Clinical and Immunologic Correlates of Vasodilatory Shock Among Ebola Virus-Infected Nonhuman Primates in a Critical Care Model.

BACKGROUND: Existing models of Ebola virus infection have not fully characterized the pathophysiology of shock in connection with daily virologic, clinical, and immunologic parameters. We implemented a nonhuman primate critical care model to investigate these associations. METHODS: Two rhesus macaques received a target dose of 1000 plaque-forming units of Ebola virus intramuscularly with supportive care initiated on day 3. High-dimensional spectral cytometry was used to phenotype neutrophils and peripheral blood mononuclear cells daily. RESULTS: We observed progressive vasodilatory shock with preserved cardiac function following viremia onset on day 5. Multiorgan dysfunction began on day 6 coincident with the nadir of circulating neutrophils. Consumptive coagulopathy and anemia occurred on days 7 to 8 along with irreversible shock, followed by death. The monocyte repertoire began shifting on day 4 with a decline in classical and expansion of double-negative monocytes. A selective loss of CXCR3-positive B and T cells, expansion of naive B cells, and activation of natural killer cells followed viremia onset. CONCLUSIONS: Our model allows for high-fidelity characterization of the pathophysiology of acute Ebola virus infection with host innate and adaptive immune responses, which may advance host-targeted therapy design and evaluation for use after the onset of multiorgan failure.

Ebola Virus Disease Features Hemophagocytic Lymphohistiocytosis/Macrophage Activation Syndrome in the Rhesus Macaque Model.

BACKGROUND: Ebola virus (EBOV) disease (EVD) is one of the most severe and fatal viral hemorrhagic fevers and appears to mimic many clinical and laboratory manifestations of hemophagocytic lymphohistiocytosis syndrome (HLS), also known as macrophage activation syndrome. However, a clear association is yet to be firmly established for effective host-targeted, immunomodulatory therapeutic approaches to improve outcomes in patients with severe EVD. METHODS: Twenty-four rhesus monkeys were exposed intramuscularly to the EBOV Kikwit isolate and euthanized at prescheduled time points or when they reached the end-stage disease criteria. Three additional monkeys were mock-exposed and used as uninfected controls. RESULTS: EBOV-exposed monkeys presented with clinicopathologic features of HLS, including fever, multiple organomegaly, pancytopenia, hemophagocytosis, hyperfibrinogenemia with disseminated intravascular coagulation, hypertriglyceridemia, hypercytokinemia, increased concentrations of soluble CD163 and CD25 in serum, and the loss of activated natural killer cells. CONCLUSIONS: Our data suggest that EVD in the rhesus macaque model mimics pathophysiologic features of HLS/macrophage activation syndrome. Hence, regulating inflammation and immune function might provide an effective treatment for controlling the pathogenesis of acute EVD.

Magnetic Resonance Imaging for Monitoring of Hepatic Disease Induced by Ebola Virus: a Nonhuman Primate Proof-of-Concept Study.

Severe liver impairment is a well-known hallmark of Ebola virus disease (EVD). However, the role of hepatic involvement in EVD progression is understudied. Medical imaging in established animal models of EVD (e.g., nonhuman primates [NHPs]) can be a strong complement to traditional assays to better investigate this pathophysiological process in vivo and noninvasively. In this proof-of-concept study, we used longitudinal multiparametric magnetic resonance imaging (MRI) to characterize liver morphology and function in nine rhesus monkeys after exposure to Ebola virus (EBOV). Starting 5 days postexposure, MRI assessments of liver appearance, morphology, and size were consistently compatible with the presence of hepatic edema, inflammation, and congestion, leading to significant hepatomegaly at necropsy. MRI performed after injection of a hepatobiliary contrast agent demonstrated decreased liver signal on the day of euthanasia, suggesting progressive hepatocellular dysfunction and hepatic secretory impairment associated with EBOV infection. Importantly, MRI-assessed deterioration of biliary function was acute and progressed faster than changes in serum bilirubin concentrations. These findings suggest that longitudinal quantitative in vivo imaging may be a useful addition to standard biological assays to gain additional knowledge about organ pathophysiology in animal models of EVD. IMPORTANCE Severe liver impairment is a well-known hallmark of Ebola virus disease (EVD), but the contribution of hepatic pathophysiology to EVD progression is not fully understood. Noninvasive medical imaging of liver structure and function in well-established animal models of disease may shed light on this important aspect of EVD. In this proof-of-concept study, we used longitudinal magnetic resonance imaging (MRI) to characterize liver abnormalities and dysfunction in rhesus monkeys exposed to Ebola virus. The results indicate that in vivo MRI may be used as a noninvasive readout of organ pathophysiology in EVD and may be used in future animal studies to further characterize organ-specific damage of this condition, in addition to standard biological assays.

