Phase I study of expanded natural killer cells in combination with cetuximab for recurrent/metastatic nasopharyngeal carcinoma.

BACKGROUND: Nasopharyngeal carcinoma (NPC) cells express high levels of epidermal growth factor receptor (EGFR). Cetuximab is an anti-EGFR monoclonal antibody that promotes natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) via engagement of CD16. We studied safety and efficacy of combining cetuximab with autologous expanded NK cells in patients with recurrent and/or metastatic NPC who had failed at least two prior lines of chemotherapy. METHODS: Seven subjects (six patients) received cetuximab every 3 weeks (six doses maximum) in the pre-trial phase. Autologous NK cells, expanded by co-culture with irradiated K562-mb15-41BBL cells, were then infused on the day after administration of cetuximab. Primary and secondary objectives were to determine safety of this combination therapy and to assess tumor responses, respectively. RESULTS: Median NK cell expansion from peripheral blood mononucleated cells after 10 days of culture with K562-mb15-41BBL was 274-fold (range, 36-534, n = 10), and the median expression of CD16 was 98.4% (range, 67.8-99.7%). Skin rash, the commonest side effect of cetuximab in the pre-trial phase, was not exacerbated by NK cell infusion. No intolerable side effects were observed. Stable disease was observed in four subjects and progressive disease in three subjects. Three patients who received NK cells twice had time to disease progression of 12, 13, and 19 months. CONCLUSION: NK cells with high ADCC potential can be expanded from patients with heavily pre-treated NPC. The safety profile and encouraging clinical responses observed after combining these cells with cetuximab warrant further studies of this approach. (clinicalTrials.gov NCT02507154, 23/07/2015). PRECIS: Engaging NK cell-mediated ADCC using cetuximab plus autologous NK cells in EGFR-positive NPC was well tolerated among heavily pre-treated recurrent NPC. Promising results were observed with 3 out of 7 subjects demonstrating durable stable disease.

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.

Enhancing the immune stimulatory effects of cetuximab therapy through TLR3 signalling in Epstein-Barr virus (EBV) positive nasopharyngeal carcinoma.

Cetuximab immunotherapy targeting the epidermal growth factor receptor (EGFR) has been used to treat nasopharyngeal cancer (NPC) with some success. Therefore, combining an immune adjuvant to boost the immune microenvironment may improve its clinical efficacy. Herein, we investigate the immune-stimulatory effects of Poly-ICLC (a TLR3 agonist) in enhancing cetuximab-based immunotherapy and correlate these responses with FcÉ£RIIIa (V158F) or TLR3 single nucleotide polymorphisms (SNPs- L412F and C829T) expressed on immune effector cells. We observed high levels of TLR3 mRNA in NPC cells; and both TLR3 and EGFR expression were unaffected by Poly-ICLC treatment. Cetuximab plus Poly-ICLC significantly enhanced NK-mediated ADCC through up-regulation of CD107a and Granzyme B expression. This effect was independent of FcÉ£RIIIa-V158F and TLR3-L412F or TLR3-C829T polymorphisms expressed on NK cells. Additionally, IFN-É£ expression and secretion were doubled following cetuximab plus poly-ICLC treatment; compared to either treatment alone. This effect was independent of TLR3 polymorphisms. Consequentially, adaptive immune responses were also seen with increased DC maturation (CD83), co-stimulatory molecules expression (CD80 and CD86) and increased frequency of EGFR-specific CD8 + T cells following Poly-ICLC treatment. The percentage of CD80+ CD83+ and CD83+ CD86+ DC was highest in the Poly-ICLC plus cetuximab group, compared to either treatment alone. These results demonstrate the effectiveness of Poly-ICLC in enhancing both cetuximab-mediated innate and adaptive anti-tumor immunity against NPC, which is independent of FcÉ£RIIIa-158, TLR3-L412F or TLR3-C829T polymorphisms. Additionally, Poly-ICLC does not downregulate EGFR expression on NPC cells and hence, will not dampen cetuximab anti-tumor activity.

Galectin-1-secreting neural stem cells elicit long-term neuroprotection against ischemic brain injury.

