Sampling and recovery of infectious SARS-CoV-2 from high-touch surfaces by sponge stick and macrofoam swab.

Effective sampling for severe acute respiratory syndrome 2 (SARS-CoV-2) is a common approach for monitoring disinfection efficacy and effective environmental surveillance. This study evaluated sampling efficiency and limits of detection (LODs) of macrofoam swab and sponge stick sampling methods for recovering infectious SARS-CoV-2 and viral RNA (vRNA) from surfaces. Macrofoam swab and sponge stick methods were evaluated for collection of SARS-CoV-2 suspended in a soil load from 6-in2 coupons composed of four materials: stainless steel (SS), acrylonitrile butadiene styrene (ABS) plastic, bus seat fabric, and Formica. Recovery of infectious SARS-CoV-2 was more efficient than vRNA recovery on all materials except Formica (macrofoam swab sampling) and ABS (sponge stick sampling). Macrofoam swab sampling recovered significantly more vRNA from Formica than ABS and SS, and sponge stick sampling recovered significantly more vRNA from ABS than Formica and SS, suggesting that material and sampling method choice can affect surveillance results. Time since initial contamination significantly affected infectious virus recovery from all materials, with vRNA recovery showing limited to no difference, suggesting that SARS-CoV-2 vRNA can remain detectable after viral infectivity has dissipated. This study showed that a complex relationship exists between sampling method, material, time from contamination to sampling, and recovery of SARS-CoV-2. In conclusion, data show that careful consideration be used when selecting surface types for sampling and interpreting SARS-CoV-2 vRNA recovery with respect to presence of infectious virus.

Efficacy of chemical disinfectants against SARS-CoV-2 on high-touch surface materials.

AIMS: This study aimed to provide operationally relevant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) surface disinfection efficacy information. METHODS AND RESULTS: Three EPA-registered disinfectants (Vital Oxide, Peroxide, and Clorox Total 360) and one antimicrobial formulation (CDC bleach) were evaluated against SARS-CoV-2 on material coupons and were tested using Spray (no touch with contact time) and Spray & Wipe (wipe immediately post-application) methods immediately and 2 h post-contamination. Efficacy was evaluated for infectious virus, with a subset tested for viral RNA (vRNA) recovery. Efficacy varied by method, disinfectant, and material. CDC bleach solution showed low efficacy against SARS-CoV-2 (log reduction < 1.7), unless applied via Spray & Wipe. Additionally, mechanical wiping increased the efficacy of treatments against SARS-CoV-2. The recovery of vRNA post-disinfection suggested that vRNA may overestimate infectious virus remaining. CONCLUSIONS: Efficacy depends on surface material, chemical, and disinfection procedure, and suggests that mechanical wiping alone has some efficacy at removing SARS-CoV-2 from surfaces. We observed that disinfectant treatment biased the recovery of vRNA over infectious virus. SIGNIFICANCE AND IMPACT OF STUDY: These data are useful for developing effective, real-world disinfection procedures, and inform public health experts on the utility of PCR-based surveillance approaches.

Neuronal ablation of mt-AspRS in mice induces immune pathway activation prior to severe and progressive cortical and behavioral disruption.

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a rare, slowly progressive white matter disease caused by mutations in the mitochondrial aspartyl-tRNA synthetase (mt-AspRS, or DARS2). While patients show characteristic MRI T2 signal abnormalities throughout the cerebral white matter, brainstem, and spinal cord, the phenotypic spectrum is broad and a multitude of gene variants have been associated with the disease. Here, Dars2 disruption in CamKIIα-expressing cortical and hippocampal neurons results in slowly progressive increases in behavioral activity at five months, and culminating by nine months as severe brain atrophy, behavioral dysfunction, reduced corpus callosum thickness, and microglial morphology indicative of neuroinflammation. Interestingly, RNAseq based gene expression studies performed prior to the presentation of this severe phenotype reveal the upregulation of several pathways involved in immune activation, cytokine production and signaling, and defense response regulation. RNA transcript analysis demonstrates that activation of immune and cell stress pathways are initiated in advance of a behavioral phenotype and cerebral deficits. An understanding of these pathways and their contribution to significant neuronal loss in CamKII-Dars2 deficient mice may aid in deciphering mechanisms of LBSL pathology.

Dendrimer-N-acetyl-L-cysteine modulates monophagocytic response in adrenoleukodystrophy.