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.

Expanded Histopathology and Tropism of Ebola Virus in the Rhesus Macaque Model: Potential for Sexual Transmission, Altered Adrenomedullary Hormone Production, and Early Viral Replication in Liver.

The pathogenesis of Ebola virus disease (EVD) is still incomplete, in spite of the availability of a nonhuman primate modelfor more than 4 decades. To further investigate EVD pathogenesis, a natural history study was conducted using 27 Chinese-origin rhesus macaques. Of these, 24 macaques were exposed intramuscularly to Kikwit Ebola virus and euthanized at predetermined time points or when end-stage clinical disease criteria were met, and 3 sham-exposed macaques were euthanized on study day 0. This study showed for the first time that Ebola virus causes uterine cervicitis, vaginitis, posthitis, and medullary adrenalitis. Not only was Ebola virus detected in the interstitial stromal cells of the genital tract, but it was also present in the epididymal and seminal vesicular tubular epithelial cells, ectocervical and vaginal squamous epithelial cells, and seminal fluid. Furthermore, as early as day 3 after exposure, Ebola virus replicative intermediate RNA was detected in Kupffer cells and hepatocytes. These findings in the nonhuman model provide additional insight into potential sexual transmission, possible disruption of sympathetic hormone production, and early virus replication sites in human EVD patients.

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.

Kikwit Ebola Virus Disease Progression in the Rhesus Monkey Animal Model.

Ongoing Ebola virus disease outbreaks in the Democratic Republic of the Congo follow the largest recorded outbreak in Western Africa (2013-2016). To combat outbreaks, testing of medical countermeasures (therapeutics or vaccines) requires a well-defined, reproducible, animal model. Here we present Ebola virus disease kinetics in 24 Chinese-origin rhesus monkeys exposed intramuscularly to a highly characterized, commercially available Kikwit Ebola virus Filovirus Animal Non-Clinical Group (FANG) stock. Until reaching predetermined clinical disease endpoint criteria, six animals underwent anesthesia for repeated clinical sampling and were compared to six that did not. Groups of three animals were euthanized and necropsied on days 3, 4, 5, and 6 post-exposure, respectively. In addition, three uninfected animals served as controls. Here, we present detailed characterization of clinical and laboratory disease kinetics and complete blood counts, serum chemistries, Ebola virus titers, and disease kinetics for future medical countermeasure (MCM) study design and control data. We measured no statistical difference in hematology, chemistry values, or time to clinical endpoint in animals that were anesthetized for clinical sampling during the acute disease compared to those that were not.

Characterizing Clinical Genetic Counselors' Countertransference Experiences: an Exploratory Study.

