William C. Reid

Human mesenchymal stem cells inhibit endothelial proliferation and angiogenesis via cell-cell contact through modulation of the VE-Cadherin/β-catenin signaling pathway.

Over the past 10 years, a great deal has been learned about the fundamental biology and therapeutic application of bone marrow-derived human mesenchymal stem cells (MSCs). Intravenous administration of these cells is the preferred route for therapeutic delivery of MSCs. Vascular endothelial cells (ECs) are the first cell type that MSCs encounter following IV administration. However, little is known about the biological consequences of interactions between MSCs and ECs, and if any therapeutic benefit results from this interaction. We show that MSCs exert potent stabilizing effects on ECs using an in vitro coculture system. Such effects include decreased EC proliferation and the reduction of EC vascular network formation in matrigel. Interestingly, these effects appear to require EC-MSC contact and result in enhanced colocalization of VE-Cadherin and β-catenin at the cell membrane. Disruption of the VE-Cadherin/β-catenin interaction abrogates the observed effects. As a functional in vivo correlate, we show that intravenously administered MSCs strongly inhibit angiogenesis in a matrigel plug assay. Taken together, these results identify a novel mechanism of action of MSCs that involves a contact-dependent EC interaction. These findings are relevant to intravenous use of MSCs and provide insight into further optimizing therapeutic strategies involving MSCs.

Diffusion tensor and volumetric magnetic resonance measures as biomarkers of brain damage in a small animal model of HIV.

Background There are currently no widely accepted neuro-HIV small animal models. We wanted to validate the HIV-1 Transgenic rat (Tg) as an appropriate neuro-HIV model and then establish in vivo imaging biomarkers of neuropathology, within this model, using MR structural and diffusion tensor imaging (DTI). Methods Young and middle-aged Tg and control rats were imaged using MRI. A subset of middle-aged animals underwent longitudinal repeat imaging six months later. Total brain volume (TBV), ventricular volume (VV) and parenchymal volume (PV = TBV–VV) were measured. Fractional anisotropy (FA) and mean diffusivity (MD) values of the corpus callosum (CC) were calculated from DTI data. Results TBV and PV were smaller in Tg compared to control rats in young and middle-aged cohorts (p<0.0001). VV increased significantly (p = 0.005) over time in the longitudinal Tg cohort. There were lower FA (p<0.002) and higher MD (p<0.003) values in the CC of middle-aged Tg rats compared to age-matched controls. Longitudinally, MD significantly decreased over time in Tg rats (p<0.03) while it did not change significantly in the control cohort over the same period of time (p>0.05). Conclusions We detected brain volume loss in the Tg rat, probably due to astrocytic dysfunction/loss, loss of structural/axonal matrix and striatal neuronal loss as suggested by immunofluorescence. Increased MD and decreased FA in the CC probably reflect microstructural differences between the Tg and Control rats which could include increased extracellular space between white matter tracts, demyelination and axonal degeneration, among other pathologies. We believe that the Tg rat is an adequate model of neuropathology in HIV and that volumetric MR and DTI measures can be potentially used as biomarkers of disease progression.

Imaging Dopaminergic Dysfunction as a Surrogate Marker of Neuropathology in a Small-Animal Model of HIV

The dopaminergic system is especially vulnerable to the effects of human immunodeficiency virus (HIV) infection, rendering dopaminergic deficits early surrogate markers of HIV-associated neuropathology. We quantified dopamine D2/3 receptors in young HIV-1 transgenic (Tg) (n = 6) and age-matched control rats (n = 7) and adult Tg (n = 5) and age-matched control rats (n = 5) using [18F]fallypride positron emission tomography (PET). Regional uptake was quantified as binding potential (BPND) using the two-tissue reference model with the cerebellum as the reference. Time-activity curves were generated for the ventral striatum, dorsal striatum, thalamus, and cerebellum. Whereas BPND values were significantly lower in the ventral striatum (p < .001) and dorsal striatum (p = .001) in the adult Tg rats compared to controls rats, they were significantly lower only in the dorsal striatum (p < .05) in the young rats. Tg rats had smaller striatal volumes on magnetic resonance imaging. We also found lower expression levels of tyrosine hydroxylase on immunohistochemistry in the Tg animals. Our findings suggest that progressive striatal D2/3 receptor deficits occur in Tg rats as they age and can be detected using small-animal PET imaging. The effectiveness of various approaches in preventing or halting this dopaminergic loss in the Tg rat can thus be measured preclinically using [18F]fallypride PET as a molecular imaging biomarker of HIV-associated neuropathology.The dopaminergic system is especially vulnerable to the effects of human immunodeficiency virus (HIV) infection, rendering dopaminergic deficits early surrogate markers of HIV-associated neuropathology. We quantified dopamine D2/3 receptors in young HIV-1 transgenic (Tg) ( n = 6) and age-matched control rats ( n = 7) and adult Tg ( n = 5) and age-matched control rats ( n = 5) using [18F]fallypride positron emission tomography (PET). Regional uptake was quantified as binding potential ( BPND) using the two-tissue reference model with the cerebellum as the reference. Time-activity curves were generated for the ventral striatum, dorsal striatum, thalamus, and cerebellum. Whereas BPND values were significantly lower in the ventral striatum ( p < .001) and dorsal striatum ( p = .001) in the adult Tg rats compared to controls rats, they were significantly lower only in the dorsal striatum ( p < .05) in the young rats. Tg rats had smaller striatal volumes on magnetic resonance imaging. We also found lower expression levels of tyrosine hydroxylase on immunohistochemistry in the Tg animals. Our findings suggest that progressive striatal D2/3 receptor deficits occur in Tg rats as they age and can be detected using small-animal PET imaging. The effectiveness of various approaches in preventing or halting this dopaminergic loss in the Tg rat can thus be measured preclinically using [18F]fallypride PET as a molecular imaging biomarker of HIV-associated neuropathology.

