The pH-triggered polyglutamate brush co-delivery of MDR1 and survivin-targeting siRNAs efficiently overcomes multi-drug resistance of NSCLC.

Multi-drug resistance (MDR) is one of the major challenges in the successful chemotherapy of non-small cell lung cancer (NSCLC). Although RNA interference (RNAi) has been widely used to silence resistance-related genes, the effect remains unsatisfactory. In this study, we attempted to overcome MDR of NSCLC by simultaneously interfering with two RNAs that have different functions. A new pH-triggered polyglutamate brush polymer dimethylmaleic anhydride-poly(ethyleneglycol) monomethyl ether-b-polyglutamate-g-spermine (DMA-mPEG-b-PG-g-spermine, DPPGS) was designed and synthesized. The DPPGS/small interfering RNA (siRNA) complex nanoparticles (DPPGSN) were prepared. The results demonstrated that DPPGSN could be transformed from a negatively charged form into a positively charged form in the slightly acidic tumor extracellular environment. The siRNA targeting MDR1 mRNA (siMDR1) and siRNA targeting survivin mRNA (siSurvivin) could be efficiently co-delivered by DPPGS to simultaneously interfere with two genes (p < 0.01). Furthermore, DPPGS co-delivery of siMDR1 and siSurvivin lowered the IC50 value of cisplatin (DDP) in A549/DDP (p < 0.01) cells and increased the apoptosis rate of the cells (p < 0.01). Therefore, co-delivery of siMDR1 and siSurvivin using DPPGS would be a promising approach for overcoming MDR of NSCLC.

Umbilical cord-derived mesenchymal stem cell transplantation for treating elderly vascular dementia.

This study aimed to determine the efficacy and safety of human umbilical cord-derived mesenchymal stem cell (HUC-MSC) transplantation for treating elderly vascular dementia (VaD). Ten VaD patients (average age, 73.88 years old) were treated. HUC-MSCs were isolated, cultured, stem cell-marked, and qualified and administered as a 3-course intravenous infusion to these patients. The Mini-Mental State Exam (MMSE) and the Activities of Daily Living Index (Barthel Index scoring system) were used to assess the cognitive function and daily living activity improvements in these patients before transplantation (T0), 3 months after transplantation (T1), and 6 months after transplantation (T2). The MMSE and Barthel Index scores were 15.80 ± 5.49 and 42.00 ± 9.33 points at T0, respectively, and were significantly different when compared with those at T1 (19.20 ± 6.39 and 49.20 ± 10.86 points, respectively, P < 0.05), whereas there was no difference when compared with those at T2 (14.00 ± 6.55 and 40.70 ± 10.37 points, respectively, P > 0.05). HUC-MSC transplantation was safe and feasible for VaD and improved early cognitive functions and daily living activities in VaD patients to a certain extent, thus improving patients' quality of life.

Immunization of stromal cell targeting fibroblast activation protein providing immunotherapy to breast cancer mouse model.

Unlike heterogeneous tumor cells, cancer-associated fibroblasts (CAF) are genetically more stable which serve as a reliable target for tumor immunotherapy. Fibroblast activation protein (FAP) which is restrictively expressed in tumor cells and CAF in vivo and plays a prominent role in tumor initiation, progression, and metastasis can function as a tumor rejection antigen. In the current study, we have constructed artificial FAP(+) stromal cells which mimicked the FAP(+) CAF in vivo. We immunized a breast cancer mouse model with FAP(+) stromal cells to perform immunotherapy against FAP(+) cells in the tumor microenvironment. By forced expression of FAP, we have obtained FAP(+) stromal cells whose phenotype was CD11b(+)/CD34(+)/Sca-1(+)/FSP-1(+)/MHC class I(+). Interestingly, proliferation capacity of the fibroblasts was significantly enhanced by FAP. In the breast cancer-bearing mouse model, vaccination with FAP(+) stromal cells has significantly inhibited the growth of allograft tumor and reduced lung metastasis indeed. Depletion of T cell assays has suggested that both CD4(+) and CD8(+) T cells were involved in the tumor cytotoxic immune response. Furthermore, tumor tissue from FAP-immunized mice revealed that targeting FAP(+) CAF has induced apoptosis and decreased collagen type I and CD31 expression in the tumor microenvironment. These results implicated that immunization with FAP(+) stromal cells led to the disruption of the tumor microenvironment. Our study may provide a novel strategy for immunotherapy of a broad range of cancer.

[Human umbilic mesenchymal stromal cells repairs diabetic foot in rats associated with VEGF expressional change].

