IL-7 promotes CD19-directed CAR-T cells proliferation through miRNA-98-5p by targeting CDKN1A.

CAR-T targeting CD19 have achieved significant effects in the treatment of B-line leukemia and lymphoma. However, the treated patients frequently relapsed and could not achieve complete remission. Therefore, improving the proliferation and cytotoxicity of CAR-T cells, reducing exhaustion and enhancing infiltration capacity are still issues to be solved. The IL-7 has been shown to enhance the memory characteristics of CAR-T cells, but the specific mechanism has yet to be elaborated. miRNAs play an important role in T cell activity. However, whether miRNA is involved in the activation of CAR-T cells by IL-7 has not yet been reported. Our previous study had established the 3rd generation CAR-T cells. The present study further found that IL-7 significantly increased the proliferation of anti-CD19 CAR-T cells, the ratio of CD4 + CAR + cells and the S phase of cell cycle. In vivo study NAMALWA xenograft model showed that IL-7-stimulated CAR-T cells possessed stronger tumoricidal efficiency. Further we validated that IL-7 induced CAR-T cells had low expression of CDKN1A and high expression of miRNA-98-5p. Additionally, CDKN1A was associated with miRNA-98-5p. Our results, for the first time, suggested IL-7 could conspicuously enhance the proliferation of CAR-T cells through miRNA-98-5p targeting CDKN1A expression, which should be applied to CAR-T production.

Andrographolide suppresses breast cancer progression by modulating tumor-associated macrophage polarization through the Wnt/ß-catenin pathway.

Andrographolide(ADE) has been demonstrated to inhibit tumor growth through direct cytotoxicity on tumor cells. However, its potential activity on tumor microenvironment (TME) remains unclear. Tumor-associated macrophages (TAMs), composed mainly of M2 macrophages, are the key cells that create an immunosuppressive TME by secretion of cytokines, thus enhancing tumor progression. Re-polarized subpopulations of macrophages may represent vital new therapeutic alternatives. Our previous studies showed that ADE possessed anti-metastasis and anoikis-sensitization effects. Here, we demonstrated that ADE significantly suppressed M2-like polarization and enhanced M1-like polarization of macrophages. Moreover, ADE inhibited the migration of M2 and tube formation in HUVECs under M2 stimulation. In vivo studies showed that ADE restrained the growth of MDA-MB-231 and HCC1806 human breast tumor xenografts and 4T-1 mammary gland tumors through TAMs. Wnt5a/ß-catenin pathway and MMPs were particularly associated with ADE's regulatory mechanisms to M2 according to RNA-seq and bioinformatics analysis. Moreover， western blot also verified the expressions of these proteins were declined with ADE exposure. Among the cytokines released by M2, PDGF-AA and CCL2 were reduced. Our current findings for the first time elucidated that ADE could modulate macrophage polarization and function through Wnt5a signaling pathway, thereby playing its role in inhibition of triple-negative breast cancer.

Evaluation of piggyBac-mediated anti-CD19 CAR-T cells after ex vivo expansion with aAPCs or magnetic beads.

Adoptive immunotherapy is a new potential method of tumour therapy, among which anti-CD19 chimeric antigen receptor T-cell therapy (CAR-T cell), is a typical treatment agent for haematological malignancies. Previous clinical trials showed that the quality and phenotype of CAR-T cells expanded ex vivo would seriously affect the tumour treatment efficacy. Although magnetic beads are currently widely used to expand CAR-T cells, the optimal expansion steps and methods have not been completely established. In this study, the differences between CAR-T cells expanded with anti-CD3/CD28 mAb-coated beads and those expanded with cell-based aAPCs expressing CD19/CD64/CD86/CD137L/mIL-15 counter-receptors were compared. The results showed that the number of CD19-specific CAR-T cells with a 4-1BB and CD28 co-stimulatory domain was much greater with stimulation by aAPCs than that with beads. In addition, the expression of memory marker CD45RO was higher, whereas expression of exhausted molecules was lower in CAR-T cells expanded with aAPCs comparing with the beads. Both CAR-T cells showed significant targeted tumoricidal effects. The CAR-T cells stimulated with aAPCs secreted apoptosis-related cytokines. Moreover, they also possessed marked anti-tumour effect on NAMALWA xenograft mouse model. The present findings provided evidence on the safety and advantage of two expansion methods for CAR-T cells genetically modified by piggyBac transposon system.

