IL-2-free tumor-infiltrating lymphocyte therapy with PD-1 blockade demonstrates potent efficacy in advanced gynecologic cancer.

BACKGROUND: Tumor-infiltrating lymphocyte (TIL) therapy has been restricted by intensive lymphodepletion and high-dose intravenous interleukin-2 (IL-2) administration. To address these limitations, we conducted preclinical and clinical studies to evaluate the safety, antitumor activity, and pharmacokinetics of an innovative modified regimen in patients with advanced gynecologic cancer. METHODS: Patient-derived xenografts (PDX) were established from a local recurrent cervical cancer patient. TILs were expanded ex vivo from minced tumors without feeder cells in the modified TIL therapy regimen. Patients underwent low-dose cyclophosphamide lymphodepletion followed by TIL infusion without intravenous IL-2. The primary endpoint was safety; the secondary endpoints included objective response rate, duration of response, and T cell persistence. RESULTS: In matched patient-derived xenografts (PDX) models, homologous TILs efficiently reduced tumor size (p < 0.0001) and underwent IL-2 absence in vivo. In the clinical section, all enrolled patients received TIL infusion using a modified TIL therapy regimen successfully with a manageable safety profile. Five (36%, 95% CI 16.3-61.2) out of 14 evaluable patients experienced objective responses, and three complete responses were ongoing at 19.5, 15.4, and 5.2 months, respectively. Responders had longer overall survival (OS) than non-responders (p = 0.036). Infused TILs showed continuous proliferation and long-term persistence in all patients and showed greater proliferation in responders which was indicated by the Morisita overlap index (MOI) of TCR clonotypes between infused TILs and peripheral T cells on day 14 (p = 0.004) and day 30 (p = 0.004). Higher alteration of the CD8+/CD4+ ratio on day 14 indicated a longer OS (p = 0.010). CONCLUSIONS: Our modified TIL therapy regimen demonstrated manageable safety, and TILs could survive and proliferate without IL-2 intravenous administration, showing potent efficacy in patients with advanced gynecologic cancer. TRIAL REGISTRATION: NCT04766320, Jan 04, 2021.

Manganese and Vanadium Co-Exposure Induces Severe Neurotoxicity in the Olfactory System: Relevance to Metal-Induced Parkinsonism.

Chronic environmental exposure to toxic heavy metals, which often occurs as a mixture through occupational and industrial sources, has been implicated in various neurological disorders, including Parkinsonism. Vanadium pentoxide (V2O5) typically presents along with manganese (Mn), especially in welding rods and high-capacity batteries, including electric vehicle batteries; however, the neurotoxic effects of vanadium (V) and Mn co-exposure are largely unknown. In this study, we investigated the neurotoxic impact of MnCl2, V2O5, and MnCl2-V2O5 co-exposure in an animal model. C57BL/6 mice were intranasally administered either de-ionized water (vehicle), MnCl2 (252 µg) alone, V2O5 (182 µg) alone, or a mixture of MnCl2 (252 µg) and V2O5 (182 µg) three times a week for up to one month. Following exposure, we performed behavioral, neurochemical, and histological studies. Our results revealed dramatic decreases in olfactory bulb (OB) weight and levels of tyrosine hydroxylase, dopamine, and 3,4-dihydroxyphenylacetic acid in the treatment groups compared to the control group, with the Mn/V co-treatment group producing the most significant changes. Interestingly, increased levels of α-synuclein expression were observed in the substantia nigra (SN) of treated animals. Additionally, treatment groups exhibited locomotor deficits and olfactory dysfunction, with the co-treatment group producing the most severe deficits. The treatment groups exhibited increased levels of the oxidative stress marker 4-hydroxynonenal in the striatum and SN, as well as the upregulation of the pro-apoptotic protein PKCδ and accumulation of glomerular astroglia in the OB. The co-exposure of animals to Mn/V resulted in higher levels of these metals compared to other treatment groups. Taken together, our results suggest that co-exposure to Mn/V can adversely affect the olfactory and nigral systems. These results highlight the possible role of environmental metal mixtures in the etiology of Parkinsonism.

High-Yield α-Synuclein Purification and Ionic Strength Modification Pivotal to Seed Amplification Assay Performance and Reproducibility.