UNLABELLED: OBJECTITVE: To explore the mechanism of human umbilic mesenchymal cells (HUMSCs) implantation for the treatment of diabetic foot in rats associate with vascular endothelia growth factor (VEGF) expression changes. METHODS: After diabetic foot model in rats were established by administration of streptozotozin (STZ) in intraperitoneal injection (2 weeks), ulceration in foot was induced by incision injury combined with swearing staphylococcus aureas. Then, HUMSCs were smeared on the ulceration of foot in diabetic rats. Ten days later, the densities of blood vessel and the level of VEGF expression were determined by using immunohistochemistry, RT-PCR and Western blot. RESULTS: HUMSC grafts reduced significantly the volume of ulceration in diabetic foot rats (P < 0.05). RT-PCR and Western blot showed that VEGF and its mRNA were significantly upregulated (P < 0.05). VEGF immunstaining was found in blood vessels and the densities of blood vessels in HUMSC group were increased significantly (P < 0.05). CONCLUSION: HUMSC implantation showed a positive role in promoting the recovery of the ulceration in foot with diabetic rats.

[Effect of human umbilici mesenchymal stromal cells implantation on the BDNF expression in diabetic foot rats].

UNLABELLED: OBJECTITVE: To investigate the effect of human umbilici mesenchymal cells (HUMSCs) implantation on the brain derived neurotrophic factor (BDNF) expression in diabetic foot rats. METHODS: SD rats were divided into three groups (n = 12): normal group, diadetic foot model group and HUMSC treatment group. Diabetic foot model in rats was established, then prepared HUMSC were implanted on the diabetic foot ulcers in rats, and control ones were administrated with saline only. The area of ulceration, sensory function, BDNF expression and its localization were determined by using morphology, physiological function measurement, RT-PCR and immunohistochemistry assay. RESULT: Siglificantly decreased area of ulceration in diabetic foot rats of HUMSC implantation group was observed. This was simultaneously companied with the sensory function improvement (P < 0.05). RT-PCR showed that BDNF mRNA expression was significantly up regulated (P < 0.05). BDNF immunstaining was located in epithelia tissue and the protein level of BDNF was markedly increased (P < 0.05). CONCLUSION: HUMSC implantation maybe an effective strategy on the treatment of ulceration in diabetic foot rats, and the possible mechanism may involve in BDNF expression.

Comprehensive analysis of transcriptome-wide expression patterns and a circRNA/lncRNA-miRNA-mRNA network in the pathogenesis of cerebral ischemia in Rattus norvegicus.

BACKGROUND: Although ischemic stroke exhibits a high prevalence in the elderly population, the involved genes and pathways are poorly understood. In this study, we proposed to identify differentially expressed genes (DEGs) and constructed a circular RAN (circRNA)/long noncoding RNA (lncRNA)/microRNA (miRNA)-mRNA network associated with the pathogenesis of ischemic stroke by using bioinformatics analysis. METHODS: We constructed a rat model of middle cerebral artery occlusion (MCAO) and conducted total RNA and microRNA sequencing in brain specimens from MCAO and normal rats. Transcriptome-wide expression patterns were analyzed and DEGs were defined by applying Ballgown and a cut of log2-transformed fold-change (log2FC) ≥ 1 (or ≤ -1) with a P value < 0.05. We exploited Pearson correlation analysis to determine the association between the circRNA/lncRNA/mRNA network and miRNAs (P < 0.05 and corr ≤ -0.6), and the competing endogenous RNAs (ceRNA) interaction network was visualized with Cytoscape software and separated into subnetworks using the Molecular Complex Detection (MCODE) algorithm. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were implemented for the pathway analysis of DEGs. RESULTS: Upregulated DEGs were significantly enhanced in positive regulation of cell migration, response to wounding, blood vessel morphogenesis, inflammatory response, and cell activation; Downregulated DEGs were associated with control of the modulation of chemical synaptic transmission, synapse organization, regulation of membrane potential, and regulation of ion transport. KEGG-pathway analysis showed that DEG-enhanced pathways were associated with the pathways of TNF signaling pathway, Fluid shear stress and atherosclerosis, NF-kappa B signaling pathway, Lipid and atherosclerosis, Human cytomegalovirus infection, Osteoclast differentiation, Chemokine signaling pathway, IL-17 signaling pathway, Viral protein interaction with cytokine and cytokine receptor, and Cytokine-cytokine receptor interaction. We uncovered several novel lncRNAs (lnc_00231, lnc_002239, lnc_004172; and a novel_circ0001704), five miRNAs (miR-200b-3p, miR-223-3p, miR-200c-3p, miR-3084a-3p, and miR-664-2-5p), and the top-10 mRNAs (upregulated mRNAs were Pdgfa, Il1b, Gdf15, Fosl1, and Cxcl2; downregulated mRNAs were Prkar2b, Olfm3, Lrrc73, Tmem38a, and Dlgap3) that were involved in ischemic stroke. CONCLUSIONS: Through bioinformatic network analysis, we identified the underlying molecular mechanisms and key central genes that may contribute to an inflammatory response after cerebral infarction.