OBJECTIVE: X-linked adrenoleukodystrophy (ALD) is a neurodegenerative disorder due to mutations in the peroxisomal very long-chain fatty acyl-CoA transporter, ABCD1, with limited therapeutic options. ALD may manifest in a slowly progressive adrenomyeloneuropathy (AMN) phenotype, or switch to rapid inflammatory demyelinating cerebral disease (cALD), in which microglia have been shown to play a pathophysiological role. The aim of this study was to determine the role of patient phenotype in the immune response of ex vivo monophagocytic cells to stimulation, and to evaluate the efficacy of polyamidoamine dendrimer conjugated to the antioxidant precursor N-acetyl-cysteine (NAC) in modulating this immune response. METHODS: Human monophagocytic cells were derived from fresh whole blood, from healthy (n = 4), heterozygote carrier (n = 4), AMN (n = 7), and cALD (n = 4) patients. Cells were exposed to very long-chain fatty acids (VLCFAs; C24:0 and C26:0) and treated with dendrimer-NAC (D-NAC). RESULTS: Ex vivo exposure to VLCFAs significantly increased tumor necrosis factor α (TNFα) and glutamate secretion from cALD patient macrophages. Additionally, a significant reduction in total intracellular glutathione was observed in cALD patient cells. D-NAC treatment dose-dependently reduced TNFα and glutamate secretion and replenished total intracellular glutathione levels in cALD patient macrophages, more efficiently than NAC. Similarly, D-NAC treatment decreased glutamate secretion in AMN patient cells. INTERPRETATION: ALD phenotypes display unique inflammatory profiles in response to VLCFA stimulation, and therefore ex vivo monophagocytic cells may provide a novel test bed for therapeutic agents. Based on our findings, D-NAC may be a viable therapeutic strategy for the treatment of cALD. Ann Neurol 2018;84:452-462.

Antioxidant Capacity and Superoxide Dismutase Activity in Adrenoleukodystrophy.

Importance: X-linked adrenoleukodystrophy (ALD) may switch phenotype to the fatal cerebral form (ie, cerebral ALD [cALD]), the cause of which is unknown. Determining differences in antioxidant capacity and superoxide dismutase (SOD) levels between phenotypes may allow for the generation of a clinical biomarker for predicting the onset of cALD, as well as initiating a more timely lifesaving therapy. Objective: To identify variations in the levels of antioxidant capacity and SOD activity between ALD phenotypes in patients with cALD or adrenomyeloneuropathy (AMN), heterozygote female carriers, and healthy controls and, in addition, correlate antioxidant levels with clinical outcome scores to determine a possible predictive value. Design, Setting, and Participants: Samples of monocytes and blood plasma were prospectively collected from healthy controls, heterozygote female carriers, and patients with AMN or cALD. We are counting each patient as 1 sample in our study. Because adrenoleukodystrophy is an X-linked disease, the affected group populations of cALD and AMN are all male. The heterozygote carriers are all female. The samples were assayed for total antioxidant capacity and SOD activity. The data were collected in an academic hospital setting. Eligibility criteria included patients who received a diagnosis of ALD and heterozygote female carriers, both of which groups were compared with age-matched controls. The prospective samples (n = 30) were collected between January 2015 to January 2016, and existing samples were collected from tissue storage banks at the Kennedy Krieger Institute (n = 30). The analyses were performed during the first 3 months of 2016. Main Outcome and Measures: Commercially available total antioxidant capacity and SOD assays were performed on samples of monocytes and blood plasma and correlated with magnetic resonance imaging severity score. Results: A reduction in antioxidant capacity was shown between the healthy controls (0.225 mmol trolox equivalent) and heterozygote carriers (0.181 mmol trolox equivalent), and significant reductions were seen between healthy controls and patients with AMN (0.102 mmol trolox equivalent; P < .01), as well as healthy controls and patients with cALD (0.042 mmol trolox equivalent; P < .01). Superoxide dismutase activity in human blood plasma mirrored these reductions between prospectively collected samples from healthy controls (2.66 units/mg protein) and samples from heterozygote female carriers (1.91 units/mg protein), patients with AMN (1.39 units/mg protein; P = .01), and patients with cALD (0.8 units/mg protein; P < .01). Further analysis of SOD activity in biobank samples showed significant reductions between patients with AMN (0.89 units/mg protein) and patients with cALD (0.18 units/mg protein) (P = .03). Plasma SOD levels from patients with cALD demonstrated an inverse correlation to brain magnetic resonance imaging severity score (R2 = 0.75, P < .002). Longitudinal plasma SOD samples from the same patients (n = 4) showed decreased activity prior to and at the time of cerebral diagnosis over a period of 13 to 42 months (mean period, 24 months). Conclusions and Relevance: Plasma SOD may serve as a potential biomarker for cerebral disease in ALD following future prospective studies.