Alpha-synuclein seed amplification assays (αSyn-SAAs) have emerged as promising diagnostic tools for Parkinson's disease (PD) by detecting misfolded αSyn and amplifying the signal through cyclic shaking and resting in vitro. Recently, our group and others have shown that multiple biospecimens, including CSF, skin, and submandibular glands (SMGs), can be used to seed the aggregation reaction and robustly distinguish between patients with PD and non-disease controls. The ultrasensitivity of the assay affords the ability to detect minute quantities of αSyn in peripheral tissues, but it also produces various technical challenges of variability. To address the problem of variability, we present a high-yield αSyn protein purification protocol for the efficient production of monomers with a low propensity for self-aggregation. We expressed wild-type αSyn in BL21 Escherichia coli, lysed the cells using osmotic shock, and isolated αSyn using acid precipitation and fast protein liquid chromatography (FPLC). Following purification, we optimized the ionic strength of the reaction buffer to distinguish the fluorescence maximum (Fmax) separation between disease and healthy control tissues for enhanced assay performance. Our protein purification protocol yielded high quantities of αSyn (average: 68.7 mg/mL per 1 L of culture) and showed highly precise and robust αSyn-SAA results using brain, skin, and SMGs with inter-lab validation.

Impact of Environmental Risk Factors on Mitochondrial Dysfunction, Neuroinflammation, Protein Misfolding, and Oxidative Stress in the Etiopathogenesis of Parkinson's Disease.

As a prevalent progressive neurodegenerative disorder, Parkinson's disease (PD) is characterized by the neuropathological hallmark of the loss of nigrostriatal dopaminergic (DAergic) innervation and the appearance of Lewy bodies with aggregated α-synuclein. Although several familial forms of PD have been reported to be associated with several gene variants, most cases in nature are sporadic, triggered by a complex interplay of genetic and environmental risk factors. Numerous epidemiological studies during the past two decades have shown positive associations between PD and several environmental factors, including exposure to neurotoxic pesticides/herbicides and heavy metals as well as traumatic brain injury. Other environmental factors that have been implicated as potential risk factors for PD include industrial chemicals, wood pulp mills, farming, well-water consumption, and rural residence. In this review, we summarize the environmental toxicology of PD with the focus on the elaboration of chemical toxicity and the underlying pathogenic mechanisms associated with exposure to several neurotoxic chemicals, specifically 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), rotenone, paraquat (PQ), dichloro-diphenyl-trichloroethane (DDT), dieldrin, manganese (Mn), and vanadium (V). Our overview of the current findings from cellular, animal, and human studies of PD provides information for possible intervention strategies aimed at halting the initiation and exacerbation of environmentally linked PD.

α-Synuclein real-time quaking-induced conversion in the submandibular glands of Parkinson's disease patients.

BACKGROUND: Identification of a peripheral biomarker is a major roadblock in the diagnosis of PD. Immunohistological identification of p-serine 129 α-synuclein in the submandibular gland tissues of PD patients has been recently reported. OBJECTIVE: We report on a proof-of-principle study for using an ultra-sensitive and specific, real-time quaking-induced conversion assay to detect pathological α-synuclein in the submandibular gland tissues of PD patients. METHODS: The α-synuclein real-time quaking-induced conversion assay was used to detect and quantify pathological α-synuclein levels in PD, incidental Lewy body disease, and control submandibular gland tissues as well as in formalin-fixed paraffin-embedded sections. RESULTS: We determined the quantitative seeding kinetics of pathological α-synuclein present in submandibular gland tissues from autopsied subjects using the α-synuclein real-time quaking-induced conversion assay. A total of 32 cases comprising 13 PD, 3 incidental Lewy body disease, and 16 controls showed 100% sensitivity and 94% specificity. Interestingly, both PD and incidental Lewy body disease tissues showed 100% concordance for elevated levels of pathological α-synuclein seeding activity compared to control tissues. End-point dilution kinetic analyses revealed that the submandibular gland had a wide dynamic range of pathological α-synuclein seeding activity. CONCLUSIONS: Our results are the first to demonstrate the utility of using the real-time quaking-induced conversion assay on peripherally accessible submandibular gland tissues and formalin-fixed paraffin-embedded tissue sections to detect PD-related pathological changes with high sensitivity and specificity. Additionally, the detection of seeding activity from incidental Lewy body disease cases containing immunohistochemically undetected pathological α-synuclein demonstrates the α-synuclein real-time quaking-induced conversion assay's potential utility for identifying prodromal PD in submandibular gland tissues. © 2019 International Parkinson and Movement Disorder Society.

Blinded RT-QuIC Analysis of α-Synuclein Biomarker in Skin Tissue From Parkinson's Disease Patients.