Characterization of neuropathology in the HIV-1 transgenic rat at different ages

The transgenic HIV-1 rat (Tg) is a commonly used neuroHIV model with documented neurologic/behavioral deficits. Using immunofluorescent staining of the Tg brain, we found astrocytic dysfunction/damage, as well as dopaminergic neuronal loss/dysfunction, both of which worsening significantly in the striatum with age. We saw mild microglial activation in young Tg brains, but this decreased with age. There were no differences in neurogenesis potential suggesting a neurodegenerative rather than a neurodevelopmental process. Gp120 CSF levels exceeded serum gp120 levels in some animals, suggesting local viral protein production in the brain. Further probing of the pathophysiology underlying astrocytic injury in this model is warranted.

Neurobehavioral Abnormalities in the HIV-1 Transgenic Rat Do Not Correspond to Neuronal Hypometabolism on 18F-FDG-PET

Motor and behavioral abnormalities are common presentations among individuals with HIV-1 associated neurocognitive disorders (HAND). We investigated whether longitudinal motor and behavioral performance in the HIV-1 transgenic rat (Tg), a commonly used neuro-HIV model, corresponded to in vivo neuronal death/dysfunction, by using rotarod and open field testing in parallel to [18F] 2-fluoro-2-deoxy-D-glucose (FDG) positron emission tomography (PET). We demonstrated that age-matched non-Tg wild type (WT) rats outperformed the HIV-1 Tg rats at most time points on rotarod testing. Habituation to rotarod occurred at 8 weeks of age (fifth weekly testing session) in the WT rats but it never occurred in the Tg rats, suggesting deficits in motor learning. Similarly, in open field testing, WT rats outperformed the Tg rats at most time points, suggesting defective exploratory/motor behavior and increased emotionality in the Tg rat. Despite the neurobehavioral abnormalities, there were no concomitant deficits in 18F-FDG uptake in Tg rats on PET compared to age-matched WT rats and no significant longitudinal loss of FDG uptake in either group. The negative PET findings were confirmed using 14C- Deoxy-D-glucose autoradiography in 32 week-old Tg and WT rats. We believe that the neuropathology in the HIV-1 Tg rat is more likely a consequence of neuronal dysfunction rather than overt neurodegeneration/neuronal cell death, similar to what is seen in HIV-positive patients in the post-ART era.

The use of 14C-FIAU to predict bacterial thymidine kinase presence: Implications for radiolabeled FIAU bacterial imaging

UNLABELLED Currently available infectious disease imaging techniques cannot differentiate between infection and sterile inflammation or between different types of infections. Recently, radiolabeled FIAU was found to be a substrate for the thymidine kinase (TK) enzyme of multiple pathogenic bacteria, leading to its translational use in the imaging of bacterial infections. Patients with immunodeficiencies, however, are susceptible to a different group of pathogenic bacteria when compared to immunocompetent subjects. In this study, we wanted to predict the usefulness of radiolabeled FIAU in the detection of bacterial infections commonly occurring in patients with immunodeficiencies, in vitro, prior to attempting in vivo imaging with (124)I-FIAU-PET. METHODS We obtained representative strains of bacterial pathogens isolated from actual patients with genetic immunodeficiencies. We evaluated the bacterial susceptibility of different strains to the effect of incubation with FIAU, which would implicate the presence of the thymidine kinase (TK) enzyme. We also incubated the bacteria with (14)C-FIAU and consequently measured its rate of incorporation in the bacterial DNA using a liquid scintillation counter. RESULTS Unlike the other bacterial strains, the growth of Pseudomonas aeruginosa was not halted by FIAU at any concentration. All the tested clinical isolates demonstrated different levels of (14)C-FIAU uptake, except for P. aeruginosa. CONCLUSION Radiolabeled FIAU has been successful in delineating bacterial infections, both in preclinical and pilot translational studies. In patients with immunodeficiencies, Pseudomonas infections are commonly encountered and are usually difficult to differentiate from fungal infections. The use of radiolabeled FIAU for in vivo imaging of those patients, however, would not be useful, considering the apparent lack of TK enzyme in Pseudomonas. One has to keep in mind that not all pathogenic bacteria possess the TK enzyme and as such will not all retain FIAU. Our technique is simple, and can be easily used to assess whether a certain bacterial strain of interest can or cannot be visualized using radiolabeled FIAU.