CTHRC1 promotes wound repair by increasing M2 macrophages via regulating the TGF-ß and notch pathways.

The healing of acute wounds is vital to humans and is a well-orchestrated process that involves systemic and local factors. However, there is a lack of effective and safe clinical therapies. The collagen triple helix repeat containing 1 (CTHRC1) protein is a type of exocrine protein that has been recently reported to contribute to tissue repair. Our aim is to validate the promoting effects of CTHRC1 on the healing of acute wounds and to elucidate the underlying molecular mechanism. Therefore, we first established acute wound healing mouse models and confirmed that CTHRC1 accelerates the healing process of acute wounds. Then, we characterized wound macrophages using a polyvinylalcohol (PVA) sponge model and used Western blotting to investigate the molecular mechanism. We found that CTHRC1 increased the M2 macrophage population and the TGF-ß expression level as a result of the activation of the TGF-ß and Notch pathways, which eventually contributed to the promotion of wound healing. Inhibition of the Notch pathway showed attenuated M2 macrophage recruitment, and it decreased the TGF-ß expression level. These results substantiate our hypothesis that CTHRC1 promotes wound healing by recruiting M2 macrophages and regulating the TGF-ß and Notch pathways.

Assessment of tumor promoting effects of amniotic and umbilical cord mesenchymal stem cells in vitro and in vivo.

PURPOSE: Human mesenchymal stem cells (hMSCs) have been applied in a variety of therapies recently. However, the role of MSCs in tumor progression remains largely elusive. Some studies demonstrated that MSCs can promote tumor growth, while others had opposite results. Therefore, the lack of evidence about the effect of MSCs on tumor cells impedes its further use. METHODS: In the current study, hMSCs from amniotic membrane (hAMSCs) and umbilical cord (hUCMSCs) were used to evaluate the effects of MSCs on tumor development in vitro and in vivo. Two different animal models based on subcutaneous xenograft bearing nude mice and a murine experimental metastatic model were established for in vivo study. Moreover, cytokines regulated by MSCs co-cultured with cancer cells SPC-A-1 were also analyzed by cytokine array. RESULTS: Our results indicated that hUCMSCs not only did not promote proliferation in cancer cells, but also inhibited migration. In addition, they inhibited tube formation in human umbilical vein endothelial cells (HUVECs). Although hAMSCs also showed inhibitory effects on cancer cell motility, the proliferation of cancer cells was indeed enhanced. The in vivo data revealed that hUCMSCs did not promote tumor progression in lung adenocarcinoma and gastric carcinoma xenografts. Nevertheless, hAMSCs could do. The results from murine experimental metastatic model also demonstrated that neither hUCMSCs nor hAMSCs significantly enhanced the lung metastasis. The data from cytokine array showed that 11 inflammatory factors, 8 growth factors and 11 chemokines were remarkably secreted and changed. CONCLUSIONS: In view of the data from in vitro and in vivo studies, the exploitation of hUCMSCs in new therapeutic strategies should be safe compared to hAMSCs under malignant conditions. Moreover, this is the first report to systematically elucidate the possible molecular mechanisms involved in UCMSC- and AMSC-affected tumor growth and metastasis.

Molecular differences in Alzheimer's disease between male and female patients determined by integrative network analysis.

Alzheimer's disease (AD) is a complex neurodegenerative disease and the most common cause of dementia among the elderly. There has been increasing recognition of sex differences in AD prevalence, clinical manifestation, disease course and prognosis. However, there have been few studies on the molecular mechanism underlying these differences. To address this issue, we carried out global gene expression and integrative network analyses based on expression profiles (GSE84422) across 17 cortical regions of 125 individuals with AD. There were few genes that were differentially expressed across the 17 regions between the two sexes, with only four (encoding glutamate metabotropic receptor 2, oestrogen-related receptor beta, kinesin family member 26B, and aspartoacylase) that were differentially expressed in three regions. A pan-cortical brain region co-expression network analysis identified pathways and genes (eg, glycogen synthase kinase 3ß) that were significantly associated with clinical characteristics of AD (such as neurofibrillary score) in males only. Similarity analyses between region-specific networks indicated that male patients exhibited greater variability, especially in the superior parietal lobule, dorsolateral prefrontal cortex and occipital visual cortex. A network module analysis revealed an association between clinical traits and crosstalk of sex-specific modules. An examination of temporal and spatial patterns of sex differences in AD showed that molecular networks were more conserved in females than in males in different cortical regions and at different AD stages. These findings provide insight into critical molecular pathways governing sex differences in AD pathology.