BACKGROUND: An unmet clinical need in Parkinson's disease (PD) is to identify biomarkers for diagnosis, preferably in peripherally accessible tissues such as skin. Immunohistochemical studies have detected pathological α-synuclein (αSyn) in skin biopsies from PD patients albeit sensitivity needs to be improved. OBJECTIVE: Our study provides the ultrasensitive detection of pathological αSyn present in the skin of PD patients, and thus, pathological αSyn in skin could be a potential biomarker for PD. METHODS: The real-time quaking-induced conversion assay was used to detect pathological αSyn present in human skin tissues. Further, we optimized this ultra-sensitive and specific assay for both frozen and formalin-fixed paraffin-embedded sections of skin tissues. We determined the seeding kinetics of the αSyn present in the skin from autopsied subjects consisting of frozen skin tissues from 25 PD and 25 controls and formalin-fixed paraffin-embedded skin sections from 12 PD and 12 controls. RESULTS: In a blinded study of skin tissues from autopsied subjects, we correctly identified 24/25 PD and 24/25 controls using frozen skin tissues (96% sensitivity and 96% specificity) compared to 9/12 PD and 10/12 controls using formalin-fixed paraffin-embedded skin sections (75% sensitivity and 83% specificity). CONCLUSIONS: Our blinded study results clearly demonstrate the feasibility of using skin tissues for clinical diagnosis of PD by detecting pathological αSyn. Moreover, this peripheral biomarker discovery study may have broader translational value in detecting misfolded proteins in skin samples as a longitudinal progression marker. © 2020 International Parkinson and Movement Disorder Society.

Prokineticin-2 promotes chemotaxis and alternative A2 reactivity of astrocytes.

Astrocyte reactivity is disease- and stimulus-dependent, adopting either a proinflammatory A1 phenotype or a protective, anti-inflammatory A2 phenotype. Recently, we demonstrated, using cell culture, animal models and human brain samples, that dopaminergic neurons produce and secrete higher levels of the chemokine-like signaling protein Prokineticin-2 (PK2) as a compensatory protective response against neurotoxic stress. As astrocytes express a high level of PK2 receptors, herein, we systematically characterize the role of PK2 in astrocyte structural and functional properties. PK2 treatment greatly induced astrocyte migration, which was accompanied by a shift in mitochondrial energy metabolism, a reduction in proinflammatory factors, and an increase in the antioxidant genes Arginase-1 and Nrf2. Overexpression of PK2 in primary astrocytes or in the in vivo mouse brain induced the A2 astrocytic phenotype with upregulation of key protective genes and A2 reactivity markers including Arginase-1 and Nrf2, PTX3, SPHK1, and TM4SF1. A small-molecule PK2 agonist, IS20, not only mimicked the protective effect of PK2 in primary cultures, but also increased glutamate uptake by upregulating GLAST. Notably, IS20 blocked not only MPTP-induced reductions in the A2 phenotypic markers SPHK1 and SCL10a6 but also elevation of the of A1 marker GBP2. Collectively, our results reveal that PK2 regulates a novel neuron-astrocyte signaling mechanism by promoting an alternative A2 protective phenotype in astrocytes, which could be exploited for development of novel therapeutic strategies for PD and other related chronic neurodegenerative diseases. PK2 signals through its receptors on astrocytes and promotes directed chemotaxis. PK2-induced astrocyte reactivity leads to an increase in antioxidant and anti-inflammatory proteins while increasing glutamate uptake, along with decreased inflammatory factors. © 2018 Wiley Periodicals, Inc.

Organophosphate pesticide chlorpyrifos impairs STAT1 signaling to induce dopaminergic neurotoxicity: Implications for mitochondria mediated oxidative stress signaling events.

The organophosphate (OP) pesticide chlorpyrifos (CPF), used in agricultural settings, induces developmental and neurological impairments. Recent studies using in vitro cell culture models have reported CPF exposure to have a positive association with mitochondria-mediated oxidative stress response and dopaminergic cell death; however, the mechanism by which mitochondrial reactive oxygen species (ROS) contribute to dopaminergic cell death remains unclear. Therefore, we hypothesized that STAT1, a transcription factor, causes apoptotic dopaminergic cell death via mitochondria-mediated oxidative stress mechanisms. Here we show that exposure of dopaminergic neuronal cells such as N27 cells (immortalized murine mesencephalic dopaminergic cells) to CPF resulted in a dose-dependent increase in apoptotic cell death as measured by MTS assay and DNA fragmentation. Similar effects were observed in CPF-treated human dopaminergic neuronal cells (LUHMES cells), with an associated increase in mitochondrial dysfunction. Moreover, CPF (10 µM) induced time-dependent increase in STAT1 activation coincided with the collapse of mitochondrial transmembrane potential, increase in ROS generation, proteolytic cleavage of protein kinase C delta (PKCδ), inhibition of the mitochondrial basal oxygen consumption rate (OCR), with a concomitant reduction in ATP-linked OCR and reserve capacity, increase in Bax/Bcl-2 ratio and enhancement of autophagy. Additionally, by chromatin immunoprecipitation (ChIP), we demonstrated that STAT1 bound to a putative regulatory sequence in the NOX1 and Bax promoter regions in response to CPF in N27 cells. Interestingly, overexpression of non-phosphorylatable STAT1 mutants (STAT1Y701F and STAT1S727A) but not STAT1 WT construct attenuated the cleavage of PKCδ and ultimately cell death in CPF-treated cells. Furthermore, small interfering RNA knockdown demonstrated STAT1 to be a critical regulator of autophagy and mitochondria-mediated proapoptotic cell signaling events after CPF treatment in N27 cells. Finally, oral administration of CPF (5 mg/kg) in postnatal rats (PNDs 27-61) induced motor deficits, and nigrostriatal dopaminergic neurodegeneration with a concomitant induction of STAT1-dependent proapoptotic cell signaling events. Conversely, co-treatment with mitoapocynin (a mitochondrially-targeted antioxidant) and CPF rescued motor deficits, and restored dopaminergic neuronal survival via abrogation of STAT1-dependent proapoptotic cell signaling events. Taken together, our study identifies a novel mechanism by which STAT1 regulates mitochondria-mediated oxidative stress response, PKCδ activation and autophagy. In this context, the phosphorylation of Tyrosine 701 and Serine 727 in STAT1 was found to be essential for PKCδ cleavage. By attenuating mitochondrial-derived ROS, mitoapocynin may have therapeutic applications for reversing CPF-induced dopaminergic neurotoxicity and associated neurobehavioral deficits as well as neurodegenerative diseases.

