Histone Deacetylase Inhibition Restores Behavioral and Synaptic Function in a Mouse Model of 16p11.2 Deletion.

BACKGROUND: Microdeletion of the human 16p11.2 gene locus confers risk for autism spectrum disorders and intellectual disability. How 16p11.2 deletion is linked to these neurodevelopmental disorders and whether there are treatment avenues for the manifested phenotypes remain to be elucidated. Emerging evidence suggests that epigenetic aberrations are strongly implicated in autism. METHODS: We performed behavioral and electrophysiological experiments to examine the therapeutic effects of epigenetic drugs in transgenic mice carrying 16p11.2 deletion (16p11del/+). RESULTS: We found that 16p11del/+ mice exhibited a significantly reduced level of histone acetylation in the prefrontal cortex (PFC). A short (3-day) treatment with class I histone deacetylase (HDAC) inhibitor MS-275 or Romidepsin led to the prolonged (3-4 weeks) rescue of social and cognitive deficits in 16p11del/+ mice. Concomitantly, MS-275 treatment reversed the hypoactivity of PFC pyramidal neurons and the hyperactivity of PFC fast-spiking interneurons. Moreover, the diminished N-methyl-D-aspartate (NMDA) receptor-mediated synaptic currents and the elevated GABAA receptor-mediated synaptic currents in PFC pyramidal neurons of 16p11del/+ mice were restored to control levels by MS-275 treatment. CONCLUSIONS: Our results suggest that HDAC inhibition provides a highly effective therapeutic strategy for behavioral deficits and excitation/inhibition imbalance in 16p11del/+ mice, likely via normalization of synaptic function in the PFC.

Human umbilical cord-derived mesenchymal stem cells suppress proliferation of PHA-activated lymphocytes in vitro by inducing CD4(+)CD25(high)CD45RA(+) regulatory T cell production and modulating cytokine secretion.

Bone marrow-derived mesenchymal stem cells (MSCs) are promising candidate cells for therapeutic application in autoimmune diseases due to their immunomodulatory properties. Unused human umbilical cords (UC) offer an abundant and noninvasive source of MSCs without ethical issues and are emerging as a valuable alternative to bone marrow tissue for producing MSCs. We thus investigated the immunomodulation effect of umbilical cord-derived MSCs (UC-MSCs) on human peripheral blood mononuclear cells (PBMCs), T cells in particular, in a co-culture system. We found that UC-MSCs efficiently suppressed the proliferation of phytohaemagglutinin (PHA)-stimulated PBMCs (p<0.01). Kinetic analysis revealed that UC-MSCs primarily inhibited the division of generation 3 (G3) and 4 (G4) of PBMCs. In addition, UC-MSCs augmented the expression of CD127(+) and CD45RA(+) but reduced the expression of CD25(+) in PBMCs stimulated by PHA (p<0.05). Furthermore, UC-MSCs inhibited PHA-resulted increase in the frequency of CD4(+)CD25(+)CD127(low/-) Tregs significantly (p<0.01) but augmented PHA-resulted increase in the frequency of CD4(+)CD25(high)CD45RA(+) Tregs to about three times in PBMCs. The levels of anti-inflammatory cytokines, PEG2, TGF-ß, and IL-10 were greatly up-regulated, accompanied by a significant down-regulation of pro-inflammatory IFN-γ in the co-culture (p<0.01). Our results showed that UC-MSCs are able to suppress mitogen-induced PBMC activation and proliferation in vitro by altering T lymphocyte phenotypes, increasing the frequency of CD4(+)CD25(high)CD45RA(+) Tregs, and modulating the associated cytokine production. Further studies are warranted to investigate the therapeutic potential of UC-MSCs in immunologically-diseased conditions.

