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.

Efficacy of Enhanced Cytokine-Induced Killer Cells as an Adjuvant Immunotherapy for Renal Cell Carcinoma: Preclinical and Clinical Studies.

Cytokine-induced killer (CIK) cells have been proved to be an effective method of tumor immunotherapy in numerous preclinical and clinical studies. In our previous study, a new method was developed to prime and propagate CIK cells by the combination of IL-2 and IL-15, and this kind of CIK cells had enhanced antitumor effect on lung cancer. For renal cell carcinoma (RCC), immunotherapy plays an important role because of the poor efficacy of radiotherapy and chemotherapy. In this study, we further evaluated the antitumor effects of these enhanced CIK cells against RCC. Enhanced CIK cells were generated by IL-2 combined with IL-15 and identified by flow cytometry. HEK-293 and ACHN cell lines were used to verify the efficiency of CIK cells in vitro, and then the ACHN tumor xenograft model was also employed for in vivo study. In addition, the secreted cytokines including IFN-γ, granzyme B, TNF-α, and perforin, as well as the local microstructure were also studied. Subsequently, 20 patients with RCC were enrolled into our study, and 11 patients were randomly divided into the autologous CIK treatment group for clinical research. The results showed that enhanced CIK cells exert better antitumor effects in RCC in vitro (p < 0.01 in HEK-293 and p < 0.05 in ACHN）and in vivo (p < 0.05). Patients benefit overall survival from enhanced CIK therapy in our clinical study. Our present preclinical and clinical studies for the first time elucidated that these enhanced CIK cells would be used as an effective adjuvant therapy in the treatment of RCC.

Umbilical Cord Mesenchymal Stem Cell-Derived Exosomes Ameliorate HaCaT Cell Photo-Aging.

Umbilical cord mesenchymal stem cells (UCMSCs) have been identified as a potentially ideal cell type for use in regenerative therapeutic contexts owing to their excellent paracrine secretory abilities and other desirable properties. Previous work has shown that stem cell-derived exosomes can effectively reduce skin aging, but few studies have specifically focused on the role of UCMSC-derived exosomes in this context. In this study, we isolated exosomes derived from UCMSCs grown in a three-dimensional culture system and explored their ability to modulate the photo-aging of HaCaT keratinocytes. Cell viability and proliferation were assessed using CCK8 assay, whereas wound healing and transwell assays were used to assess cell migratory capabilities. UVB irradiation (60 mJ/cm2) was used to induce photo-aging of HaCaT cells. TUNEL and SA-ß-Gal staining were used to explore HaCaT cell apoptosis and senescence, respectively, whereas real-time quantitative PCR was used to assess the expression of relevant genes at the mRNA level. We found that UCMSC-derived exosomes were able to enhance normal HaCaT cell proliferation and migration while also inhibiting UVB-induced damage to these cells. These exosomes also reduced HaCaT cell apoptosis and senescence, increasing collagen type I expression and reducing matrix metalloproteinase (MMP1) expression in photo-aged HaCaT cells. Together, these findings indicate that UCMSC-derived exosomes have the potential to be used therapeutically to suppress skin aging.

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.

Prevalence of Congenital Heart Disease Amongst Schoolchildren in Southwest China.

OBJECTIVE: To investigate the prevalence and risk factors of congenital heart disease in Yunnan, China which has diverse ethnic groups. METHODS: This cross-sectional study enrolled 244,023 children from 2010 to 2015. To diagnose CHD, a conventional physical examination was used to screen suspicious cases, which were further confirmed by echocardiography. RESULTS: A total of 1695 children were diagnosed with CHD. The estimated prevalence was 6.94%. Atrial septal defects were the most common cardiac abnormalities. A higher prevalence of CHD was observed with preterm birth, low birth weight, maternal age ≥35 years, and high-altitude regions. The prevalence also showed differences between diverse ethnic groups. CONCLUSIONS: The prevalence of CHD in China may have ethnic differences.

Generation of human iPSC line from a patient with Tetralogy of Fallot, YAHKMUi001-A, carrying a mutation in TBX1 gene.

The human induced pluripotent stem cell (iPSC) line YAHKMUi001-A was derived from the dermal fibroblasts of a patient with Tetralogy of Fallot (TOF), with a mutation in the TBX1 gene (c.928G > A). The skin fibroblasts were obtained from a 4-year-old boy, and were infected with Sendai virus expressing the Yamanaka factors. The YAHKMUi001-A iPSC line expresses pluripotent stem cell markers, displays a normal karyotype, and has the capacity to differentiate into 3 germ layers. This cell line model can be a good tool to study the pathological mechanism of the TBX1 gene mutations associated with TOF.

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.

Novel Genetic Variants of Sporadic Atrial Septal Defect (ASD) in a Chinese Population Identified by Whole-Exome Sequencing (WES).