Engineered CAR T cells targeting mesothelin by piggyBac transposon system for the treatment of pancreatic cancer.

Patients with pancreatic cancer have a poor prognosis largely due to the poor efficacy of the available treatment modalities. In this study, we engineered mesothelin-targeting chimeric antigen receptor T cells (mesoCAR T) using the piggyBac transposon based plasmid electroporation technique for specific targeting of pancreatic cancer cells expressing mesothelin. In vitro, mesoCAR T cells exhibited rapid and robust killing effect against ASPC1 cells with high expression levels of mesothelin with high production of IFN-γ; the cytotoxic effect on PANC1 cells with low expressions of mesothelin was relatively attenuated. In the ASPC1 xenograft mice model, mesoCAR T cells significantly suppressed the tumor growth accompanied with higher-level IFN-γ secretion as compared to control T cells. Besides, more mesoCAR T cells differentiated into memory T cells after tumor remission, whilst causing minimal lesions in major organs. Our study suggests promising efficacy of piggyBac transposon-based mesoCAR T cell therapy for pancreatic cancer, which is a potential candidate for clinical translation.

Molecular mechanisms underlying protective effects of quercetin against mitochondrial dysfunction and progressive dopaminergic neurodegeneration in cell culture and MitoPark transgenic mouse models of Parkinson's Disease.

Quercetin, one of the major flavonoids in plants, has been recently reported to have neuroprotective effects against neurodegenerative processes. However, since the molecular signaling mechanisms governing these effects are not well clarified, we evaluated quercetin's effect on the neuroprotective signaling events in dopaminergic neuronal models and further tested its efficacy in the MitoPark transgenic mouse model of Parkinson's disease (PD). Western blot analysis revealed that quercetin significantly induced the activation of two major cell survival kinases, protein kinase D1 (PKD1) and Akt in MN9D dopaminergic neuronal cells. Furthermore, pharmacological inhibition or siRNA knockdown of PKD1 blocked the activation of Akt, suggesting that PKD1 acts as an upstream regulator of Akt in quercetin-mediated neuroprotective signaling. Quercetin also enhanced cAMP response-element binding protein phosphorylation and expression of the cAMP response-element binding protein target gene brain-derived neurotrophic factor. Results from qRT-PCR, Western blot analysis, mtDNA content analysis, and MitoTracker assay experiments revealed that quercetin augmented mitochondrial biogenesis. Quercetin also increased mitochondrial bioenergetics capacity and protected MN9D cells against 6-hydroxydopamine-induced neurotoxicity. To further evaluate the neuroprotective efficacy of quercetin against the mitochondrial dysfunction underlying PD, we used the progressive dopaminergic neurodegenerative MitoPark transgenic mouse model of PD. Oral administration of quercetin significantly reversed behavioral deficits, striatal dopamine depletion, and TH neuronal cell loss in MitoPark mice. Together, our findings demonstrate that quercetin activates the PKD1-Akt cell survival signaling axis and suggest that further exploration of quercetin as a promising neuroprotective agent for treating PD may offer clinical benefits.

Mesothelin-targeting chimeric antigen receptor-modified T cells by piggyBac transposon system suppress the growth of bile duct carcinoma.