Lack of neuroinflammation in the HIV-1 transgenic rat: an [(18)F]-DPA714 PET imaging study.

BackgroundHIV-associated neuroinflammation is believed to be a major contributing factor in the development of HIV-associated neurocognitive disorders (HAND). In this study, we used micropositron emission tomography (PET) imaging to quantify neuroinflammation in HIV-1 transgenic rat (Tg), a small animal model of HIV, known to develop neurological and behavioral problems.MethodsDynamic [18F]DPA-714 PET imaging was performed in Tg and age-matched wild-type (WT) rats in three age groups: 3-, 9-, and 16-month-old animals. As a positive control for neuroinflammation, we performed unilateral intrastriatal injection of quinolinic acid (QA) in a separate group of WT rats. To confirm our findings, we performed multiplex immunofluorescent staining for Iba1 and we measured cytokine/chemokine levels in brain lysates of Tg and WT rats at different ages.Results[18F]DPA-714 uptake in HIV-1 Tg rat brains was generally higher than in age-matched WT rats but this was not statistically significant in any age group. [18F]DPA-714 uptake in the QA-lesioned rats was significantly higher ipsilateral to the lesion compared to contralateral side indicating neuroinflammatory changes. Iba1 immunofluorescence showed no significant differences in microglial activation between the Tg and WT rats, while the QA-lesioned rats showed significant activation. Finally, cytokine/chemokine levels in brain lysates of the Tg rats and WT rats were not significantly different.ConclusionMicroglial activation might not be the primary mechanism for neuropathology in the HIV-1 Tg rats. Although [18F]DPA-714 is a good biomarker of neuroinflammation, it cannot be reliably used as an in vivo biomarker of neurodegeneration in the HIV-1 Tg rat.

Elevated suppressor of cytokine signaling-1 (SOCS-1): a mechanism for dysregulated osteoclastogenesis in HIV transgenic rats.

Accelerated bone loss leading to osteopenia, osteoporosis, and bone fracture is a major health problem that is increasingly common in human immunodeficiency virus (HIV)-infected patients. The underlying pathogenesis is unclear but occurs in both treatment naïve and individuals receiving antiretroviral therapies. We developed an HIV-1 transgenic rat that exhibits many key features of HIV disease including HIV-1-induced changes in bone mineral density (BMD). A key determinant in the rate of bone loss is the differentiation of osteoclasts, the cells responsible for bone resorption. We found HIV-1 transgenic osteoclast precursors (OCP) express higher levels of suppressor of cytokine signaling-1 (SOCS-1) and TNF receptor-associated factor 6 (TRAF6) and are resistant to interferon-gamma (IFN-γ) mediated suppression of osteoclast differentiation. Our data suggest that dysregulated SOCS-1 expression by HIV-1 transgenic OCP promotes osteoclastogenesis leading to the accelerated bone loss observed in this animal model. We propose that elevated SOCS-1 expression in OCP antagonizes the inhibitory effects of IFN-γ and enhances receptor activator of NF-kB ligand (RANKL) signaling that drives osteoclast differentiation and activation. Understanding the molecular mechanisms of HIV-associated BMD changes has the potential to detect and treat bone metabolism disturbances early and improve the quality of life in patients.

MR brain volumetric measurements are predictive of neurobehavioral impairment in the HIV-1 transgenic rat

Introduction HIV infection is known to be associated with brain volume loss, even in optimally treated patients. In this study, we assessed whether dynamic brain volume changes over time are predictive of neurobehavorial performance in the HIV-1 transgenic (Tg) rat, a model of treated HIV-positive patients. Materials and methods Cross-sectional brain MRI imaging was first performed comparing Tg and wild type (WT) rats at 3 and 19 months of age. Longitudinal MRI and neurobehavioral testing of another group of Tg and WT rats was then performed from 5 to 23 weeks of age. Whole brain and subregional image segmentation was used to assess the rate of brain growth over time. We used repeated-measures mixed models to assess differences in brain volumes and to establish how predictive the volume differences are of specific neurobehavioral deficits. Results Cross-sectional imaging showed smaller whole brain volumes in Tg compared to WT rats at 3 and at 19 months of age. Longitudinally, Tg brain volumes were smaller than age-matched WT rats at all time points, starting as early as 5 weeks of age. The Tg striatal growth rate delay between 5 and 9 weeks of age was greater than that of the whole brain. Striatal volume in combination with genotype was the most predictive of rota-rod scores and in combination with genotype and age was the most predictive of total exploratory activity scores in the Tg rats. Conclusion The disproportionately delayed striatal growth compared to whole brain between 5 and 9 weeks of age and the role of striatal volume in predicting neurobehavioral deficits suggest an important role of the dopaminergic system in HIV associated neuropathology. This might explain problems with motor coordination and executive decisions in this animal model. Smaller brain and subregional volumes and neurobehavioral deficits were seen as early as 5 weeks of age, suggesting an early brain insult in the Tg rat. Neuroprotective therapy testing in this model should thus target this early stage of development, before brain damage becomes irreversible.