Stem cell factor and granulocyte colony-stimulating factor exhibit therapeutic effects in a mouse model of CADASIL.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a Notch3 dominant mutation-induced cerebral small vascular disease, is characterized by progressive degeneration of vascular smooth muscle cells (vSMCs) of small arteries in the brain, leading to recurrent ischemic stroke, vascular dementia and death. To date, no treatment can stop or delay the progression of this disease. Herein, we determined the therapeutic effects of stem cell factor (SCF) in combination with granulocyte colony-stimulating factor (G-CSF) (SCF+G-CSF) in a mouse model of CADASIL carrying the human mutant Notch3 gene. SCF+G-CSF was subcutaneously administered for 5 days and repeated 4 times with 1-4 month intervals. We found through water maze testing that SCF+G-CSF treatment improved cognitive function. SCF+G-CSF also attenuated vSMC degeneration in small arteries, increased cerebral blood vascular density, and inhibited apoptosis in CADASIL mice. We also discovered that loss of cerebral capillary endothelial cells and neural stem cells/neural progenitor cells (NSCs/NPCs) occurred in CADASIL mice. SCF+G-CSF treatment inhibited the CADASIL-induced cell loss in the endothelia and NSCs/NPCs and promoted neurogenesis. In an in vitro model of apoptosis, SCF+G-CSF prevented apoptotic cell death in vSMCs through AKT signaling and by inhibiting caspase-3 activity. These data suggest that SCF+G-CSF restricts the pathological progression of CADASIL. This study offers new insights into developing therapeutic strategies for CADASIL.

The association between expressions of Ras and CD68 in the angiogenesis of breast cancers.

OBJECTIVE: Angiogenesis is a critical step of breast cancer metastasis. Oncogenic Ras promotes the remodeling of cancer microenviroment. Tumor-associated macrophages (TAMs) are a prominent inflammatory cell population emerging in the microenviroment and facilitating the angiogenesis and metastasis. In the present study, we tried to investigate the relationship between the expression of Ras and infiltration of TAM, both of which could further promote angiogenesis. METHODS: Expressions of Ras, CD68 and CD34 were assessed by immunohistochemistry. The infiltration of macrophages was evaluated by counting the number of CD68(+) cells. Vessel endothelial cells were defined as CD34(+) cells. Angiogenesis vascularity was defined by microvessel density (MVD) assay through counting the number of vessels per field counted in the area of highest vascular density. The Kaplan-Meier survival analysis was used to estimate the overall survival (OS). Macrophages were derived from monocytes in the presence of macrophage colony-stimulating-factor (MCSF). Breast cancer cells were treated with macrophage-conditioned medium (MCM) and tested the expressions of K-, H- and N-Ras by using realtime-PCR. RESULTS: Ras positive status was correlated with ER, PR and Her-2 positivity, larger tumour size and lymph node metastasis, as well as higher TNM stages. A higher number of CD68(+) cells was correlated with larger tumour size, higher TNM stages and Her-2 positivity. Both Ras positivity and infiltration of CD68(+) macrophages correlated with poor OS. The number of CD68(+) cells was positively correlated with the expression of Ras. Treatment with MCM did not up-regulate but repressed the expression of Ras. Both up-regulation of Ras and infiltration of TAMs correlated with increased MVD. CONCLUSION: Expression of Ras and infiltration of TAM were positively correlated, and both participated in angiogenesis. Elevated Ras could be responsible for the infiltration of TAM.

Artesunate induces G2/M cell cycle arrest through autophagy induction in breast cancer cells.

We found that artesunate (ART) inhibited the growth of MCF-7 and MDA-MB-231 breast cancer cells. ART arrested the cell cycle in the G2/M phase, which was accompanied by an upregulation of p21. ART upregulated the expression of Beclin1, an initiator of autophagy (type II programmed cell death). In addition, ART stimulated the aggregation of LC3, which is considered to be a marker of autophagosome formation. We further verified the transformation of LC3 from type I into type II. 3-MA, a classical autophagy inhibitor, attenuated ART-induced autophagosome formation, cell growth repression, G2/M arrest, and p21 upregulation. Autophagy induction and p21 upregulation were also repressed by knockdown of Beclin1. Furthermore, ART sensitized breast cancer cells to the chemotherapeutic agent epirubicin through an autophagy-dependent cascade. Our study showed that ART induced autophagy in breast cancer cells and indicated that the anticancer effects of ART were exerted through an autophagy pathway. Moreover, ART sensitized breast cancer cells to epirubicin chemotherapy. Our results provide a basis for further development of ART as a novel therapeutic agent for the treatment of breast cancer.