Chimeric antigen receptor modified T cell-based immunotherapy is revolutionizing the field of cancer treatment. However, its potential in treating bile duct carcinoma has not been fully explored. Herein, we developed the second-generation mesothelin-targeting chimeric antigen receptor-modified T cells with the 4-1BB co-stimulatory module by the piggyBac transposon system. Mesothelin-targeting chimeric antigen receptor was expressed by 66.0% of mesothelin-targeting chimeric antigen receptor-modified T cells post electrophoretic transfection and stimulation with K562-meso cells; the expressions of activation markers were tested by flow cytometry assay and showed greater activation of mesothelin-targeting chimeric antigen receptor-modified T cells than control T cells (CD107α: 71.9% vs 48.6%; CD27: 92.1% vs 61.8%; CD137: 55.5% vs 8.4%; CD28: 98.0% vs 82.1%; CD134: 37.5% vs 10.4%). Furthermore, mesothelin-targeting chimeric antigen receptor-modified T cells exerted cytotoxicity toward mesothelin-expressing EH-CA1b and EH-CA1a cells in an effector-to-target ratio-dependent manner, while leaving mesothelin-negative GSC-SD and EH-GB1 cells and normal liver L02 cells almost unharmed. Mesothelin-targeting chimeric antigen receptor-modified T cells secreted cytokines at higher levels when co-cultured with mesothelin-positive EH-CA1a and EH-CA1b cells than with mesothelin-negative GSC-SD and EH-GB1 cells. Enhanced cytotoxicity and cytokine secretion of mesothelin-targeting chimeric antigen receptor-modified T cells compared to control T cells were also observed when co-cultured with 293-meso cells (interferon γ: 85.1% ± 1.47% vs 8.3% ± 2.50%, p = 0.000; tumor necrosis factor α: 90.9% ± 4.67% vs 18.5% ± 3.62%, p = 0.0004; interleukin 2: 60.8% ± 2.00% vs 15.6% ± 2.06%, p = 0.002; interleukin 6: 6.4% ± 2.95% vs 1.7% ± 0.63%, p = 0.055). In addition, mesothelin-targeting chimeric antigen receptor-modified T cells showed greater inhibitory and proliferative capability than control T cells within EH-CA1a cell xenografts. This study shows the potential of mesothelin-targeting chimeric antigen receptor-modified T cells in treating bile duct carcinoma.

Fyn Kinase Regulates Microglial Neuroinflammatory Responses in Cell Culture and Animal Models of Parkinson's Disease.

Sustained neuroinflammation mediated by resident microglia is recognized as a key pathophysiological contributor to many neurodegenerative diseases, including Parkinson's disease (PD), but the key molecular signaling events regulating persistent microglial activation have yet to be clearly defined. In the present study, we examined the role of Fyn, a non-receptor tyrosine kinase, in microglial activation and neuroinflammatory mechanisms in cell culture and animal models of PD. The well-characterized inflammogens LPS and TNFα rapidly activated Fyn kinase in microglia. Immunocytochemical studies revealed that activated Fyn preferentially localized to the microglial plasma membrane periphery and the nucleus. Furthermore, activated Fyn phosphorylated PKCδ at tyrosine residue 311, contributing to an inflammogen-induced increase in its kinase activity. Notably, the Fyn-PKCδ signaling axis further activated the LPS- and TNFα-induced MAP kinase phosphorylation and activation of the NFκB pathway, implying that Fyn is a major upstream regulator of proinflammatory signaling. Functional studies in microglia isolated from wild-type (Fyn(+/+)) and Fyn knock-out (Fyn(-/-)) mice revealed that Fyn is required for proinflammatory responses, including cytokine release as well as iNOS activation. Interestingly, a prolonged inflammatory insult induced Fyn transcript and protein expression, indicating that Fyn is upregulated during chronic inflammatory conditions. Importantly, in vivo studies using MPTP, LPS, or 6-OHDA models revealed a greater attenuation of neuroinflammatory responses in Fyn(-/-) and PKCδ (-/-) mice compared with wild-type mice. Collectively, our data demonstrate that Fyn is a major upstream signaling mediator of microglial neuroinflammatory processes in PD. SIGNIFICANCE STATEMENT: Parkinson's disease (PD) is a complex multifactorial disease characterized by the progressive loss of midbrain dopamine neurons. Sustained microglia-mediated neuroinflammation has been recognized as a major pathophysiological contributor to chronic degenerative processes in PD; however, the key molecular signaling mechanisms underlying microglial activation are not entirely clear. Herein, we identified a novel role for the non-receptor tyrosine kinase Fyn in regulating neuroinflammatory responses in microglia. Our data clearly suggest that the Fyn-PKCδ signaling axis acts as a major upstream signaling mediator of the sustained neuroinflammatory processes in cell culture and animal models of PD. Our finding has important clinical significance to PD because it identifies Fyn as a potential translational target for intervention of progressive neurodegenerative processes in PD.