Prevalence of the methylenetetrahydrofolate reductase 677C>T polymorphism in the pregnant women of Yunnan Province, China.

Mutations in the methylenetetrahydrofolate reductase (MTHFR) gene can result in a reduced ability to utilize folic acid. The MTHFR 677C>T polymorphism in particular has been linked to both birth defects and pregnancy-associated diseases. This study aimed to evaluate the prevalence of the MTHFR 677C>T mutation among pregnant women in Yunnan Province so as to collect baseline data that may be utilized to guide folic acid supplementation efforts and to support related disease prevention programs. We retrospectively reviewed 3387 pregnant women from Yunnan Province. The MTHFR 677C>T polymorphism was identified using polymerase chain reaction (PCR) and DNA sequencing. In total, 1350 (39.9%) subjects were homozygous for the C allele (CC), 1540 (45.4%) subjects were heterozygous (CT), and 497 (14.7%) subjects were homozygous for the T allele (TT). The MTHFR 677C>T polymorphism was found to be present within the studied population, with ∼60% of these patients being either heterozygous or homozygous for the mutant allele and with an overall T allele frequency of 0.37. The frequency of the T allele was significantly higher among pregnant women with complications relative to women with healthy pregnancies, particularly among women <30 years old. As such, the maternal MTHFR 677C>T polymorphism may be a genetic risk factor associated with pregnancy complications and may help identify pregnant women at a high risk of such complications.

The differential neuroprotection of HSP70-hom gene single nucleotide polymorphisms: In vitro (neuronal hypoxic injury model) and in vivo (rat MCAO model) studies.

To investigate the effect of HSP70-hom+2437 single nucleotide polymorphisms (SNPs) on hypoxia and ischemia condition, we constructed the neuronal hypoxic injury model and the rat middle cerebral artery occlusion (MCAO) model to compare the inhibition rate of neurons and detect the infarct volume as well as the expression of related apoptotic proteins in order to explore the possible mechanisms. The neuroblastoma cells SHSY5Y were divided into the OE (transfected with the C allele) group, OEmu (transfected with the T allele) group and negative control (NC, transfected with the empty lentiviral vector CON195) group. Varying degrees of hypoxia were induced by deferoxamine (DFO). The inhibition rate of hypoxic neurons and the expression of related apoptotic proteins were detected in the three genotype groups. While in the rat MCAO model, we built five groups including the sham group, the blank control group (injected with physiological saline), the negative control group (injected with lentivirus and physiological saline), the C allele group and the T allele group (injected with lentivirus overexpressing C and T allele). The MCAO model operation was then underwent in all five groups, the infarct volume by TTC staining and the expression of related apoptotic proteins were detected after 24 h. The results in neuronal hypoxic injury model showed a significant difference in the inhibition rate between the three groups (P < 0.05), and the average inhibition rates for the OEmu, OE and NC groups were 13.2%, 19.2% and 23.3%, respectively. The inhibition rates also differed between lower and higher DFO concentrations (P < 0.05). Compared with the NC group, Bax decreased significantly in the OE and OEmu groups, whereas PI3K and HSPA1L (HSP70-hom) increased. However, the expression of Bax in the OEmu group decreased significantly more than in the OE group, whereas PI3K and HSPA1L levels showed no difference between the two groups. Corresponding with the results above, overexpressing HSP70-hom could reduce the infarct volume of ischemic injury by TTC staining in rat MCAO model and the T allele group also had less infarct volume than C allele group. Compared with the sham group, blank control group and negative control group, Bax decreased significantly in the C and T allele groups, while HSPA1L and p- AKT increased. Furthermore, the expression of Bax in the T allele group decreased significantly more than that in the C allele group, while there were no significant differences in HSPA1L and p-AKT levels between the two groups. Therefore, the overexpression of HSP70-hom+2437 could play a protective role in hypoxic neurons and ischemic brain tissue by upregulating the expression of HSPA1L and PI3K/p-AKT and downregulating the expression of BAX. The neuroprotective effect of the T allele was stronger than that of the C allele, which may be related to the strengthened downregulation of BAX.

