B) of SIV-infected animals with SIV encephalitis (SIVE) compared to those without SIVE (p = 0.014). There were no longitudinal changes in SERT gene promoter region percentage methylation between baselines and last time points in group A animals. Conclusion: Upregulated SERT leading to lower synaptic levels of serotonin is a possible mechanism of depression in HIV+ patients, and extrapolating our conclusions from SIV to HIV should be sought using translational human studies.

Neuroinflammatory Changes in Relation to Cerebrospinal Fluid Viral Load in Simian Immunodeficiency Virus Encephalitis.

The exact cause of neurocognitive dysfunction in HIV-positive patients despite successful control of the infection in the periphery is not completely understood. One suggested mechanism is a vicious cycle of microglial activation and release of proinflammatory chemokines/cytokines that eventually leads to neuronal loss and dysfunction. However, the exact role of microglial activation in the earliest stages of the infection with high cerebrospinal fluid (CSF) viral loads (VL) is unclear. In this study, we imaged the translocator protein (TSPO), a mitochondrial membrane receptor known to be upregulated in activated microglia and macrophages, in rhesus macaques before and multiple times after inoculation with a neurotropic simian immunodeficiency virus (SIV) strain (SIVsm804E), using 18F-DPA714 positron emission tomography (PET). The whole-brain standardized uptake values of TSPO at equilibrium reflecting total binding (SUVT) and binding potentials (BPND) were calculated and correlated with CSF and serum markers of disease, and a corresponding postmortem immunostaining analysis was also performed. SUVT was found to be inversely correlated with both CSF VL and monocyte chemoattractant protein 1 (MCP-1) levels. In SIV-infected macaques with very high CSF VL at necropsy (>106 copies/ml), we found decreased TSPO binding by PET, and this was supported by immunostaining which showed glial and neuronal apoptosis rather than microglial activation. On the other hand, with only moderately elevated CSF VL (∼104 copies/ml), we found increased TSPO binding as well as focal and diffuse microglial activation on immunostaining. Our results in the SIV-infected macaque model provide insights into the relationship between HIV neuropathology and CSF VL at various stages of the disease.IMPORTANCE Neurological and cognitive problems are a common complication of HIV infection and are prevalent even in treated individuals. Although the molecular processes underlying brain involvement with HIV are not completely understood, inflammation is suspected to play a significant role. Our work presents an in vivo assessment of neuroinflammation in an animal model of HIV, the simian immunodeficiency virus (SIV)-infected rhesus macaque. Using positron emission tomography (PET) imaging, we identified changes in brain inflammation after inoculation with SIV over time. Interestingly, we found decreased binding of the PET ligand in the presence of very high cerebrospinal fluid (CSF) viral loads. These findings were supported by immunostaining which showed marked glial loss instead of inflammation. This study provides insight into glial and neuronal changes associated with very high CSF viral load and could reflect similar changes occurring in HIV-infected patients.

A transposon-mediated system for flexible control of transgene expression in stem and progenitor-derived lineages.

Precise methods for transgene regulation are important to study signaling pathways and cell lineages in biological systems where gene function is often recycled within and across lineages. We engineered a genetic toolset for flexible transgene regulation in these diverse cellular contexts. Specifically, we created an optimized piggyBac transposon-based system, allowing for the facile generation of stably transduced cell lineages in vivo and in vitro. The system, termed pB-Tet-GOI (piggyBac-transposable tetracycline transactivator-mediated flexible expression of a genetic element of interest), incorporates the latest generation of tetracycline (Tet) transactivator and reverse Tet transactivator variants--along with engineered mutants--in order to provide regulated transgene expression upon addition or removal of doxycycline (dox). Altogether, the flexibility of the system allows for dox-induced, dox-suppressed, dox-resistant (i.e., constitutive), and dox-induced/constitutive regulation of transgenes. This versatile strategy provides reversible temporal regulation of transgenes with robust inducibility and minimal leakiness.