Countertransference (CT) refers to conscious and unconscious emotions, fantasies, behaviors, perceptions, and psychological defenses genetic counselors experience in response to any aspect of genetic counseling situations (Weil 2010). Some authors theorize about the importance of recognizing and managing CT, but no studies solely aim to explore genetic counselors' experiences of the phenomenon. This study examined the extent to which clinical genetic counselors' perceive themselves as inclined to experience CT, gathered examples of CT encountered in clinical situations, and assessed their CT management strategies. An anonymous online survey, sent to NSGC members, yielded 127 usable responses. Participants completed Likert-type items rating their CT propensities; 57 of these individuals also provided examples of CT they experienced in their practice. Factor analysis of CT propensities tentatively suggested four factors: Control, Conflict Avoidance, Directiveness, and Self-Regulation, accounting for 38.5% of response variance. Thematic analysis of CT examples yielded five common triggers: general similarity to patient, medical/genetic similarity, angry patients, patient behaves differently from counselor expectations, and disclosing bad news; six common manifestations: being self-focused, projecting feelings onto the patient, intense emotional reaction to patient, being overly invested, disengagement, and physical reaction; five CT effects: disruption in rapport building, repaired empathy, over-identification, conversation does not reach fullest potential, and counselor is drained emotionally; and three management strategies: recognizing CT as it occurs, self-reflection, and consultation. Results suggest CT is a common experience, occurring in both "routine" and emotionally complex cases. Training programs, continuing education, and peer supervision might include discussion of CT, informed by examples from the present study, to increase genetic counselor awareness and skills for managing the phenomenon.

Comparison of respiratory inductive plethysmography versus head-out plethysmography for anesthetized nonhuman primates in an animal biosafety level 4 facility.

For inhalational studies and aerosol exposures to viruses, head-out plethysmography acquisition has been traditionally used for the determination of estimated inhaled dose in anesthetized nonhuman primates prior to or during an aerosol exposure. A pressure drop across a pneumotachograph is measured within a sealed chamber during inspiration/exhalation of the nonhuman primate, generating respiratory values and breathing frequencies. Due to the fluctuation of depth of anesthesia, pre-exposure respiratory values can be variable, leading to less precise and accurate dosing calculations downstream. Although an anesthesia infusion pump may help stabilize the depth of sedation, pumps are difficult to use within a sealed head-out plethysmography chamber. Real-time, head-out plethysmography acquisition could increase precision and accuracy of the measurements, but the bulky equipment needed for head-out plethysmography precludes real-time use inside a Class III biological safety cabinet, where most aerosol exposures occur. However, the respiratory inductive plethysmography (RIP) acquisition method measures the same respiratory parameters by detecting movement of the chest and abdomen during breathing using two elastic bands within the Class III biological safety cabinet. As respiratory values are relayed to a computer for software integration and analysis real-time, adjustment of aerosol exposure duration is based on the depth of sedation of the animal. The objective of this study was to compare values obtained using two methodologies (pre-exposure head-out plethysmography and real-time RIP). Transitioning to RIP technology with real-time acquisition provides more consistent, precise, and accurate aerosol dosing by reducing reported errors in respiratory values from anesthesia variability when using pre-exposure head-out plethysmography acquisition.

Characterization of clinical and environmental Mycobacterium avium spp. isolates and their interaction with human macrophages.

Members of the Mycobacterium avium complex (MAC) are naturally occurring bacteria in the environment. A link has been suggested between M. avium strains in drinking water and clinical isolates from infected individuals. There is a need to develop new screening methodologies that can identify specific virulence properties of M. avium isolates found in water that predict a level of risk to exposed individuals. In this work we have characterized 15 clinical and environmental M. avium spp. isolates provided by the US Environmental Protection Agency (EPA) to improve our understanding of the key processes involved in the binding, uptake and survival of these isolates in primary human macrophages. M. avium serovar 8 was predominant among the isolates studied. Different amounts and exposure of mannose-capped lipoarabinomannan (ManLAM) and glycopeptidolipids (GPLs), both major mycobacterial virulence factors, were found among the isolates studied. Reference clinical isolate 104 serovar 1 and clinical isolates 11 and 14 serovar 8 showed an increased association with macrophages. Serum opsonization increased the cell association and survival at 2 h post infection for all isolates. However, only the clinical isolates 104 and 3 among those tested showed an increased growth in primary human macrophages. The other isolates varied in their survival in these cells. Thus we conclude that the amounts of cell envelope ManLAM and GPL, as well as GPL serovar specificity are not the only important bacterial factors for dictating the early interactions of M. avium with human macrophages.