[Comparison of 3 hand-carried ultrasound devices with comprehensive echocardiographic devices in elderly cardiac inpatient examinations].

In order to test the clinical capacity of hand-carried ultrasound (HCU) devices in elderly inpatients with heart disease, chamber sizes of heart structure, ventricular wall thickness and motion abnormality (WMA), mitral valve and tricuspid regurgitation evaluated by HCU devices in 401 elderly inpatients with heart disease were compared with those evaluated by comprehensive echocardiography (CE) devices. As a result, there was no significant difference in measurements of cardiac chamber dimensions or left ventricular ejection fraction between the two techniques. The HCU's WMA detection rate relative to the CE was 92.15%. Their conformable rates for detection of mitral and tricuspid regurgitation was 93% and 91.4% respectively. Therefore, we conclude that HCU is one of the practical modalities for diagnosis and monitoring in elderly inpatients with heart disease.

MicroRNA profiling of CD3+ CD56+ cytokine-induced killer cells.

Studies have proven that IL-2 and IL-15 showed contrasting roles during CIK cells preparation. By employing microarray, we analyzed miRNA expression profiles of PBMC, CIKIL-2 and CIKIL-15. Advanced bioinformatic analyses were performed to explore the key miRNAs which may regulate cell proliferation and anti-tumor activity of CIK. We identified 261 differentially expressed miRNAs (DEMs) between PBMC and CIKIL-2, and 249 DEMs between PBMC and CIKIL-15. MiR-143-3p/miR-145-5p was miRNA cluster which may positively regulate cell proliferation. In contrast, miR-340-5p/miR-340-3p cluster may negatively regulate cell proliferation via induction apoptosis, which may cause decreased cell proliferation capacity of CIKIL-2. MiRNA-target interaction analysis indicated that 10 co-downregulated miRNAs may synergistically turn on the expression of a pool of tumor cytotoxic genes in CIK cells. The DEMs between CIKIL-2 and CIKIL-15 may contribute to enhanced tumor cytotoxic capacity of CIKIL-2. Importantly, we found that repressed miR-193a-5p may regulate the expressions of inhibitory receptor KLRD1. The results of the validation assay have shown that KLRD1 were upregulated in CIK cells. Our findings have provided new insights into mechanisms of CIK cells production and tumor cytotoxic function, and shed light on their safety for clinical trial.

Correlation of lymphocyte heat shock protein 70 levels with neurologic deficits in elderly patients with cerebral infarction.

PURPOSE: To assess the association between heat shock protein 70 (HSP70) levels and the severity of ischemic stroke in elderly patients. METHODS: We conducted a case-control study to investigate the changes in lymphocyte HSP70 levels by immunoblot in 65 elderly patients with mild (n = 22), intermediate (n = 21), or severe (n = 22) stroke, and in 34 healthy controls. We analyzed correlations between HSP70 levels and neurologic deficit scores on days 1, 15, and 30 after the onset of stroke. RESULTS: Mean (+/- SD) HSP70 levels were higher in all stroke patients compared with controls (mild stroke: 709 +/- 194 units; intermediate: 585 +/- 165 units; severe: 421 +/- 124 units; controls: 86 +/- 34 units on day 1). Patients with mild stroke had higher levels at day 1 and 15 than did patients with severe stroke. HSP70 levels decreased rapidly from days 1 to 30 in all patients, except in patients with severe stroke, in whom levels decreased slowly between days 15 and 30. There were significant negative correlations between HSP70 levels and neurologic deficit scores in patients on days 1 (r = -0.53, P < 0.001) and 15 (r = -0.54, P < 0.001), but a positive correlation on day 30 (r = 0.49, P < 0.001). CONCLUSION: These data suggest that HSP70 may be a marker for neuroprotection in the early stage of ischemic stroke and a marker for a crisis in the later stages of severe cerebral infarction. Further studies on the use of lymphocyte HSP70 levels in predicting clinical outcomes and underlying mechanisms in cerebral infarction are warranted.

Serum and lymphocyte levels of heat shock protein 70 in aging: a study in the normal Chinese population.