Galectin-1 (gal-1), a special lectin with high affinity to ß-galactosides, is implicated in protection against ischemic brain injury. The present study investigated transplantation of gal-1-secreting neural stem cell (s-NSC) into ischemic brains and identified the mechanisms underlying protection. To accomplish this goal, secretory gal-1 was stably overexpressed in NE-4C neural stem cells. Transient cerebral ischemia was induced in mice by middle cerebral artery occlusion for 60 minutes and s-NSCs were injected into the striatum and cortex within 2 hours post-ischemia. Brain infarct volume and neurological performance were assessed up to 28 days post-ischemia. s-NSC transplantation reduced infarct volume, improved sensorimotor and cognitive functions, and provided more robust neuroprotection than non-engineered NSCs or gal-1-overexpressing (but non-secreting) NSCs. White matter injury was also ameliorated in s-NSC-treated stroke mice. Gal-1 modulated microglial function in vitro, by attenuating secretion of pro-inflammatory cytokines (TNF-α and nitric oxide) in response to LPS stimulation and enhancing production of anti-inflammatory cytokines (IL-10 and TGF-ß). Gal-1 also shifted microglia/macrophage polarization toward the beneficial M2 phenotype in vivo by reducing CD16 expression and increasing CD206 expression. In sum, s-NSC transplantation confers robust neuroprotection against cerebral ischemia, probably by alleviating white matter injury and modulating microglial/macrophage function.

n-3 PUFA supplementation benefits microglial responses to myelin pathology.

Microglia represent rational but challenging targets for improving white matter integrity because of their dualistic protective and toxic roles. The present study examines the effect of Omega-3 polyunsaturated fatty acids (n-3 PUFAs) on microglial responses to myelin pathology in primary cultures and in the cuprizone mouse model of multiple sclerosis (MS), a devastating demyelination disease. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), the two main forms of n-3 PUFAs in the brain, inhibited the release of nitric oxide and tumor necrosis factor-α from primary microglia upon IFN-γ and myelin stimulation. DHA and EPA also enhanced myelin phagocytosis in vitro. Therefore, n-3 PUFAs can inhibit inflammation while at the same time enhancing beneficial immune responses such as microglial phagocytosis. In vivo studies demonstrated that n-3 PUFA supplementation reduced cuprizone-induced demyelination and improved motor and cognitive function. The positive effects of n-3 PUFAs were accompanied by a shift in microglial polarization toward the beneficial M2 phenotype both in vitro and in vivo. These results suggest that n-3 PUFAs may be clinically useful as immunomodulatory agents for demyelinating diseases through a novel mechanism involving microglial phenotype switching.

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.

GST P1, a novel downstream regulator of LRRK2, G2019S-induced neuronal cell death.

The enhanced neurotoxicity of the Parkinson's disease-associated LRRK2 mutant, G2019S, than its wild-type counter-part has recently been reported. Overexpression of LRRK2 (G2019S) in cultured neural cells results in caspase-3-dependent apoptosis via a yet undefined signaling pathway. Elucidation of the mechanism underlying LRRK2 (G2019S) neurotoxicity may offer new insights into the pathogenesis of Parkinson's disease. In this study, we identified glutathione s-transferase P1 (GSTP1) as a selective target whose expression is negatively regulated at the transcriptional levels via promoter hyper-methylation by LRRK2 (G2019S). Overexpression of LRRK2 (G2019S) in the human neuronal cell line SH-SY5Y markedly suppressed the expression of GSTP1 prior to any manifestation of cell death. Moreover, shRNA-mediated knockdown of endogenous GSTP1 expression exacerbated LRRK2 (G2019S) neurotoxicity, whereas overexpression of GSTP1 protected against LRRK2 (G2019S)-induced caspase-3 activation and neuronal apoptosis. In conclusion, the results suggest a previously undefined signaling mechanism underlying the neurotoxic effect of LRRK2 (G2019S), in which LRRK2 (G2019S) triggers oxidative stress in cells and, in turn, results in caspase-dependent apoptosis at least in part by suppressing the expression of GSTP1.

Wild-type LRRK2 but not its mutant attenuates stress-induced cell death via ERK pathway.

Leucine-rich repeat kinase 2 (LRRK2) is a recently identified gene that, when mutated at specific locations, results in the onset of parkinsonian symptoms with clinical features indistinguishable from idiopathic Parkinson's disease. Based on structural and domain analysis, LRRK2 is predicted to function as a stress-responsive protein scaffold mediating the regulation of mitogen activating protein kinase (MAPK) pathways. Consistent with this notion, our results supported the notion that expression of wild-type LRRK2 but not Y1699C or G2019S mutants enhanced the tolerance of HEK293 and SH-SY5Y cells towards H(2)O(2)-induced oxidative stress. This increase in stress tolerance was dependent on the presence of the kinase domain of the LRRK2 gene and manifested through the activation of the ERK pathway. Collectively, our results indicated that cells expressing LRRK2 mutants suffer a loss of protection normally derived from wild-type LRRK2, making them more vulnerable to oxidative stress.