Establishment and validation of a novel predictive model to quantify the risk of bone metastasis in patients with prostate cancer.

BACKGROUND: Patients with prostate cancer (PCa) commonly suffer from bone metastasis during disease progression. This study aims to construct and validate a nomogram to quantify bone metastasis risk in patients with PCa. METHODS: Clinicopathological data of patients diagnosed with PCa between 2010 and 2015 were retrospectively retrieved from the Surveillance, Epidemiology, and End Results (SEER) database. Predictors for bone metastasis were identified by logistic regression analyses to establish a nomogram. The concordance index (c-index) and calibration plots were generated to assess the nomogram's discrimination, and the area under the receiver operating characteristic curve (AUC) was used to compare the precision of the nomogram with routine staging systems. The nomogram's clinical performance was evaluated by decision curve analysis (DCA) and clinical impact curves (CIC). Independent prognostic factors were identified by Cox regression analysis. RESULTS: A total of 168,414 eligible cases were randomly assigned to the training cohort or validation cohort at a ratio of 1:1. The nomogram, which was established based on independent factors, showed good accuracy, with c-indexes of 0.911 in the training set and 0.910 in the validation set. Calibration plots also approached 45 degrees. After other distant metastatic sites were included in the predictive model, the new nomogram displayed superior prediction performance. The AUCs and net benefit of the nomograms were both higher than those of other routine staging systems. Furthermore, bone metastasis prediction points were shown to be a new risk factor for overall survival. CONCLUSIONS: Novel validated nomograms can effectively predict the risk of bone metastasis in patients with PCa and help clinicians improve cancer management.

Identification of key genes and pathways at the downstream of S100PBP in pancreatic cancer cells by integrated bioinformatical analysis.

BACKGROUND: The aim of the present study was to identify key genes and pathways downstream of S100PPBP in pancreatic cancer cells. METHODS: The microarray datasets GSE35196 (S100PBP knockdown) and GSE35198 (S100PBP overexpression) were downloaded from the Gene Expression Omnibus (GEO). Differentially expressed genes (DEGs) were obtained separately from GEO2R, and heatmaps showing clustering analysis of DEGs were generated using R software. Gene Ontology and pathway enrichment analyses were performed for identified DEGs using the Database for Annotation, Visualization, and Integrated Discovery and Kyoto Encyclopedia of Genes and Genomes, respectively. A protein-protein interaction (PPI) network was created using the Search Tool for the Retrieval of Interacting Genes and Cytoscape software. Relevant expression datasets of key identified genes were downloaded from The Cancer Genome Atlas, and overall survival (OS) analysis was performed with R software. Finally, Gene Expression Profiling Interactive Analysis was used to evaluate the expression of key DEGs in pancreatic cancer tissues. RESULTS: A total of 34 DEGs (11 upregulated and 23 downregulated) were screened out from the two datasets. Gene Ontology enrichment analysis revealed that the identified DEGs were mainly functionally enriched in ATPase activity, production of siRNA involved in RNA interference, and production of miRNAs involved in gene silencing by miRNA. The pathway enrichment analysis of the identified DEGs showed enrichment mainly in apoptosis, non-homologous end-joining, and miRNA pathways in cancer. The protein-protein interaction network was composed of 21 nodes and 30 edges. After survival analysis and gene expression analysis, 4 genes associated with poor prognosis were selected, including LMNB1, PRKRA, SEPT2, and XRCC5. CONCLUSIONS: LMNB1, PRKRA, SEPT2, and XRCC5 could be key downstream genes of the S100PBP gene in the inhibition of pancreatic cancer cell adhesion.

