Implementing Pool-Based Surveillance Testing for SARS-CoV-2 at the Army Public Health Center Laboratory and across the Army Public Health Laboratory Enterprise.

With limited clinical resources, burgeoning testing requests from Army and other Service units to clinical laboratories, and the continued spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) throughout the military population, the Army Public Health Laboratory (APHL) Enterprise was tasked to establish surveillance testing capabilities for active duty military populations in an expedient manner. Following a proof-of-concept study conducted by Public Health Command-Pacific, Public Health Command-Europe was the first public health laboratory to offer the capability to assess for SARS-CoV-2 in pooled samples, followed closely by the Army Public Health Center (APHC) at Aberdeen Proving Grounds, MD, paralleling the spread of the SARS-CoV-2 virus from China to Europe to the continental US. The APHLs have selected pool sizes of up to 10 samples per pool based on the best evidence available at the time of method development and validation. Real-Time quantitative Reverse Transcriptase-Polymerase Chain Reaction (qRT-PCR) assays using RNA extracts from pooled nasopharyngeal swabs preserved in viral transport media were selected to assess the presence of SARS-CoV-2. The rapid development of initial surveillance testing capabilities depended on existing equipment in each laboratory, with a plan to implement full operational capability using additional staff and common high-throughput platforms. APHL Enterprise has successfully used existing resources to begin to address the changing and complex needs for COVID-19 testing within the Army population. Successful implementation of pooled surveillance testing at the APHC Laboratory has enabled more than 8,600 Soldiers to avoid clinical testing to date. The APHC Laboratory alone has tested over 10,000 samples and prevented approximately 8,600 soldiers from seeking testing with clinical diagnostic assays.

NNZ-2566 treatment inhibits neuroinflammation and pro-inflammatory cytokine expression induced by experimental penetrating ballistic-like brain injury in rats.

BACKGROUND: Inflammatory cytokines play a crucial role in the pathophysiology of traumatic brain injury (TBI), exerting either deleterious effects on the progression of tissue damage or beneficial roles during recovery and repair. NNZ-2566, a synthetic analogue of the neuroprotective tripeptide Glypromate, has been shown to be neuroprotective in animal models of brain injury. The goal of this study was to determine the effects of NNZ-2566 on inflammatory cytokine expression and neuroinflammation induced by penetrating ballistic-like brain injury (PBBI) in rats. METHODS: NNZ-2566 or vehicle (saline) was administered intravenously as a bolus injection (10 mg/kg) at 30 min post-injury, immediately followed by a continuous infusion of NNZ-2566 (3 mg/kg/h), or equal volume of vehicle, for various durations. Inflammatory cytokine gene expression from the brain tissue of rats exposed to PBBI was evaluated using microarray, quantitative real time PCR (QRT-PCR), and enzyme-linked immunosorbent assay (ELISA) array. Histopathology of the injured brains was examined using hematoxylin and eosin (H&E) and immunocytochemistry of inflammatory cytokine IL-1beta. RESULTS: NNZ-2566 treatment significantly reduced injury-mediated up-regulation of IL-1beta, TNF-alpha, E-selectin and IL-6 mRNA during the acute injury phase. ELISA cytokine array showed that NZ-2566 treatment significantly reduced levels of the pro-inflammatory cytokines IL-1beta, TNF-alpha and IFN-gamma in the injured brain, but did not affect anti-inflammatory cytokine IL-6 levels. CONCLUSION: Collectively, these results suggest that the neuroprotective effects of NNZ-2566 may, in part, be functionally attributed to the compound's ability to modulate expression of multiple neuroinflammatory mediators in the injured brain.

NNZ-2566, a glypromate analog, improves functional recovery and attenuates apoptosis and inflammation in a rat model of penetrating ballistic-type brain injury.

Glycine-proline-glutamate (GPE) is an N-terminal tripeptide endogenously cleaved from insulin-like growth factor-1 in the brain and is neuroprotective against hypoxic-ischemic brain injury and neurodegeneration. NNZ-2566 is an analog of GPE designed to have improved bioavailability. In this study, we tested NNZ-2566 in a rat model of penetrating ballistic-type brain injury (PBBI) and assessed its effects on injury-induced histopathology, behavioral deficits, and molecular and cellular events associated with inflammation and apoptosis. In the initial dose-response experiments, NNZ-2566 (0.01-3 mg/kg/h x 12 h intravenous infusion) was given at 30 min post-injury and the therapeutic time window was established by delaying treatments 2-4 h post-injury, but with the addition of a 10- or 30-mg/kg bolus dose. All animals survived 72 h. Neuroprotection was evaluated by balance beam testing and histopathology. The effects of NNZ-2566 on injury-induced changes in Bax and Bcl-2 proteins, activated microgliosis, neutrophil infiltration, and astrocyte reactivity were also examined. Behavioral results demonstrated that NNZ-2566 dose-dependently reduced foot faults by 19-66% after acute treatments, and 35-55% after delayed treatments. Although gross lesion volume was not affected, NNZ-2566 treatment significantly attenuated neutrophil infiltration and reduced the number of activated microglial cells in the peri-lesion regions of the PBBI. PBBI induced a significant upregulation in Bax expression (36%) and a concomitant downregulation in Bcl-2 expression (33%), both of which were significantly reversed by NNZ-2566. Collectively, these results demonstrated that NNZ-2566 treatment promoted functional recovery following PBBI, an effect related to the modulation of injury-induced neural inflammatory and apoptotic mechanisms.