Inhibition of MiRNA-125b Decreases Cerebral Ischemia/Reperfusion Injury by Targeting CK2α/NADPH Oxidase Signaling.

BACKGROUND/AIMS: Cerebral ischemia-reperfusion (I/R) injury involves multiple independently fatal terminal pathways. CK2α/NADPH oxidase is an important signaling pathway associated with ischemia-reperfusion injury, and miR-125b can regulate oxidative stress-related injury. In this study, we investigated whether the effect of miR-125b in rat brain I/R injury occurs through its modulation of the CK2α/NADPH oxidase pathway. METHODS: Rats were subjected to 2 h of cerebral ischemia followed by 24 h of reperfusion to establish an I/R injury model. Neurological deficit was evaluated using a five-point score. Infarct volume was evaluated with 2, 3, 5-triphenyltetrazolium chloride (TTC) staining, and RT-PCR was used to detect expressions of miR125b and CK2α. We then examined the association between miR-125b expression and the CK2α/NADPH oxidative signaling pathway in a PC-12 cell oxygen-glucose deprivation and reoxygenation (OGD/R) injury model. Transfection with miR-125b mimics, an miR-125b inhibitor, and luciferase reporter gene plasmid was accomplished using commercial kits. In these cells, Western blots were used to detect the levels of expression of CK2α, cleaved caspase-3, NOX2, and NOX4. RT-PCR was used to detect the expressions of CK2α, miR125b, NOX2, and NOX4. We evaluated Lactate Dehydrogenase (LDH) level, NADPH oxidase activity, and caspase-3 activity using commercial kits. Mitochondrial reactive oxygen species (ROS) were measured by fluorescence microscopy. For both PC-12 cells and rat brains, histological analyses were conducted to observe morphological changes, and apoptosis was measured using a commercial kit. RESULTS: I/R rats exhibited an increase in neurological deficit score, infarct volume, and cellular apoptosis, along with miR-125b elevation and CK2α downregulation. OGD/R treatment increased PC-12 cells' injuries, cellular apoptosis, and ROS levels. These changes were associated with miR-125b elevation, CK2α downregulation and activations of NOX2 and NOX4, mimicking our in vivo findings. All of these effects were reversed by the inhibition of miR-125b, confirming a strong correlation between miR-125b activity and the CK2α/NADPH oxidase signaling pathway. CONCLUSIONS: Based on these observations, we conclude that inhibition of miR-125b protects the rat brain from I/R injury by regulating the CK2α/NADPH oxidative signaling pathway.

Effect of long-term pulsed electromagnetic field exposure on hepatic and immunologic functions of rats.

BACKGROUND: In this report, the effects of long-term pulsed electromagnetic field (PEMF) exposure on hepatic and immunologic functions were examined. METHODS: Male rats were randomly divided into four groups: a control group and three experimental groups exposed to a 50-Hz PEMF at 5, 10, or 20 mT for 10 weeks. RESULTS: Compared with the control group, activities of serum alanine aminotransferase and aspartate aminotransferase and concentrations of serum, liver, and spleen Metabolism of lipid peroxidation (MDA) in the 10- and 20-mT PEMF groups were significantly increased. The activities of Glutathione peroxidase (GSH-Px) and Superoxide Dismutase (SOD) in the serum, liver, and spleen and concentrations of serum immunoglobulins were significantly decreased. CONCLUSION: These results demonstrate that long-term exposure to PEMF can lead to oxidative damage of the liver and spleen.

Emodin has a protective effect in cases of severe acute pancreatitis via inhibition of nuclear factor­κB activation resulting in antioxidation.