A bioinformatics analysis to evaluate the prognostic value of stemness-related genes in gastric cancer.

BACKGROUND: This study aimed to identify potential stemness-related targets in gastric cancer (GC) in order to support the development of new treatment strategies and improve patient survival. METHODS: Using the edgeR package, we identified stemness-related differentially expressed genes (DEGs) using GSE112631 and the stemness-related signaling pathways in the Gene Set Enrichment Analysis (GSEA) database. Lasso-penalized Cox regression analysis and multivariate Cox regression analysis tested by Akaike Information Criterion (AIC) were used to screen out survival genes in order to construct a prognostic model. We verified the accuracy of our prognostic model using a nomogram and receiver operating characteristic (ROC) curve analysis. Patients were divided into two groups based on the median risk score, and functional enrichment analysis was used to explore the differences between the two groups. RESULTS: Eight genes were selected to establish a prognostic model of The Cancer Genome Atlas (TCGA) and a validation model of the GSE84437 dataset from the Genome Expression Omnibus (GEO). In both models, we found that the low risk score group had better overall survival (OS) than the high-risk score group. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways between the two risk groups were totally different. CONCLUSIONS: We used eight stemness-related genes to build a prognostic model. The high-risk score group had a worse prognosis compared to the low-risk score group.

A novel nomogram based on SEER database for the prediction of liver metastasis in patients with small-cell lung cancer.

BACKGROUND: To establish and validate a nomogram to predict liver metastasis in patients with small-cell lung cancer (SCLC). METHODS: Information on patients diagnosed with SCLC between 2010 and 2015 was retrospectively retrieved from the Surveillance, Epidemiology, and End Results (SEER) database. Risk factors for liver metastasis were identified by logistic regression analyses to construct a nomogram. The predictive accuracy was evaluated by concordance indexes (c-index) and calibration plots, and the comparison of discrimination between the nomogram and other routine staging systems was achieved with the area under receiver operating characteristic curve (AUC) analysis. Decision curve analysis (DCA) was performed to measure the clinical performance of the nomogram. RESULTS: A total of 12,957 patients met our inclusion criteria and were randomly assigned to training (n=6,479) and validation (n=6,478) sets. The nomogram which was established based on independent clinicopathological factors had poor accuracy, and after other distant metastatic sites were added into the predictive model, the new nomogram displayed better discrimination power, with c-indexes of 0.703 in the training set and 0.712 in the validation set. Both internal and external calibration plots approached 45 degrees. The AUCs and net benefit of the predictive model were both higher than those of routine staging systems. CONCLUSIONS: The validated nomogram might be a practical tool for clinicians to quantify the risk of liver metastasis in patients with SCLC and improve cancer management.

The prognostic roles of red blood cell-associated indicators in patients with resectable gastric cancers.

BACKGROUND: Gastric cancer (GC) is one of the leading causes of cancer-related death worldwide. This study was designed to investigate the prognostic values of red blood cell (RBC)-associated indicators, including RBC, hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and RBC distribution width (RDW) in resectable GC patients. METHODS: In this retrospective study, a total of 104 pathologically confirmed GC patients were recruited. These cases were divided into two groups according to the median values of pretreatment RBC, HGB, HCT, MCV, MCH, MCHC, or RDW. To evaluate the changes in RBC-associated indicators values after treatment, we introduced the concept of post-/pre-treatment ratios (≤1 suggested RBC, HGB, HCT, MCV, MCH, MCHC, or RDW values were not increased after therapy, while >1 represented those in increased levels). RESULTS: The lower pretreatment MCHC levels were correlated with worse overall survival (OS), while pretreatment levels of RBC, HGB, HCT, MCV, MCH, or RDW were not. The whole course of treatment (surgery plus adjuvant chemotherapy) significantly decreased the values of MCHC, and increased the values of MCV and RDW, whereas it had no obvious effects on the values of RBC, HGB, HCT, or MCH. Patients with post-/pre-treatment MCV ratio >1 had an increased survival ratio. Meanwhile, post-/pre-treatment RBC, HGB, HCT, MCH, MCHC, or RDW ratios were not correlated with outcomes. Multivariate Cox regression analysis revealed that the American Joint Committee on Cancer (AJCC) stage (III), and lower pretreatment MCHC levels were independent risk factors affecting OS. The receiver operating characteristic (ROC) curve analysis showed that an MCHC value of 341.98 g/L was the optimal cutoff value for prognosis, with a sensitivity of 58.3% and a specificity of 75.0%. CONCLUSIONS: Pretreatment MCHC levels could become a potential prognostic factor for resectable GC.

