Elevated IL18 levels in Nasopharyngeal carcinoma induced PD-1 expression on NK cells in TILS leading to poor prognosis.

OBJECTIVES: Characterisation of tumor-infiltrating lymphocytes (TILS) population for cancer prognostication has enabled deeper understanding of tumor immune interactions in cancer immunology. We aim to examine the significance of both the density and functional status of NK cells in a cohort of Epstein Barr Virus (EBV) associated Nasopharyngeal Cancer (NPC) patients. METHODS: NK TILS of 50 NPC samples were quantified on immunohistochemistry and the density of NK TILS was correlated with clinical outcomes. Next, NK cells and a panel of cytokines of 10 newly diagnosed NPC patients were characterized in both NPC tissue and peripheral circulation. Exhausted NK cells were identified using co-expression of PD-1 and/or Tim-3. Comparison of percentage of NK cells in NPC and healthy controls was performed using student t-test for two groups; and a p value of less than 0.05 values was considered significant. RESULTS: NK TILS exhibited a bimodal distribution; with the NKhigh cohort demonstrating a poorer 2-year overall survival rate (p < 0.035). In-vitro studies revealed a higher proportion of infiltrated NK cells in the NKhigh cohort co-expressed PD-1. Additionally, IL-18 levels in NPC tissue were significantly higher than in healthy nasopharynx; and IL-18 alone induced PD-1 expression on NK cells. Expectedly, plasma IL-18 concentration and percentage of circulating PD-1-expressing NK cells were similar among NPC patients and healthy controls. CONCLUSION: The cytotoxic function of NK TILS is mitigated by an elevated IL-18 levels within the NPC microenvironment. Hence, the functional status, and the density of NK cells in TILS should be considered when prognosticating NPC.

The neuroprotective mechanism of erythropoietin-TAT fusion protein against neurodegeneration from ischemic brain injury.

AIMS: To compare the neuroprotection of erythropoietin (EPO) and EPO fusion protein containing transduction domain derived from HIV TAT (EPO-TAT) against ischemic brain injury, inclusive of the side effect, and explore the mechanism underlying the role of EPO-TAT in a transient focal cerebral ischemia model in rats. METHODS: Transient focal ischemia was induced by middle cerebral artery occlusion (MCAO) in rats. Rats were treated, respectively, with following regimens: saline, 1000 U/kg EPO, 5000 U/kg EPO, 1000 U/kg EPO-TAT, 1000 U/kg EPOTAT+5 µl of 10 mM LY294002 (or/plus 5 µl of 5 mM PD98059). Neurological deficit scores, infarct volume, and hematologic side effect were assessed at 72 hours after MCAO. Apoptotic cells were determined with TUNEL staining. The expression and localization of phosphorylated AKT (pAKT) and phosphorylated ERK (pERK) were detected with Western blot, immunohistochemistry, and immunofluorescence, respectively. RESULTS: 1000 U/kg EPO-TAT exhibited a comparable neuroprotection to 5000 U/kg EPO, as evidenced by a comparable attenuation in neurological deficit, infarct volume, and number of apoptotic cells in the rat ischemic cortex after MCAO. The pAKT and pERK levels were significantly elevated solely in neurons of rodents receiving EPO or EPO-TAT treatments, suggesting the concurrent activation of these two pathways. Specific inhibition of either AKT or ERK pathway partially abolished EPO-TAT protection, but exhibited no influence on the activation status of its counterpart, suggesting no cross-modulation between these two protective pathways. CONCLUSION: Our study indicates that EPO-TAT at 1000 U/kg displays neuroprotection with no detectable side effects. The mechanism for neuroprotection may be attributable to the simultaneous activation of the AKT and ERK pathways, which preserve neuronal cell viability and attenuate behavioral deficits.

Microglia/macrophage polarization dynamics in white matter after traumatic brain injury.