Adaptation to chronic MG132 reduces oxidative toxicity by a CuZnSOD-dependent mechanism.

To study whether and how cells adapt to chronic cellular stress, we exposed PC12 cells to the proteasome inhibitor MG132 (0.1 microM) for 2 weeks and longer. This treatment reduced chymotrypsin-like proteasome activity by 47% and was associated with protection against both 6-hydroxydopamine (6-OHDA; 100 microM) and higher dose MG132 (40 microM). Protection developed slowly over the course of the first 2 weeks of exposure and was chronic thereafter. There was no change in total GSH levels after MG132. Buthionine sulfoximine (100 microM) reduced GSH levels by 60%, but exacerbated 6-OHDA toxicity to the same extent in both MG132-treated and control cells and failed to reduce MG132-induced protection. Chronic MG132 resulted in elevated antioxidant proteins CuZn superoxide dismutase (SOD; +55%), MnSOD (+21%), and catalase (+15%), as well as chaperone heat-shock protein 70 (+42%). Examination of SOD enzyme activity revealed higher levels of CuZnSOD (+40%), with no change in MnSOD. We further assessed the mechanism of protection by reducing CuZnSOD levels with two independent siRNA sequences, both of which successfully attenuated protection against 6-OHDA. Previous reports suggested that artificial over-expression of CuZnSOD in dopaminergic cells is protective. Our data complement such observations, revealing that dopaminergic cells are also able to use endogenous CuZnSOD in self-defensive adaptations to chronic stress, and that they can even do so in the face of extensive GSH loss.

Critical role of calpain I in mitochondrial release of apoptosis-inducing factor in ischemic neuronal injury.

Loss of mitochondrial membrane integrity and release of apoptogenic factors are a key step in the signaling cascade leading to neuronal cell death in various neurological disorders, including ischemic injury. Emerging evidence has suggested that the intramitochondrial protein apoptosis-inducing factor (AIF) translocates to the nucleus and promotes caspase-independent cell death induced by glutamate toxicity, oxidative stress, hypoxia, or ischemia. However, the mechanism by which AIF is released from mitochondria after neuronal injury is not fully understood. In this study, we identified calpain I as a direct activator of AIF release in neuronal cultures challenged with oxygen-glucose deprivation and in the rat model of transient global ischemia. Normally residing in both neuronal cytosol and mitochondrial intermembrane space, calpain I was found to be activated in neurons after ischemia and to cleave intramitochondrial AIF near its N terminus. The truncation of AIF by calpain activity appeared to be essential for its translocation from mitochondria to the nucleus, because neuronal transfection of the mutant AIF resistant to calpain cleavage was not released after oxygen-glucose deprivation. Adeno-associated virus-mediated overexpression of calpastatin, a specific calpain-inhibitory protein, or small interfering RNA-mediated knockdown of calpain I expression in neurons prevented ischemia-induced AIF translocation. Moreover, overexpression of calpastatin or knockdown of AIF expression conferred neuroprotection against cell death in neuronal cultures and in hippocampal CA1 neurons after transient global ischemia. Together, these results define calpain I-dependent AIF release as a novel signaling pathway that mediates neuronal cell death after cerebral ischemia.

Effect of sublethal 6-hydroxydopamine on the response to subsequent oxidative stress in dopaminergic cells: evidence for preconditioning.

Exposure to sublethal stress can trigger endogenous protection against subsequent, higher levels of stress. We tested for this preconditioning phenomenon in a model of Parkinson's disease by applying 6-hydroxydopamine to the dopaminergic MN9D cell line. Exposure to sublethal concentrations of 6-hydroxydopamine (5-10 microM) protected against the toxic effects of a subsequent exposure to a higher concentration (50 microM), as measured by the Hoechst assay for nuclear viability. This was accompanied by little or no protection against 6-hydroxydopamine-induced lactate dehydrogenase release, decline in ATP, or reduction in (3)H-dopamine uptake. The antioxidant, N-acetyl cysteine (20 mM), when applied during preconditioning, abolished protection, as did the protein synthesis inhibitor, cycloheximide (0.2 microM). Preconditioning did not affect superoxide dismutase or glutathione peroxidase enzymes, or levels of heat shock protein-72. However, Bcl-2 protein levels rose with preconditioning. Preconditioning rapidly increased phosphorylation of kinases ERK1/2, Akt and JNK, and was abolished by pharmacological inhibitors of their activity. Finally, sublethal 6-hydroxydopamine preconditioned against the toxicity of proteasome inhibitor, MG-132 (1 microM). Thus, exposure of a dopaminergic cell line to sublethal oxidative stress can protect against additional oxidative stress due to translational and post-translational modifications, as well as confer 'cross-tolerance' against a different insult, proteasome inhibition.