Severe acute pancreatitis (SAP) accounts for up to 20% of acute pancreatitis (AP) cases. The absence of effective treatment options has resulted in a high rate of morbidity and mortality. Emodin is a major component of the Chinese herb rhubarb, which has been widely used in the treatment of numerous diseases, including inflammation and cancer. There are a limited number of studies however, that have investigated the effectiveness of emodin in the treatment of SAP. The present study used a rat model of SAP, to investigate the effect and molecular mechanisms of emodin treatment. Administration of emodin was identified to significantly attenuate SAP, as determined by serum amylase analysis and histological assessment of edema, vacuolization, inflammation and necrosis (P<0.01). Furthermore, treatment with emodin markedly inhibited nuclear factor (NF)­κB DNA­binding activity (P<0.01) and the serum expression levels of tumor necrosis factor­α, interleukin (IL)­6 and IL­1ß (P<0.05). This attenuation was associated with decreased malondialdehyde and increased superoxide dismutase levels in the pancreatic tissues and serum (P<0.05). This study indicated that administration of exogenous emodin had therapeutic effects on the severity of SAP. The mechanism may be due to inhibition of NF­κB activation resulting in an antioxidation response, which can subsequently suppress the expression of cytokines.

Suppressive effects of induced pluripotent stem cell-conditioned medium on in vitro hypertrophic scarring fibroblast activation.

Hypertrophic scarring (HS) is a type of fibrosis that occurs in the skin, and is characterized by fibroblast activation and excessive collagen production. However, at present, therapeutic strategies for this condition are ineffective. Previous studies have identified that the mutual regulation of chronic inflammation, mechanical force and fibroblast activation leads to the formation of HS. Induced pluripotent stem cells (iPSCs) are novel bioengineered embryonic­like stem cells, initially created from mouse adult fibroblasts. The current study demonstrated that iPSC­conditioned medium (iPSC­CM) may significantly suppress hypertrophic scar fibroblast activation. It was observed that in the presence of iPSC­CM, the level of collagen I was markedly reduced and α­smooth muscle actin, a marker for myofibroblasts (activated fibroblasts that mediate mechanical force­induced HS formation), exhibited a significantly lower level of expression in human dermal fibroblasts (HDFs) activated with transforming growth factor­ß1. Additionally, iPSC­CM attenuated the local inflammatory cell response by blocking the adhesion of human acute monocytic leukemia cell monocytes and fibroblasts in vitro. In addition, the contractile ability of HDFs may be reduced by iPSC­CM. These observations suggest that iPSC­CM may protect against processes leading to hypertrophic scarring by attenuating fibroblast activation, blocking inflammatory cell recruitment and adhesion and reducing the contractile ability of fibroblasts.

Lumican Accelerates Wound Healing by Enhancing α2ß1 Integrin-Mediated Fibroblast Contractility.

Lumican is a dermatan sulfate proteoglycan highly expressed in connective tissue and has the ability to regulate collagen fibril assembly. Previous studies have shown that lumican is involved in wound healing, but the precise effects of lumican on reepithelialization and wound contraction, the two pivotal aspects of skin wound healing, have not been investigated. Here we explored the roles of lumican in fibroblast contractility, a main aspect of skin wound healing, by adopting mice skin wound healing model and the corresponding in vitro cellular experiments. Our results showed that lumican can promote skin wound healing by facilitating wound fibroblast activation and contraction but not by promoting keratinocyte proliferation and migration. Silencing of integrin α2 completely abolished the pro-contractility of lumican, indicating lumican enhances fibroblast contractility via integrin α2. Our study for the first time demonstrated that lumican can affect fibroblast's mechanical property, which is pivotal for many important pathological processes, such as wound healing, fibrosis, and tumor development, suggesting that lumican might have a potential to be used to modulate these processes.

Identification and characterization of an anti-fibrotic benzopyran compound isolated from mangrove-derived Streptomyces xiamenensis.