Glial-Restricted Precursors Protect Neonatal Brain Slices from Hypoxic-Ischemic Cell Death Without Direct Tissue Contact.

Glial-Restricted Precursors (GRPs) are tripotential progenitors that have been shown to exhibit beneficial effects in several preclinical models of neurological disorders, including neonatal brain injury. The mechanisms of action of these cells, however, require further study, as do clinically relevant questions such as timing and route of cell administration. Here, we explored the effects of GRPs on neonatal hypoxia-ischemia during acute and subacute stages, using an in vitro transwell co-culture system with organotypic brain slices exposed to oxygen-glucose deprivation (OGD). OGD-exposed slices that were then co-cultured with GRPs without direct cell contact had decreased tissue injury and cortical cell death, as evaluated by lactate dehydrogenase (LDH) release and propidium iodide (PI) staining. This effect was more pronounced when cells were added during the subacute phase of the injury. Furthermore, GRPs reduced the amount of glutamate in the slice supernatant and changed the proliferation pattern of endogenous progenitor cells in brain slices. In summary, we show that GRPs exert a neuroprotective effect on neonatal hypoxia-ischemia without the need for direct cell-cell contact, thus confirming the rising view that beneficial actions of stem cells are more likely attributable to trophic or immunomodulatory support rather than to long-term integration.

Transplanted glial restricted precursor cells improve neurobehavioral and neuropathological outcomes in a mouse model of neonatal white matter injury despite limited cell survival.

OBJECTIVE: Neonatal white matter injury (NWMI) is the leading cause of cerebral palsy and other neurocognitive deficits in prematurely-born children, and no restorative therapies exist. Our objective was to determine the fate and effect of glial restricted precursor cell (GRP) transplantation in an ischemic mouse model of NWMI. METHODS: Neonatal CD-1 mice underwent unilateral carotid artery ligation on postnatal-Day 5 (P5). At P22, intracallosal injections of either enhanced green fluorescent protein (eGFP) + GRPs or saline were performed in control and ligated mice. Neurobehavioral and postmortem studies were performed at 4 and 8 weeks post-transplantation. RESULTS: GRP survival was comparable at 1 month but significantly lower at 2 months post-transplantation in NWMI mice compared with unligated controls. Surviving cells showed better migration capability in controls; however, the differentiation capacity of transplanted cells was similar in control and NWMI. Saline-treated NWMI mice showed significantly altered response in startle amplitude and prepulse inhibition (PPI) paradigms compared with unligated controls, while these behavioral tests were completely normal in GRP-transplanted animals. Similarly, there was significant increase in hemispheric myelin basic protein density, along with significant decrease in pathologic axonal staining in cell-treated NWMI mice compared with saline-treated NWMI animals. INTERPRETATION: The reduced long-term survival and migration of transplanted GRPs in an ischemia-induced NWMI model suggests that neonatal ischemia leads to long-lasting detrimental effects on oligodendroglia even months after the initial insult. Despite limited GRP-survival, behavioral, and neuropathological outcomes were improved after GRP-transplantation. Our results suggest that exogenous GRPs improve myelination through trophic effects in addition to differentiation into mature oligodendrocytes.

Derivation of glial restricted precursors from E13 mice.

This is a protocol for derivation of glial restricted precursor (GRP) cells from the spinal cord of E13 mouse fetuses. These cells are early precursors within the oligodendrocytic cell lineage. Recently, these cells have been studied as potential source for restorative therapies in white matter diseases. Periventricular leukomalacia (PVL) is the leading cause of non-genetic white matter disease in childhood and affects up to 50% of extremely premature infants. The data suggest a heightened susceptibility of the developing brain to hypoxia-ischemia, oxidative stress and excitotoxicity that selectively targets nascent white matter. Glial restricted precursors (GRP), oligodendrocyte progenitor cells (OPC) and immature oligodendrocytes (preOL) seem to be key players in the development of PVL and are the subject of continuing studies. Furthermore, previous studies have identified a subset of CNS tissue that has increased susceptibility to glutamate excitotoxicity as well as a developmental pattern to this susceptibility. Our laboratory is currently investigating the role of oligodendrocyte progenitors in PVL and use cells at the GRP stage of development. We utilize these derived GRP cells in several experimental paradigms to test their response to select stresses consistent with PVL. GRP cells can be manipulated in vitro into OPCs and preOL for transplantation experiments with mouse PVL models and in vitro models of PVL-like insults including hypoxia-ischemia. By using cultured cells and in vitro studies there would be reduced variability between experiments which facilitates interpretation of the data. Cultured cells also allows for enrichment of the GRP population while minimizing the impact of contaminating cells of non-GRP phenotype.