BimEL up-regulation potentiates AIF translocation and cell death in response to MPTP.

This study attempted to elucidate the signaling mechanism underlying dopaminergic cell death in the MPP+ model for Parkinson's disease. In neuronal-differentiated PC12 cells, through the regulation by activated JNK and c-jun, BimEL expression was markedly increased in response to MPP+ treatment, which led to the cell degeneration. In lieu of Smac translocation as seen in other paradigms, up-regulation of BimEL effected an increase in calpain I activity that, in turn, mediated AIF release from the mitochondria. In support, we found that knocking down BimEL expression resulted in a decrease in calpain I activity, as well as AIF release from the mitochondria and cell death. Finally, inhibition of calpain activity mitigated AIF release from the mitochondria and cell death. Under cell-free conditions, activated purified calpain I could induce the release of AIF from isolated mitochondria without the participation of BimEL or activated JNK, suggesting that AIF release is a direct consequence of calpain I activity. In concert, the results suggest a novel signaling pathway for dopaminergic cell degeneration, in which MPP+ induces the up-regulation of BimEL, which in turn potentiates an elevation in calpain I activity that mediates AIF release and cell death in a caspase-independent manner.

6-Hydroxydopamine induces dopaminergic cell degeneration via a caspase-9-mediated apoptotic pathway that is attenuated by caspase-9dn expression.

This study showed that primary dopaminergic neurons or the dopaminergic cell line MN9D, when exposed to 15 min of the parkinsonian toxin 6-hydroxydopamine (6-OHDA) in the range of 30-100 microM, underwent delayed degeneration and exhibited hallmarks of apoptosis. These results, along with the absence of any increase in lactate dehydrogenase (LDH) release from the degenerated cells, imply that apoptosis was the dominant mode of cell death. Moreover, a distinct elevation in the measured cellular activities of caspase-9 and -3 but not of caspase-8 points to the caspase-9/caspase-3 cascade as the predominant apoptotic pathway in the degeneration of dopaminergic neurons and MN9D cells. In addition, the presence of caspase-9 or -3 peptide inhibitors but not of caspase-8 inhibitor attenuated cell death significantly, supporting the notion that only the intrinsic apoptotic pathway is utilized to achieve cell death. Finally, overexpression of a mutant caspase-9 with dominant negative phenotype (caspase-9dn) in MN9D cells and primary dopaminergic neurons via the adenovirus and adenoassociated virus gene delivery system, respectively, conferred marked increases in tolerance to the toxicity of 6-OHDA. These results point to the intrinsic caspase-9/caspase-3 cascade as the predominant signaling pathway underlying dopaminergic cell death induced by 6-OHDA and suggest that gene delivery of caspase-9dn can attenuate this pathway and its degenerative consequences.

To die or not to die for neurons in ischemia, traumatic brain injury and epilepsy: a review on the stress-activated signaling pathways and apoptotic pathways.

After a severe episode of ischemia, traumatic brain injury (TBI) or epilepsy, it is typical to find necrotic cell death within the injury core. In addition, a substantial number of neurons in regions surrounding the injury core have been observed to die via the programmed cell death (PCD) pathways due to secondary effects derived from the various types of insults. Apart from the cell loss in the injury core, cell death in regions surrounding the injury core may also contribute to significant losses in neurological functions. In fact, it is the injured neurons in these regions around the injury core that treatments are targeting to preserve. In this review, we present our cumulated understanding of stress-activated signaling pathways and apoptotic pathways in the research areas of ischemic injury, TBI and epilepsy and that gathered from concerted research efforts in oncology and other diseases. However, it is obvious that our understanding of these pathways in the context of acute brain injury is at its infancy stage and merits further investigation. Hopefully, this added research effort will provide a more detailed knowledge from which better therapeutic strategies can be developed to treat these acute brain injuries.