Heat shock proteins (Hsps) have been reported to play an important role in both physiological and pathological processes. Hsps also may serve as biomarkers for evaluating disease states and exposure to environmental stresses. Whether Hsp levels in serum and lymphocytes are correlated with age and sex is largely unknown. In this study, we analyzed serum Hsp70 (the most abundant mammalian Hsp) levels by using Western dot blot in 327 healthy male donors aged between 15 and 50 years. We also investigated the association between Hsp70 levels and age in lymphocytes of 80 normal individuals aged between 40 and 77 years because various chronic diseases increase after the age of 40 years. Our data showed that serum Hsp70 levels were positively correlated with age in subjects aged between 15 and 30 years (P < 0.05) but negatively correlated with age in subjects aged between 30 and 50 years (P < 0.05). Serum Hsp70 levels were the highest in individuals aged between 25 and 30 years among all age groups. In the lymphocyte study there also was a significant age-related decrease in Hsp70 levels in lymphocytes of individuals older than 40 years. The Hsp70 levels were negatively correlated with age (r = -3.708, P < 0.0001) but not with sex (r = -10.536, P = 0.452). This suggests that both serum and lymphocyte Hsp70 levels are age-related and that these may be linked to age-related stress. Thus, age is an important factor in using serum and lymphocyte Hsp70 as biomarkers to evaluate the disease states or exposure to environmental stresses (or both).

Global transcriptome-wide analysis of CIK cells identify distinct roles of IL-2 and IL-15 in acquisition of cytotoxic capacity against tumor.

BACKGROUND: Cytokine-induced killer (CIK) cells are an emerging approach of cancer treatment. Our previous study have shown that CIK cells stimulated with combination of IL-2 and IL-15 displayed improved proliferation capacity and tumor cytotoxicity. However, the mechanisms of CIK cell proliferation and acquisition of cytolytic function against tumor induced by IL-2 and IL-15 have not been well elucidated yet. METHODS: CIK(IL-2) and CIK(IL-15) were generated from peripheral blood mononuclear cells primed with IFN-γ, and stimulated with IL-2 and IL-15 in combination with OKT3 respectively. RNA-seq was performed to identify differentially expressed genes, and gene ontology and pathways based analysis were used to identify the distinct roles of IL-2 and IL-15 in CIK preparation. RESULTS: The results indicated that CIKIL-15 showed improved cell proliferation capacity compared to CIK(IL-2). However, CIK(IL-2) has exhibited greater tumor cytotoxic effect than CIKIL-15. Employing deep sequencing, we sequenced mRNA transcripts in CIK(IL-2) and CIK(IL-15). A total of 374 differentially expressed genes (DEGs) were identified including 175 up-regulated genes in CIK(IL-15) and 199 up-regulated genes in CIK(IL-2)). Among DEGs in CIK(IL-15), Wnt signaling and cell adhesion were significant GO terms and pathways which related with their functions. In CIK(IL-2, type I interferon signaling and cytokine-cytokine receptor interaction were significant GO terms and pathways. We found that the up-regulation of Wnt 4 and PDGFD may contribute to enhanced cell proliferation capacity of CIK(IL-15), while inhibitory signal from interaction between CTLA4 and CD80 may be responsible for the weak proliferation capacity of CIK(IL-2). Moreover, up-regulated expressions of CD40LG and IRF7 may make for improved tumor cytolytic function of CIK(IL-2) through type I interferon signaling. CONCLUSIONS: Through our findings, we have preliminarily elucidated the cells proliferation and acquisition of tumor cytotoxicity mechanism of CIK(IL-15) and CIK(IL-2). Better understanding of these mechanisms will help to generate novel CIK cells with greater proliferation potential and improved tumor cytolytic function.

Interaction between hypertension and HSP70 variants increase the risk of cerebral ischemia in Chinese Han population: an association study.

Cerebral infarction has become one of the leading diseases and a major mortality factor around the world. Atherosclerosis is recognized as one of the important causes of ischemic stroke. Recently, accumulating evidences have indicated that the anti-inflammatory and anti-apoptotic functions of the HSP70 family play an important role in cerebral ischemia. However, the association between HSP70 SNPs and ischemic stroke was also not well established. We chose 101 cases of cerebral ischemia and 100 healthy people from the Chinese Han population as our study subjects, and PCR-RFLP was employed to analyze HSP70 polymorphisms: HSP70-1+190G/C, HSP70-2+1267A/G and HSP70-hom+2437T/C. There were no significant differences in +1267A/G allele or genotype frequencies between patients with stroke and healthy controls. However, genotypes of +190CG and +2437TT were differentially distributed between the patients and controls. A significant difference of T allele distribution in the HSP70-hom+2437T/C site was observed. Logistic regression analysis indicated that genotypes of +190CG, +2437TT and T allele in HSP70-hom were risk factors of ischemic stroke. Moreover, the study has formulated that the interactions between hypertension and +190CG or +2437TT may increase the risks of ischemic stroke. The results from this study have suggested a clinical indicator for assessing the possibilities of cerebral stroke, and supply basis to clinicians to give precaution to people who are at risk of stroke.