Prognostic values of platelet-associated indicators in advanced breast cancer.

BACKGROUND: Breast cancer is the leading cause of cancer death in the female population. Platelet-related indicators can be used as prognostic markers of cancers. The present study investigated the potential values of platelet count (PLT), plateletcrit (PCT), mean platelet volume (MPV), platelet distribution width (PDW), and platelet to lymphocyte ratio (PLR) in the prognosis of advanced breast cancer (ABC). METHODS: This retrospective study recruited 94 locally advanced or metastatic breast cancer cases who had histologic and cytologic evidence. The patients were divided into two groups according to the median baseline values of PLT, PCT, MPV, PDW, and PLR. To evaluate the individual value changes after treatment, we introduced the concept of post/pre-treatment ratio (≤1 indicated value was not increased after treatment, while >1 suggested increased value). Responses to chemotherapy, including partial response (PR), stable disease (SD), and progressive disease (PD), were recorded. Kaplan-Meier curves were constructed to show overall survival (OS). Univariate and multivariate Cox regression analysis models were employed to identify the independent risk factors. RESULTS: A lower baseline PCT level was correlated with a better OS. A lower baseline PLT level and a higher baseline PDW level were related to better chemotherapeutic efficacy of breast cancer patients. Univariate analysis and multivariate analysis both revealed that a higher baseline PCT level was an independent prognostic factor for OS. CONCLUSIONS: PCT level may be a potential prognosis factor for ABC.

Discovery of 2,4,6-trisubstitued pyrido[3,4-d]pyrimidine derivatives as new EGFR-TKIs.

Targeting acquired drug resistance is the major challenge in the treatment of EGFR-driven non-small cell lung cancer (NSCLC). In this study, a novel class of compounds containing pyrido[3,4-d]pyrimidine scaffold was designed as new generation EGFR-TKIs to overcome this challenge. The most promising compound B30 inhibited HCC827 and H1975 cells growth with the IC50 values of 0.044 µM and 0.40 µM, respectively. Meanwhile, B30 displayed potent inhibitory activity against the EGFRL858R (IC50 = 1.1 nM) and EGFRL858R/T790M/C797S (IC50 = 7.2 nM). B30 could suppress EGFR phosphorylation in a dose-dependent manner in HCC827 cell line and significantly induce the apoptosis of HCC827 cells. Molecular docking indicated that the hydroxyl in B30 could form additional hydrogen bond with mutant Ser797. These findings strongly support our assumption that 2,4,6-trisubstitued pyrido[3,4-d] pyrimidine derivatives can serve as EGFR-TKIs. The predicted hydrogen bond interaction formed by a small molecule inhibitor with mutant Ser797 is available to design the fourth-generation EGFR-TKIs.

Brain repair by hematopoietic growth factors in a rat model of stroke.

