Recombinant annexin A2 inhibits peripheral leukocyte activation and brain infiltration after traumatic brain injury

Background Traumatic brain injury (TBI) is a significant cause of death and disability worldwide. The TLR4-NFκB signaling cascade is the critical pro-inflammatory activation pathway of leukocytes after TBI, and modulating this signaling cascade may be an effective therapeutic target for treating TBI. Previous studies indicate that recombinant annexin A2 (rA2) might be an interactive molecule modulating the TLR4-NFκB signaling; however, the role of rA2 in regulating this signaling pathway in leukocytes after TBI and its subsequent effects have not been investigated. Methods C57BL/6 mice were subjected to TBI and randomly divided into groups that received intraperitoneal rA2 or vehicle at 2 h after TBI. The peripheral leukocyte activation and infiltrating immune cells were examined by flow cytometry, RT-qPCR, and immunostaining. The neutrophilic TLR4 expression on the cell membrane was examined by flow cytometry and confocal microscope, and the interaction of annexin A2 with TLR4 was assessed by co-immunoprecipitation coupled with Western blotting. Neuroinflammation was measured via cytokine proteome profiler array and RT-qPCR. Neurodegeneration was determined by Western blotting and immunostaining. Neurobehavioral assessments were used to monitor motor and cognitive function. Brain tissue loss was assessed via MAP2 staining. Results rA2 administration given at 2 h after TBI significantly attenuates neutrophil activation and brain infiltration at 24 h of TBI. In vivo and in vitro data show that rA2 binds to and reduces TLR4 expression on the neutrophil surface and suppresses TLR4/NFκB signaling pathway in neutrophils at 12 h after TBI. Furthermore, rA2 administration also reduces pro-inflammation of brain tissues within 24 h and neurodegeneration at 48 h after TBI. Lastly, rA2 improves long-term sensorimotor ability and cognitive function, and reduces brain tissue loss at 28 days after TBI. Conclusions Systematic rA2 administration at 2 h after TBI significantly inhibits activation and brain infiltration of peripheral leukocytes, especially neutrophils at the acute phase. Consequently, rA2 reduces the detrimental brain pro-inflammation-associated neurodegeneration and ultimately ameliorates neurological deficits after TBI. The underlying molecular mechanism might be at least in part attributed to rA2 bindings to pro-inflammatory receptor TLR4 in peripheral leukocytes, thereby blocking NFκB signaling activation pathways following TBI.

Regulation of the acetylcholine/α7nAChR anti-inflammatory pathway in COVID-19 patients

The cholinergic system has been proposed as a potential regulator of COVID-19-induced hypercytokinemia. We investigated whole-blood expression of cholinergic system members and correlated it with COVID-19 severity. Patients with confirmed SARS-CoV-2 infection and healthy aged-matched controls were included in this non-interventional study. A whole blood sample was drawn between 9–11 days after symptoms onset, and peripheral leukocyte phenotyping, cytokines measurement, RNA expression and plasma viral load were determined. Additionally, whole-blood expression of native alpha-7 nicotinic subunit and its negative dominant duplicate (CHRFAM7A), choline acetyltransferase and acetylcholine esterase (AchE) were determined. Thirty-seven patients with COVID-19 (10 moderate, 11 severe and 16 with critical disease) and 14 controls were included. Expression of CHRFAM7A was significantly lower in critical COVID-19 patients compared to controls. COVID-19 patients not expressing CHRFAM7A had higher levels of CRP, more extended pulmonary lesions and displayed more pronounced lymphopenia. COVID-19 patients without CHRFAM7A expression also showed increased TNF pathway expression in whole blood. AchE was also expressed in 30 COVID-19 patients and in all controls. COVID-19-induced hypercytokinemia is associated with decreased expression of the pro-inflammatory dominant negative duplicate CHRFAM7A. Expression of this duplicate might be considered before targeting the cholinergic system in COVID-19 with nicotine.

0031 Photoperiodic Effects on Diurnal Rhythms in the Immune System, Rest-Activity Behavior, and Cortisol Revealed in a Diurnally Active Large Animal Model - The Domestic Pig