Protein kinase Cδ upregulation in microglia drives neuroinflammatory responses and dopaminergic neurodegeneration in experimental models of Parkinson's disease.

Chronic microglial activation has been linked to the progressive degeneration of the nigrostriatal dopaminergic neurons evidenced in Parkinson's disease (PD) pathogenesis. The exact etiology of PD remains poorly understood. Although both oxidative stress and neuroinflammation are identified as co-contributors in PD pathogenesis, signaling mechanisms underlying neurodegenerative processes have yet to be defined. Indeed, we recently identified that protein kinase C delta (PKCδ) activation is critical for induction of dopaminergic neuronal loss in response to neurotoxic stressors. However, it remains to be defined whether PKCδ activation contributes to immune signaling events driving microglial neurotoxicity. In the present study, we systematically investigated whether PKCδ contributes to the heightened microglial activation response following exposure to major proinflammatory stressors, including α-synuclein, tumor necrosis factor α (TNFα), and lipopolysaccharide (LPS). We report that exposure to the aforementioned inflammatory stressors dramatically upregulated PKCδ with a concomitant increase in its kinase activity and nuclear translocation in both BV-2 microglial cells and primary microglia. Importantly, we also observed a marked upregulation of PKCδ in the microglia of the ventral midbrain region of PD patients when compared to age-matched controls, suggesting a role for microglial PKCδ in neurodegenerative processes. Further, shRNA-mediated knockdown and genetic ablation of PKCδ in primary microglia blunted the microglial proinflammatory response elicited by the inflammogens, including ROS generation, nitric oxide production, and proinflammatory cytokine and chemokine release. Importantly, we found that PKCδ activated NFκB, a key mediator of inflammatory signaling events, after challenge with inflammatory stressors, and that transactivation of NFκB led to translocation of the p65 subunit to the nucleus, IκBα degradation and phosphorylation of p65 at Ser536. Furthermore, both genetic ablation and siRNA-mediated knockdown of PKCδ attenuated NFκB activation, suggesting that PKCδ regulates NFκB activation subsequent to microglial exposure to inflammatory stimuli. To further investigate the pivotal role of PKCδ in microglial activation in vivo, we utilized pre-clinical models of PD. We found that PKCδ deficiency attenuated the proinflammatory response in the mouse substantia nigra, reduced locomotor deficits and recovered mice from sickness behavior in an LPS-induced neuroinflammation model of PD. Likewise, we found that PKCδ knockout mice treated with MPTP displayed a dampened microglial inflammatory response. Moreover, PKCδ knockout mice exhibited reduced susceptibility to the neurotoxin-induced dopaminergic neurodegeneration and associated motor impairments. Taken together, our studies propose a pivotal role for PKCδ in PD pathology, whereby sustained PKCδ activation drives sustained microglial inflammatory responses and concomitant dopaminergic neurotoxicity consequently leading to neurobehavioral deficits. We conclude that inhibiting PKCδ activation may represent a novel therapeutic strategy in PD treatment.

Therapeutic Cancer Vaccines.

Cancer is one of the major leading death causes of diseases. Prevention and treatment of cancer is an important way to decrease the incidence of tumorigenesis and prolong patients' lives. Subversive achievements on cancer immunotherapy have recently been paid much attention after many failures in basic and clinical researches. Based on deep analysis of genomics and proteomics of tumor antigens, a variety of cancer vaccines targeting tumor antigens have been tested in preclinical and human clinical trials. Many therapeutic cancer vaccines alone or combination with other conventional treatments for cancer obtained spectacular efficacy, indicating the tremendously potential application in clinic. With the illustration of underlying mechanisms of cancer immune regulation, valid, controllable, and persistent cancer vaccines will play important roles in cancer treatment, survival extension and relapse and cancer prevention. This chapter mainly summarizes the recent progresses and developments on cancer vaccine research and clinical application, thus exploring the existing obstacles in cancer vaccine research and promoting the efficacy of cancer vaccine.

Histone hyperacetylation up-regulates protein kinase Cδ in dopaminergic neurons to induce cell death: relevance to epigenetic mechanisms of neurodegeneration in Parkinson disease.