BACKGROUND AND PURPOSE: Stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) are essential growth factors in hematopoiesis. We determined whether receptors for SCF and G-CSF exist in the brain and whether exogenous SCF and G-CSF are beneficial to brain repair after brain ischemia. METHODS: A well-established rat model of experimental stroke was used in this study. SCF, G-CSF, SCF+G-CSF, or saline was subcutaneously administered 3 hours to 7 days after brain ischemia. Bromodeoxyuridine was administered simultaneously. Sensorimotor function was evaluated with a limb placement test and foot fault test over time. RESULTS: We observed that receptors for SCF and G-CSF were expressed in both neurogenic regions and neurons. SCF-treated rats showed the best functional restoration at 1 week that was maintained 4, 7, and 10 weeks after the final injection. G-CSF-induced functional recovery was limited and unstable. Interestingly, stable but delayed functional improvement was seen in SCF+G-CSF-treated rats. Infarction size was significantly reduced in all growth factor-treated rats. In addition, SCF and SCF+G-CSF enhanced neural progenitor cell proliferation in the subventricular zone bilaterally, whereas G-CSF and SCF+G-CSF treatment increased bromodeoxyuridine -positive cells in periinfarct areas. CONCLUSIONS: SCF and G-CSF are neuroprotective and beneficial to functional restoration when administered during the acute phase after brain ischemia, indicating hematopoietic growth factors play a role in brain repair.

Beneficial effects of hematopoietic growth factor therapy in chronic ischemic stroke in rats.

BACKGROUND AND PURPOSE: Stroke is the leading cause of adult disability worldwide. Currently, there is no effective treatment for stroke survivors. Stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) are the growth factors regulating hematopoiesis. We have previously observed that SCF and G-CSF have neuroprotective and functional effects on acute brain ischemia. In the present study, the beneficial effects of SCF and G-CSF on chronic brain ischemia were determined. METHODS: SCF, G-CSF, or SCF+G-CSF was administered subcutaneously to rats 3.5 months after induction of ischemic stroke by middle cerebral artery occlusion. Neurological deficits were evaluated by limb placement test and foot fault test over time. Field-evoked potential was performed 19 weeks after treatment. Infarct volume was histologically determined using serial coronal sections. RESULTS: Significant functional improvement was seen in SCF+G-CSF-treated rats 1, 5, and 17 weeks after injections. SCF alone also improved functional outcome, but it did not show as stable improvement as SCF+G-CSF. No functional benefit was seen in G-CSF-treated rats. Field-evoked potential studies further confirmed the behavioral data that the normal pattern of neuronal activity was reestablished in the lesioned brain of the rats with good functional outcome. Interestingly, infarction volume was also significantly reduced in SCF+G-CSF-treated rats. CONCLUSIONS: These data provide first evidence that functional restoration in chronic brain ischemia can be attained using hematopoietic growth factors.

Long-term protective effects of AAV9-mesencephalic astrocyte-derived neurotrophic factor gene transfer in parkinsonian rats.

Intrastriatal injection of mesencephalic astrocyte-derived neurotrophic factor (MANF) protein has been shown to provide neuroprotective and neurorestorative effects in a 6-hydroxydopamine (6-OHDA) - lesioned rat model of Parkinson's disease. Here, we used an adeno-associated virus serotype 9 (AAV9) vector to deliver the human MANF (hMANF) gene into the rat striatum 10days after a 6-OHDA lesion to examine long-term effects of hMANF on nigral dopaminergic neurons and mechanisms underlying MANF neuroprotection. Intrastriatal injection of AAV9-hMANF vectors led to a robust and widespread expression of the hMANF gene in the injected striatum up to 24weeks. Increased levels of hMANF protein were also detected in the ipsilateral substantia nigra. The hMANF gene transfer promoted the survival of nigral dopaminergic neurons, regeneration of striatal dopaminergic fibers and an upregulation of striatal dopamine levels, resulting in a long-term improvement of rotational behavior up to 16weeks after viral injections. By using SH-SY5Y cells, we found that intra- and extracellular application of MANF protected cells against 6-OHDA-induced toxicity via inhibiting the endoplasmic reticulum stress and activating the PI3K/Akt/mTOR pathway. Our results suggest that AAV9-mediated hMANF gene delivery into the striatum exerts long-term neuroprotective and neuroregenerative effects on the nigrostriatal dopaminergic system in parkinsonian rats, and provide insights into mechanisms responsible for MANF neuroprotection.