An anti-fibrotic compound produced by Streptomycesn xiamenensis, found in mangrove sediments, was investigated for possible therapeutic effects against fibrosis. The compound, N-[[3,4-dihydro-3S-hydroxy-2S-methyl-2-(4'R-methyl-3'S-pentenyl)-2H-1-benzopyran-6-yl]carbonyl]-threonine (1), was isolated from crude extracts and its structure, including the absolute configuration was determined by extensive spectroscopic data analyses, Mosher's method, Marfey's reagent and quantum mechanical calculations. In terms of biological effects, this compound inhibits the proliferation of human lung fibroblasts (WI26), blocks adhesion of human acute monocytic leukemia cells (THP-1) to a monolayer of WI26 cells, and reduces the contractile capacity of WI26 cells in three-dimensional free-floating collagen gels. Altogether, these data indicate that we have identified a bioactive alkaloid (1) with multiple inhibitory biological effects on lung excessive fibrotic characteristics, that are likely involved in fibrosis, suggesting that this molecule might indeed have therapeutic potential against fibrosis.

[Retrospective analysis of ocular cicatricial pemphigoid].

OBJECTIVE: To analyze the clinical characterization of ocular cicatricial pemphigoid (OCP). METHODS: It was a retrospective series case study. Five consecutive patients referred for the evaluation of possible OCP from January 2005 to October 2008 in Departments of Ophthalmology and Dermatology of Peking University First Hospital. History and clinical characterization of 5 cases (10 eyes) OCP having been misdiagnosed were analyzed to find the causes of misdiagnosis. RESULTS: All of cases were diagnosed as chronic conjunctivitis during the early stages of the diseases, one case was diagnosed as Stevens-Johnson syndrome and one as Sjögren syndrome during the later stage. It was two to five years from the first time to see a doctor to definite diagnosis. All of cases have been prescribed antibiotic eye drops for a long times, one case has been undergone three times trichiasis operation and made the disease progression. Among the five patients with OCP, 3 eyes were diagnosed Stage II, 5 eyes Stage III, 2 eyes Stage IV. Three cases were positive of bacterial culture. Only in 1 case, there was slight increase of iron protein as tumor mark. Inflammation was controlled by the end of the study, but cicatrization of 2 cases still progressed. CONCLUSION: Manifestation of OCP can mimic chronic conjunctivitis during the early stages, it is important to pay high attention to OCP, misdiagnosis may be stopped.

KZn(4)SbO(7) and KZn(4)Sb(3)O(12): syntheses, structures and photophysics of Sb(5+) control materials.

Two novel compounds KZn(4)SbO(7) (1) and KZn(4)Sb(3)O(12) (2) having quasi-white light emission and second-harmonic generation are prepared, and their single-crystal structures are characterized as P6(3)mc and R3 space groups, respectively. The crystal structure transition from high symmetry P6(3)mc of 1 to low symmetry R3 of 2 arises from the large contents of Sb(5+) diluter in wurtzite-type ZnO crystals (ionic ratio of Sb(5+)/Zn(2+) = 1/4 and 3/4 for crystals 1 and 2). The lifetime and quantum efficiency of luminescence are measured, and the density of states and frequency doubling coefficients are calculated for the two compounds. The strong charge transfers from O(2-) to Sb(5+), and blue-shifts of absorption edge and emission bands originate from a large Sb(5+) ion content for compound 2. Further, large charge transfer from O(2-) to Sb(5+) lead to high quantum efficiency and low lifetime, and weak second-harmonic generation intensity for compound 2.

[Study on quantitative model for suspended sediment concentration in Taihu Lake].

The complicated compositions of Case II waters result in the complex properties of spectral curves. The present paper analyzed the in situ measurements data of spectral curves, and further realized the relationships between the properties of spectral curves and suspended sediment concentration. The study found that the max peak of spectral curves was moving to the direction of shortwavelength as increasing suspended sediment concentration, namely the blue shift of wavelength; the area enclosed by spectral curve and coordinate axis in the range of sensitive bands had preferably linear relationship with the suspended sediment concentration (curve area model); the trapezoidal area model which was an approximation of curve area model could also excellently reflect those relationships, and be greatly suitable for multi-spectral satellite imagery retrieval such as LandSat/TM, MODIS and so on. The inversion results of trapezoidal area model for LandSat/TM imagery on October 27, 2003 in Taihu Lake showed that the suspended sediment concentration ranged from 30 to 80 mg x L(-1), the distribution pattern was higher in the west, south and central lake and lower in the east lake; compared with the in situ measurements in the regions, and the relative error of retrieval model was 6.035%.

[Retrospective analysis of clinical characteristics of toxic anterior segment syndrome].