Mononuclear phagocytes are a population of multi-phenotypic cells and have dual roles in brain destruction/reconstruction. The phenotype-specific roles of microglia/macrophages in traumatic brain injury (TBI) are, however, poorly characterized. In the present study, TBI was induced in mice by a controlled cortical impact (CCI) and animals were killed at 1 to 14 days post injury. Real-time polymerase chain reaction (RT-PCR) and immunofluorescence staining for M1 and M2 markers were performed to characterize phenotypic changes of microglia/macrophages in both gray and white matter. We found that the number of M1-like phagocytes increased in cortex, striatum and corpus callosum (CC) during the first week and remained elevated until at least 14 days after TBI. In contrast, M2-like microglia/macrophages peaked at 5 days, but decreased rapidly thereafter. Notably, the severity of white matter injury (WMI), manifested by immunohistochemical staining for neurofilament SMI-32, was strongly correlated with the number of M1-like phagocytes. In vitro experiments using a conditioned medium transfer system confirmed that M1 microglia-conditioned media exacerbated oxygen glucose deprivation-induced oligodendrocyte death. Our results indicate that microglia/macrophages respond dynamically to TBI, experiencing a transient M2 phenotype followed by a shift to the M1 phenotype. The M1 phenotypic shift may propel WMI progression and represents a rational target for TBI treatment.

Scriptaid, a novel histone deacetylase inhibitor, protects against traumatic brain injury via modulation of PTEN and AKT pathway : scriptaid protects against TBI via AKT.

Traumatic brain injury (TBI) is a leading cause of motor and cognitive deficits in young adults for which there is no effective therapy. The present study characterizes the protective effect of a new histone deacetylase inhibitor, Scriptaid (Sigma-Aldrich Corporation, St. Louis, MO), against injury from controlled cortical impact (CCI). Scriptaid elicited a dose-dependent decrease in lesion size at 1.5 to 5.5 mg/kg and a concomitant attenuation in motor and cognitive deficits when delivered 30 minutes postinjury in a model of moderate TBI. Comparable protection was achieved even when treatment was delayed to 12 h postinjury. Furthermore, the protection of motor and cognitive functions was long lasting, as similar improvements were detected 35 days postinjury. The efficacy of Scriptaid (Sigma-Aldrich Corporation) was manifested as an increase in surviving neurons, as well as the number/length of their processes within the CA3 region of the hippocampus and the pericontusional cortex. Consistent with other histone deacetylase inhibitors, Scriptaid treatment prevented the decrease in phospho-AKT (p-AKT) and phosphorylated phosphatase and tensin homolog deleted on chromosome 10 (p-PTEN) induced by TBI in cortical and CA3 hippocampal neurons. Notably, the p-AKT inhibitor LY294002 attenuated the impact of Scriptaid, providing mechanistic evidence that Scriptaid functions partly by modulating the prosurvival AKT signaling pathway. As Scriptaid offers long-lasting neuronal and behavioral protection, even when delivered 12 h after controlled cortical impact, it is an excellent new candidate for the effective clinical treatment of TBI.

Enhanced Delivery of Erythropoietin Across the Blood-Brain Barrier for Neuroprotection against Ischemic Neuronal Injury.

Due to limited penetration of the BBB, many therapeutic agents in clinical use require higher doses in order to reach effective concentrations in brain. In some instances, these high doses elicit severe side effects. In the case of erythropoietin (EPO), an established neuroprotectant against ischemic brain injury, its low BBB permeability requires such a high therapeutic dose that it can induce dangerous complications such as polycythmia and secondary stroke. The purpose of this study is to generate a modified EPO that has increased facility crossing the BBB without losing its neuroprotective element. We have engineered a fusion protein (EPO-TAT) by tagging a protein transduction domain derived from HIV TAT to the EPO protein. This sequence enhanced the capacity of EPO to cross the BBB in animals at least twofold when IP administered and up to five-fold when IV administered. In vitro experiments showed that this EPO fusion protein retained all its protective properties against neuronal death elicited by oxygen-glucose deprivation and NMDA insults. The needed therapeutic dose of the EPO-TAT was decreased by ~10-fold compared to that of regular EPO to achieve equivalent neuroprotection in terms of reducing volume of infarction induced by middle cerebral artery occlusion in mice. Our results support the approach of using a protein transduction domain coupled to therapeutic agents. In this way, not only can the therapeutic doses be lowered, but agents without BBB permeability may now be available for clinical applications.