Acute and delayed neuroinflammatory response following experimental penetrating ballistic brain injury in the rat.

BACKGROUND: Neuroinflammation following acute brain trauma is considered to play a prominent role in both the pathological and reconstructive response of the brain to injury. Here we characterize and contrast both an acute and delayed phase of inflammation following experimental penetrating ballistic brain injury (PBBI) in rats out to 7 days post-injury. METHODS: Quantitative real time PCR (QRT-PCR) was used to evaluate changes in inflammatory gene expression from the brain tissue of rats exposed to a unilateral frontal PBBI. Brain histopathology was assessed using hematoxylin and eosin (H&E), silver staining, and immunoreactivity for astrocytes (GFAP), microglia (OX-18) and the inflammatory proteins IL-1beta and ICAM-1. RESULTS: Time course analysis of gene expression levels using QRT-PCR indicated a peak increase during the acute phase of the injury between 3-6 h for the cytokines TNF-alpha (8-11 fold), IL-1beta (11-13 fold), and IL-6 (40-74 fold) as well as the cellular adhesion molecules VCAM (2-3 fold), ICAM-1 (7-15 fold), and E-selectin (11-13 fold). Consistent with the upregulation of pro-inflammatory genes, peripheral blood cell infiltration was a prominent post-injury event with peak levels of infiltrating neutrophils (24 h) and macrophages (72 h) observed throughout the core lesion. In regions of the forebrain immediately surrounding the lesion, strong immunoreactivity for activated astrocytes (GFAP) was observed as early as 6 h post-injury followed by prominent microglial reactivity (OX-18) at 72 h and resolution of both cell types in cortical brain regions by day 7. Delayed thalamic inflammation (remote from the primary lesion) was also observed as indicated by both microglial and astrocyte reactivity (72 h to 7 days) concomitant with the presence of fiber degeneration (silver staining). CONCLUSION: In summary, PBBI induces both an acute and delayed neuroinflammatory response occurring in distinct brain regions, which may provide useful diagnostic information for the treatment of this type of brain injury.

PAN-811 provides neuroprotection against glutamate toxicity by suppressing activation of JNK and p38 MAPK.

In an earlier study, we demonstrated that PAN-811 (3-aminopyridine-2-carboxaldehyde thiosemicarbazone), a novel neuroprotectant, provides protection against glutamate, staurosporine, veratridine, or hypoxia/hypoglycemia toxicities in primary cortical neuronal cultures by upregulating Bcl-2 expression [R.-W. Chen, C. Yao, X.C. Lu, Z.-G. Jiang, R. Whipple, Z. Liao, H.A. Ghanbari, B. Almassian, F.C. Tortella, J.R. Dave. PAN-811 (3-aminopyridine-2-carboxaldehyde thiosemicarbazone), a novel neuroprotectant, elicits its function in primary neuronal cultures by upregulating Bcl-2 expression. Neuroscience 135 (2005) 191-201]. Both JNK (c-Jun N-terminal kinase) and p38 MAP (mitogen-activated protein) kinase activation have a direct inhibitory action on Bcl-2 by phosphorylation. In the present study, we continued to explore the mechanism of PAN-811 neuroprotection. Our results indicate that treatment of cultured cortical neurons with glutamate (100 microM) induces phosphorylation of both JNK and p38 MAPK. Specifically, pretreatment of neurons with 10 microM PAN-811 (an optimal neuroprotective concentration) for 1h, 4h, or 24h significantly suppresses glutamate-mediated activation of both JNK and p38 MAPK. Furthermore, the p38 MAPK-specific inhibitor SB203580 and the JNK-specific inhibitor SP600125 prevented glutamate-induced neuronal death in these primary cultures. Our results demonstrate that glutamate-induced phosphorylation of JNK and p38 MAPK is suppressed by PAN-811, which might contribute to Bcl-2 upregulation and PAN-811 neuroprotection.