OBJECTIVE: To investigate the etiology, clinical features, treatment and prognosis of toxic anterior segment syndrome (TASS). METHODS: It was a retrospective series case study. The clinical data of eight definite diagnosed TASS cases were retrospectively analyzed. RESULTS: Among eight TASS cases, seven were post cataract surgery cases and one was post cornea penetrating injury. Three cases were caused by residual povidone iodine on instruments, 2 cases resulted from the misuse of distilled water as intraocular irrigating liquid during cataract surgery, 2 cases were produced by the countercurrent of antibiotic solution via the cornea-scleral incision into anterior chamber during subconjunctival injection at the end of the surgery, and 1 case was induced by the injection of the distilled water into the anterior chamber at the end of the surgery. Three TASS cases occurred during operation and 5 cases occurred at 1 day after operation. All eight cases suffered from the painless blurred vision. Three cases occurred during operation presented with decrease of corneal transparency and depigmentation of iris. On the first day after operation, all cases had diffuse corneal stroma edema and severe anterior uveitis. Dexamethasone 0.1% or prednisolone acetate 1% eye drops, three times per day or one time per hour was used in all cases. Carteolol 2% eye drop, two times per day, was used for the cases with ocular hypertension. The cornea was clear in 6 cases, but corneal endothelial decompensation in 2 cases after therapy. CONCLUSION: Various toxic agents injected into anterior chamber by misuse can result in TASS. All these misuse can be avoided. Early diagnosis and proper management may be important to improve the prognosis of TASS.

KMBP2O8 (M = Sr, Ba): a new kind of noncentrosymmetry borophosphate with the three-dimensional diamond-like framework.

Two new isotypic anhydrous borophosphates, KMBP(2)O(8) (M = Sr, Ba), have been prepared under high temperature solution growth (HTSG). Investigation of single-crystal X-ray diffraction shows that they crystallize in noncentrosymmetric space group I42d with the following lattice parameters: a = 7.1095(18), c = 13.882(5)A for KSrBP(2)O(8) and a = 7.202(2), c = 14.300(6)A for KBaBP(2)O(8). The fundamental building block (FBB) of title borophosphates with B:P ratio = 1:2 is 12square:<12square> (symbol square represents BO(4) and PO(4) tetrahedra). The FBBs are further interlocked together to form the final 3D diamond-like architecture. Second-harmonic generation (SHG) on powder samples has been measured using Kurtz and Perry technique, which indicates that they are phase-matchable materials, and their SHG coefficients are measured to be about 1/5 (for KSrBP(2)O(8)) and 1/3 (for KBaBP(2)O(8)) times as large as that of KDP.

[Effect of recombinant Sp1 gene on the collagen expression of hypertrophic scar fibroblasts in vitro].

OBJECTIVE: To explore the feasibility of transfecting recombinant Sp1 into hypertrophic scar fibroblasts and investigate the proliferation and collagen I, III synthesis in the transfected cells. METHODS: Recombinant human Sp1 was transfected into hypertrophic scar fibroblasts with the karyocyte expressive vector. The expression of Sp1, collagen I, III mRNA was tested by real time PCR. The change of cell proliferation was observed with CCK8 colorimeter. RESULTS: About 30% of transfected hypertrophic scar fibroblasts showed green fluorescence positive. The relative expression of Sp1 mRNA in transfected cells, empty-vector cell or untransfected cells group was 5.26 +/- 0.76, 1.08 +/- 0.18, 1.09 +/- 0.15, respectively, showing a significant difference between thansfected and untransfected cells or between the transfected cells and empty-vector group (P <0.01, n = 5). Expression of collagen I, III mRNA was 2.49 +/- 0.40 and 1.88 +/- 0.30 in transfected cells, 0.96 +/- 0.18 and 0.95 +/- 0.18 in empty-vector cell, and 0.97 +/- 0.15 and 0.93 +/- 0.13 in untransfected cells, respectively, showing a significant difference between thansfected and untransfected cells or between the transfected cells and empty-vector group (P < 0.01, n = 5). CONCLUSIONS: The hypertrophic scar fibroblasts could be as the target cells of Sp1 gene transfection. Sp1 gene may play an important role in abnormal collagen metabolism in hypertrophic scar.