Introduction Diurnal variations in immune cell number and function are regarded important for immune competence and are thought to be mediated by rest-activity rhythms and hormones. Moreover, the photoperiod is also known to modulate the immune system and considered to affect seasonal disease susceptibility. Whereas few studies investigated seasonal effects, the present study is the first investigating the specific effect of the photoperiod on diurnal rhythms in cell numbers of peripheral leukocyte types in any species. Methods Domestic pigs were held either under long day (LD, 16L:8D, lights-on 07:00-23:00, n=9) or short day conditions (SD, 8L:16D, lights-on 07:00-15:00, n=11) and fed concentrate at 07:30 + 14:00 (ad libitum hay/water). Blood samples were taken every 2 h over periods of 50 h via indwelling vein catheters. Rest-activity behavior of the pigs was analyzed using continuous camera recordings. Results Cosinor analyses (p<.05) revealed photoperiodic differences in diurnal rhythms of cell numbers of various peripheral leukocyte subtypes, rest-activity behavior, and cortisol concentration. Cell numbers of total leukocytes, NK cells, T cells, and eosinophils in blood, rest-activity behavior, and cortisol concentration peaked earlier relative to lights-on under SD (p<.05). Relative amplitudes in rest-activity behavior and cell counts of total leukocytes, NK cells, T cells, and monocytes were higher under SD (p<.05). However, there was no photoperiodic effect on diurnal rhythms in neutrophil counts and mesor in any leukocyte type. Generalized linear mixed models revealed associations of leukocyte counts with rest-activity behavior and cortisol concentration in most cell types (p<.05). Moreover, the found photoperiodic effects on diurnal rhythms in rest-activity behavior and cortisol concentration are in agreement with research in humans and primates. Conclusion The present study revealed photoperiodic effects on diurnal rhythms in the immune system, rest-activity behavior, and cortisol concentration in pigs and strengthens the importance of the domestic pig as suitable model for chronoimmunology. Support German Research Foundation DFG (grant provided to SS, grant number SCHM3162/1-1), Federal Ministry of Education and Research, Germany (grant number 01PL16003), Faculty of Agricultural Sciences, University of Hohenheim (scholarship provided to LE).

Relation between carotid vulnerable plaques and peripheral leukocyte: A case-control study of comparison utilizing multi-parametric CEUS

Background: This study evaluates carotid vulnerable plaques using contrast-enhanced ultrasound (CEUS) and explores the relationship between vulnerable plaques and leukocytes.Methods: Sixty-two symptomatic and 54 asymptomatic patients underwent CEUS. The images were analyzed using time-intensity and fitting curves, and peak (PTIC), mean (MTIC), peak (PFC), sharpness (SFC), and area under the curve (AUCFC) were obtained. The relations between CEUS parameters and leukocytes were analyzed.Results: In the symptomatic group, total leukocytes and neutrophils were higher, while lymphocyte was decreased; PTIC, MTIC, PFC, SFC,, and AUCFC were significantly higher; MTIC and AUCFC were negatively correlated with lymphocytes, and MTIC was positively correlated with neutrophils. Classification and regression tree analysis showed that MTIC at a cutoff of 20.8 and AUCFC at a cutoff of 8.8 resulted in a predictive of acute cerebral infarction, accuracy of 84.3%, sensitivity of 87.1%, and specificity of 81.5%.Conclusions: The variation in the perivascular leucocyte is significantly related to intraplaque inflammatory activities, CEUS is a feasible monitor of intraplaque neovascularization, so CEUS combined with perivascular leucocyte could be helpful as a warning for vulnerable plaques.

2587. Etiology and Outcome of Acute Neonatal Infectious Encephalitis

Background There are very few studies on acute encephalitis with onset during the neonatal period. The objectives of this study were to investigate the etiology and salient clinical features of neonatal encephalitis. Methods Neonates with possible infectious encephalitis (IE) were prospectively enrolled. Inclusion criteria included encephalopathy (altered/fluctuating level of consciousness ≥24 hours) plus ≥2 of: fever/temperature instability; seizure(s); focal neurologic findings; CSF pleocytosis; EEG abnormalities consistent with encephalitis; neuroimaging abnormalities consistent with encephalitis. Neonates with a clear diagnosis of post-perinatal asphyxial encephalopathy or culture proven bacterial meningitis were excluded. Results shown as absolute numbers, proportions or medians [interquartile range] as appropriate. Results Fifty-nine neonates fulfilled the inclusion/exclusion criteria (June 2013–November 2018). Empiric acyclovir was initiated in 49 (83.1%) cases. An infectious etiology was identified in 25 (42.4%): enteroviruses (n = 15), HSV (n = 5), HHV6 (n = 2), parainfluenza 3 (n = 1), influenza A (n = 1), CMV (n = 1). A noninfectious cause was confirmed in 20 (33.9%): missed hypoxic-ischemic encephalopathy (n = 10), genetic/metabolic disorders (n = 7), ischemic/hemorrhagic stroke (n = 3). No specific etiology was identified in 14 (23.7%). Thirteen (52%) neonates with IE either died (n = 7) or suffered neurologic sequelae (n = 6). Deaths were attributable to HSV (n = 4), enteroviruses (n = 2) and HHV6 (n = 1). Neurocognitive sequelae were documented in one case each of enterovirus, HSV2, HHV6, CMV, parainfluenza 3 and influenza A. Differences between neonates with and without IE, respectively, included age in days of symptom onset (7 [6, 10] vs. 1 [0, 3]; P < 0.001), gestational age (37.0 [36.0, 39.0] vs. 38.6 [37.6, 40.0]; P = 0.045), peripheral leukocyte count (10.5 [IQR 5.9, 14.6] vs. 14.3 [IQR 10.7, 21.7]; P = 0.008) and CSF glucose (2.80 [IQR 2.3, 3.2] vs. 3.10 [2.8, 3.8]; P = 0.003). Conclusion Enteroviruses and HSV are the predominant causes of neonatal IE. Outcome of neonatal IE is poor with approximately half dying or suffering neurologic sequelae. Disclosures All authors: No reported disclosures.