A new method to effectively and rapidly generate neurons from SH-SY5Y cells.

It is well known that neurons differentiated from SH-SY5Y cells can serve as cell models for neuroscience research; i.e., neurotoxicity and tolerance to morphine in vitro. To differentiate SH-SY5Y cells into neurons, RA (retinoic acid) is commonly used to produce the inductive effect. However, the percentage of neuronal cells produced from SH-SY5Y cells is low, either from the use of RA treatment alone or from the combined application of RA and other chemicals. In the current study, we used CM-hNSCs (conditioned medium of human neural stem cells) as the combinational inducer with RA to prompt neuronal differentiation of SH-SY5Y cells. We found that neuronal differentiation was improved and that neurons were greatly increased in the differentiated SH-SY5Y cells using a combined treatment of CM-hNSCs and RA compared to RA treatment alone. The neuronal percentage was higher than 80% (about 88%) on the 3rd day and about 91% on the 7th day examined after a combined treatment with CM-hNSCs and RA. Cell maturation and neurite growth of these neuronal cells were also improved. In addition, the use of CM-hNSCs inhibited the apoptosis of RA-treated SH-SY5Y cells in culture. We are the first to report the use of CM-hNSCs in combination with RA to induce neuronal differentiation of RA-treated SH-SY5Y cells. Our method can rapidly and effectively promote the neuronal production of SH-SY5Y cells in culture conditions.

Sequential Adeno-Associated Viral Vector Serotype 9-Green Fluorescent Protein Gene Transfer Causes Massive Inflammation and Intense Immune Response in Rat Striatum.

Green fluorescent protein (GFP) is a broadly used live cell reporter for gene transduction although side effects associated with GFP in gene transfer are reported. The present study was designed to systematically examine host responses, including inflammatory and immune responses, induced by persistent overexpression of the GFP gene mediated by adeno-associated viral vector serotype 9 (AAV9), and their effects on GFP gene transduction in rat striatum. Our results show that host responses against AAV9-GFP transduction, and GFP transgene expression in the striatum exhibited a temporal and dose-dependent pattern. Both muscular and striatal delivery of AAV9-GFP increased levels of inflammation and immune reactions against sequential AAV9-GFP transduction in the striatum, leading to reduced levels of GFP expression. We also observed that rat sera from sequential administrations of AAV9-GFP group had significantly higher levels of neutralizing antibody against AAV9 vectors when compared with the age-matched rats. As excessive GFP can trigger vigorous inflammation and intense immune response after GFP gene transduction, the use of GFP as a live cell marker protein should be deliberated, especially in repeated administration studies.

Intraventricular administration of endoneuraminidase-N facilitates ectopic migration of subventricular zone-derived neural progenitor cells into 6-OHDA lesioned striatum of mice.

Polysialic acid (PSA), a carbohydrate polymer associated with the neural cell adhesion molecule (NCAM), plays an important role in the migration, differentiation and maturation of neuroblasts. Endoneuraminidase-N (Endo-N) can specifically cleave PSA from NCAM. The objective of the present study was to examine: the effect of Endo-N on characteristics of subventricular zone (SVZ)-derived neural progenitor cells (NPCs) in vitro; whether intraventricular administration of Endo-N could increase ectopic migration of SVZ-derived NPCs into 6-hydroxydopamine (6-OHDA)-lesioned striatum, and whether migrated NPCs could differentiate into neuronal and glial cells. In in vitro study, Endo-N was found to inhibit the migration of NPCs, and to enhance the differentiation of NPCs. In in vivo study, mice sequentially received injections of 6-OHDA into the right striatum, Endo-N into the right lateral ventricle, and bromodeoxyuridine (BrdU) intraperitoneally. The data showed that intraventricular injections of Endo-N disorganized the normal structure of the rostral migratory stream (RMS), and drastically increased the number of BrdU-immunoreactive (IR) cells in 6-OHDA-lesioned striatum. In addition, a number of BrdU-IR cells were double labeled for doublecortin (DCX), NeuN or glial fibrillary acidic protein (GFAP). The results suggest that interruption of neuroblast chain pathway with Endo-N facilitates ectopic migration of SVZ-derived NPCs into the lesioned striatum, and migrated NPCs can differentiate into neurons and astrocytes.