The oxidative stress-sensitive protein kinase Cδ (PKCδ) has been implicated in dopaminergic neuronal cell death. However, little is known about the epigenetic mechanisms regulating PKCδ expression in neurons. Here, we report a novel mechanism by which the PKCδ gene can be regulated by histone acetylation. Treatment with histone deacetylase (HDAC) inhibitor sodium butyrate (NaBu) induced PKCδ expression in cultured neurons, brain slices, and animal models. Several other HDAC inhibitors also mimicked NaBu. The chromatin immunoprecipitation analysis revealed that hyperacetylation of histone H4 by NaBu is associated with the PKCδ promoter. Deletion analysis of the PKCδ promoter mapped the NaBu-responsive element to an 81-bp minimal promoter region. Detailed mutagenesis studies within this region revealed that four GC boxes conferred hyperacetylation-induced PKCδ promoter activation. Cotransfection experiments and Sp inhibitor studies demonstrated that Sp1, Sp3, and Sp4 regulated NaBu-induced PKCδ up-regulation. However, NaBu did not alter the DNA binding activities of Sp proteins or their expression. Interestingly, a one-hybrid analysis revealed that NaBu enhanced transcriptional activity of Sp1/Sp3. Overexpression of the p300/cAMP-response element-binding protein-binding protein (CBP) potentiated the NaBu-mediated transactivation potential of Sp1/Sp3, but expressing several HDACs attenuated this effect, suggesting that p300/CBP and HDACs act as coactivators or corepressors in histone acetylation-induced PKCδ up-regulation. Finally, using genetic and pharmacological approaches, we showed that NaBu up-regulation of PKCδ sensitizes neurons to cell death in a human dopaminergic cell model and brain slice cultures. Together, these results indicate that histone acetylation regulates PKCδ expression to augment nigrostriatal dopaminergic cell death, which could contribute to the progressive neuropathogenesis of Parkinson disease.

Mitochondria-targeted antioxidants for treatment of Parkinson's disease: preclinical and clinical outcomes.

Parkinson's disease is a progressive neurodegenerative disease in the elderly, and no cure or disease-modifying therapies exist. Several lines of evidence suggest that mitochondrial dysfunction and oxidative stress have a central role in the dopaminergic neurodegeneration of Parkinson's disease. In this context, mitochondria-targeted therapies that improve mitochondrial function may have great promise in the prevention and treatment of Parkinson's disease. In this review, we discuss the recent developments in mitochondria-targeted antioxidants and their potential beneficial effects as a therapy for ameliorating mitochondrial dysfunction in Parkinson's disease.

Molecular cloning, epigenetic regulation, and functional characterization of Prkd1 gene promoter in dopaminergic cell culture models of Parkinson's disease.

We recently identified a compensatory survival role for protein kinase D1 (PKD1) in protecting dopaminergic neurons from oxidative insult. To investigate the molecular mechanism of Prkd1 gene expression, we cloned the 5'-flanking region (1620-bp) of the mouse Prkd1 gene. Deletion analyses revealed that the -250/+113 promoter region contains full promoter activity in MN9D dopaminergic neuronal cells. In silico analysis of the Prkd1 promoter uncovered binding sites for key redox transcription factors including Sp1 and NF-κB. Over-expression of Sp1, Sp3, and NF-κB-p65 proteins stimulated Prkd1 promoter activity. Binding of Sp3 and NF-κB-p65 to the Prkd1 promoter was confirmed using chromatin immunoprecipitation. Treatment with the Sp inhibitor mithramycin A significantly attenuated Prkd1 promoter activity and PKD1 mRNA and protein expression. Further mechanistic studies revealed that inhibition of histone deacetylation and DNA methylation up-regulated PKD1 mRNA expression. Importantly, negative modulation of PKD1 signaling by pharmacological inhibition or shRNA knockdown increased dopaminergic neuronal sensitivity to oxidative damage in a human mesencephalic neuronal cell model. Collectively, our findings demonstrate that Sp1, Sp3, and NF-κB-p65 can transactivate the mouse Prkd1 promoter and that epigenetic mechanisms, such as DNA methylation and histone modification, are key regulatory events controlling the expression of pro-survival kinase PKD1 in dopaminergic neuronal cells. Previously, we demonstrated that protein kinase D1 (PKD1) plays a survival role during the early stage of oxidative stress in dopaminergic neuronal cells.

Argonaute 2 promotes angiogenesis via the PTEN/VEGF signaling pathway in human hepatocellular carcinoma.

AIM: Argonaute2 (AGO2) protein is the active part of RNA-induced silencing complex, cleaving the target mRNA strand complementary to their bound siRNA. An increasing number of miRNAs has been identified as essential to angiogenesis of hepatocellular carcinoma (HCC). In this study we investigated how AGO2 affected HCC angiogenesis. METHODS: Human HCC cell lines HepG2, Hep3B, Huh7, SMMC-7721, Bel-7404, MHCC97-H and LM-3, and human umbilical vein endothelial cells (HUVEC) were tested. The expression of AGO2 in HCC cells was knocked down with siRNA and restored using recombinant adenovirus expressing Ago2. The levels of relevant mRNAs and proteins were examined using RT-PCR, Western blot and EILSA. Nude mice were implanted with Huh7 or SMMC-7721 cells, and tumor volumes were measured. After the mice were euthanized, the xenograft tumors were used for immunohistological analysis. RESULTS: In 6 HCC cell lines, AGO2 protein expression was significantly correlated with VEGF expression (r=+0.79), and with VEGF secretion (r=+0.852). Knockdown of Ago2 in Huh7 cells and SMMC-7721 cells substantially decreased VEGF expression, whereas the restoration of AGO2 reversed both VEGF expression and secretion. Furthermore, knockdown of Ago2 significantly up-regulated the expression of PTEN (a tumor suppressor involved in the inhibition of HCC angiogenesis), and vice versa. Moreover, the specific PTEN inhibitor bisperoxovanadate (7, 14, 28 nmol/L) dose-dependently restored the expression of VEGF and the capacity of HCC cells to induce HUVECs to form capillary tubule structures. In the xenograft nude mice, knockdown of Ago2 markedly suppressed the tumor growth and decreased PTEN expression and CD31-positive microvascular in the xenograft tumors. CONCLUSION: A direct relationship exists between the miRNA processing machinery AGO2 and HCC angiogenesis that is mediated by the AGO2/PTEN/VEGF signaling pathway. The results suggest the high value of Ago2 knockdown in anti-angiogenesis therapy for HCC.

Nanoneuromedicines for degenerative, inflammatory, and infectious nervous system diseases.

Interest in nanoneuromedicine has grown rapidly due to the immediate need for improved biomarkers and therapies for psychiatric, developmental, traumatic, inflammatory, infectious and degenerative nervous system disorders. These, in whole or in part, are a significant societal burden due to growth in numbers of affected people and in disease severity. Lost productivity of the patient and his or her caregiver, and the emotional and financial burden cannot be overstated. The need for improved health care, treatment and diagnostics is immediate. A means to such an end is nanotechnology. Indeed, recent developments of health-care enabling nanotechnologies and nanomedicines range from biomarker discovery including neuroimaging to therapeutic applications for degenerative, inflammatory and infectious disorders of the nervous system. This review focuses on the current and future potential of the field to positively affect clinical outcomes. From the clinical editor: Many nervous system disorders remain unresolved clinical problems. In many cases, drug agents simply cannot cross the blood-brain barrier (BBB) into the nervous system. The advent of nanomedicines can enhance the delivery of biologically active molecules for targeted therapy and imaging. This review focused on the use of nanotechnology for degenerative, inflammatory, and infectious diseases in the nervous system.

The peptidyl-prolyl isomerase Pin1 up-regulation and proapoptotic function in dopaminergic neurons: relevance to the pathogenesis of Parkinson disease.

Parkinson disease (PD) is a chronic neurodegenerative disease characterized by a slow and progressive degeneration of dopaminergic neurons in substantia nigra. The pathophysiological mechanisms underlying PD remain unclear. Pin1, a major peptidyl-prolyl isomerase, has recently been associated with certain diseases. Notably, Ryo et al. (Ryo, A., Togo, T., Nakai, T., Hirai, A., Nishi, M., Yamaguchi, A., Suzuki, K., Hirayasu, Y., Kobayashi, H., Perrem, K., Liou, Y. C., and Aoki, I. (2006) J. Biol. Chem. 281, 4117-4125) implicated Pin1 in PD pathology. Therefore, we sought to systematically characterize the role of Pin1 in PD using cell culture and animal models. To our surprise we observed a dramatic up-regulation of Pin1 mRNA and protein levels in dopaminergic MN9D neuronal cells treated with the parkinsonian toxicant 1-methyl-4-phenylpyridinium (MPP(+)) as well as in the substantia nigra of the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. Notably, a marked expression of Pin1 was also observed in the substantia nigra of human PD brains along with a high co-localization of Pin1 within dopaminergic neurons. In functional studies, siRNA-mediated knockdown of Pin1 almost completely prevented MPP(+)-induced caspase-3 activation and DNA fragmentation, indicating that Pin1 plays a proapoptotic role. Interestingly, multiple pharmacological Pin1 inhibitors, including juglone, attenuated MPP(+)-induced Pin1 up-regulation, α-synuclein aggregation, caspase-3 activation, and cell death. Furthermore, juglone treatment in the MPTP mouse model of PD suppressed Pin1 levels and improved locomotor deficits, dopamine depletion, and nigral dopaminergic neuronal loss. Collectively, our findings demonstrate for the first time that Pin1 is up-regulated in PD and has a pathophysiological role in the nigrostriatal dopaminergic system and suggest that modulation of Pin1 levels may be a useful translational therapeutic strategy in PD.