Synthetic fibronectin peptide exerts neuroprotective effects on transient focal brain ischemia in rats.

Leukocytes have been investigated during the past decade for their roles in secondary tissue damage after ischemia/reperfusion injury. Peptide PRARIY, a synthetic fibronectin peptide, has shown an anti-adhesion effect in in vitro studies. Previous studies have demonstrated that anti-adhesion agents lead to reductions in apoptosis. The purpose of the present study was to determine whether the peptide PRARIY displays anti-inflammatory, anti-apoptotic, and neuroprotective effects following transient focal brain ischemia in rats. Twenty-six male Sprague-Dawley rats (300-350 g) were randomly divided into three groups: phosphate-buffered saline (PBS) controls, PRARI controls, and PRARIY treatments. The right middle cerebral artery was transiently occluded using a 4-0 nylon suture. One hour later, the occluder was withdrawn, and reperfusion was maintained for 48 h. Immediately after reperfusion, the peptides (20 mg/kg, dissolved in PBS) and the same volume of PBS were continuously infused through the right external carotid artery using an osmotic minipump for 24 h. Neurological deficits were examined at 3, 24, and 48 h after ischemia. Forty-eight hours after reperfusion, the rats were sacrificed for determining infarction size, leukocyte infiltration, and apoptosis in the ischemia area. Unexpectedly, PRARIY did not influence leukocyte infiltration. However, PRARIY-treated rats showed significantly functional outcome, reduction of infarction size, decrease of TUNEL positive cells, and increase of Bcl-2 (anti-apoptotic protein) positive cells in the ischemic areas when compared to the controls. These data indicate that the peptide PRARIY exerts its neuroprotective effects via supporting neural cell survival rather than anti-leukocyte recruitment following brain ischemia/reperfusion injury.

Inducible Lentivirus-Mediated Expression of the Oct4 Gene Affects Multilineage Differentiation of Adult Human Bone Marrow-Derived Mesenchymal Stem Cells.

The octamer-binding transcription factor 4 (Oct4) gene plays an important role in maintaining the undifferentiated state of embryonic stem cells (ESCs) and reprogramming adult somatic cells into induced pluripotent stem cells (iPSCs). In the present study, we transduced human bone marrow-derived mesenchymal stem cells (hMSCs) using tetracycline-on (Tet-On) lentiviruses carrying human Oct4 to examine the effects of regulated expression of human Oct4 on the proliferation and differentiation of hMSCs. hMSCs were efficiently transduced by Tet-On lentiviruses to express regulated levels of human Oct4 with doxycycline (Dox), as examined by immunofluorescent staining, flow cytometry, and quantitative real time-PCR (qRT-PCR) assays. Ectopic expression of Oct4 in transduced hMSCs increased the ability of colony formation. Continued expression of Oct4 further enhanced adipogenic differentiation of hMSCs, and transient expression of Oct4 sufficiently enhanced osteogenic differentiation of hMSCs. qRT-PCR analysis showed that ectopic expression of Oct4 in transduced hMSCs temporally increased the expression of Sox2 and c-Myc. Interestingly, ectopic expression of Oct4 reduced neuronal differentiation of hMSCs when incubated under neuronal differentiation conditions. Our results suggest that ectopic expression of human Oct4 leads to temporal changes in multilineage differentiation of hMSCs and may inhibit neuronal differentiation of hMSCs.