BACKGROUND Recently, mutations in several genes have been described to be associated with sporadic ASD, but some genetic variants remain to be identified. The aim of this study was to use whole-exome sequencing (WES) combined with bioinformatics analysis to identify novel genetic variants in cases of sporadic congenital ASD, followed by validation by Sanger sequencing. MATERIAL AND METHODS Five Han patients with secundum ASD were recruited, and their tissue samples were analyzed by WES, followed by verification by Sanger sequencing of tissue and blood samples. Further evaluation using blood samples included 452 additional patients with sporadic secundum ASD (212 male and 240 female patients) and 519 healthy subjects (252 male and 267 female subjects) for further verification by a multiplexed MassARRAY system. Bioinformatic analyses were performed to identify novel genetic variants associated with sporadic ASD. RESULTS From five patients with sporadic ASD, a total of 181,762 genomic variants in 33 exon loci, validated by Sanger sequencing, were selected and underwent MassARRAY analysis in 452 patients with ASD and 519 healthy subjects. Three loci with high mutation frequencies, the 138665410 FOXL2 gene variant, the 23862952 MYH6 gene variant, and the 71098693 HYDIN gene variant were found to be significantly associated with sporadic ASD (P<0.05); variants in FOXL2 and MYH6 were found in patients with isolated, sporadic ASD (P<5×10^-4). CONCLUSIONS This was the first study that demonstrated variants in FOXL2 and HYDIN associated with sporadic ASD, and supported the use of WES and bioinformatics analysis to identify disease-associated mutations.

Transplantation of umbilical cord and bone marrow-derived mesenchymal stem cells in a patient with relapsing-remitting multiple sclerosis.

There is currently great interest in the use of mesenchymal stem cells as a therapy for multiple sclerosis with potential to both ameliorate inflammatory processes as well as improve regeneration and repair. Although most clinical studies have used autologous bone marrow-derived mesenchymal stem cells, other sources such as allogeneic umbilical cord-derived cells may provide a more accessible and practical supply of cells for transplantation. In this case report we present the treatment of aggressive multiple sclerosis with multiple allogenic human umbilical cord-derived mesenchymal stem cell and autologous bone marrow-derived mesenchymal stem cells over a 4 y period. The treatments were tolerated well with no significant adverse events. Clinical and radiological disease appeared to be suppressed following the treatments and support the expansion of mesenchymal stem cell transplantation into clinical trials as a potential novel therapy for patients with aggressive multiple sclerosis.

[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.

Stemness gene expression profile analysis in human umbilical cord mesenchymal stem cells.

Umbilical cord mesenchymal stem cells (UC-MSCs) have several advantages for clinical therapy: the material is easily obtainable, the donation procedure is painless and there is low risk of viral contamination. UC-MSCs play important roles in tissue regeneration, tissue damage repair, autoimmune disease and graft-versus-host disease. In this study, we investigated the normal mRNA expression profile of UC-MSCs, and analyzed the candidate proteins responsible for the signaling pathway that may affect the differentiation characteristics of UC-MSCs. UC-MSCs were isolated by mincing UC samples into fragments and placing them in growth medium in a six-well plate. The immunophenotype characteristics and multilineage differentiation potential of the UC-MSCs were measured by flow cytometry and immunohistochemical assays. In addition, the pathway-focused gene expression profile of UC-MSCs was compared with those of normal or tumorous cells by realtime quantitative polymerase chain reaction. We successfully isolated and cultured UC-MSCs and analyzed the appropriate surface markers and their capacity for osteogenic, adipogenic and neural differentiation. In total, 168 genes focusing on signal pathways were examined. We found that the expression levels of some genes were much higher or lower than those of control cells, either normal or tumorous. UC-MSCs exhibit a unique mRNA expression profile of pathway-focused genes, especially some stemness genes, which warrants further investigation.

[Analysis on molecular characteristic of VP7 and NSP4].

OBJECTIVE: To explore the molecular characteristics and molecular variation of human rotavirus (HRV) strains and to understand the relationship between clinical characteristics and epidemiology of different HRV-VP7 and NSP4. METHODS: Double-strand RNA of rotavirus extracted from stool samples was used as the template for reverse transcription of gene VP7, which was followed by nested PCR for VP7 typing. NSP4 genes from 22 epidemic strains of human rotavirus isolated in Kunming in 2002 and 2003 were amplified with RT-PCR. Then cDNAs were sequenced and compared with 4 human rotavirus NSP4 (Wa, KUN, AU-1, Hochi)) and 3 animal rotavirus NSP4 (EW, OSU, SA11) available in the GenBank while the epidemic strains of human rotavirus isolated in different areas of China were compared, using the Clustal-mp, DNAssist, MEGA2 software. The G serotype of VP7 was analysed by PCR. RESULTS: Serotype G1 was prevalent in 2002 while serotype G3 was the prevalent in Kumming in 2003. The NSP4 genes from 22 epidemic strains of human rotavirus isolated in Kunming in 2002 and 2003 belonged to Wa with highly conservative amino acid. Samples isolated in the same years but not in the same area shared higher homology. Symptoms associated with heavy diarrhea did not seem to be associated with NSP4 molecular variation (P > 0.05). CONCLUSION: Obvious variations of VP7 typing were seen in the same season, as well as in different areas and years. Due to the stable nature of NSP4, it seem to be a better candidate for vaccine production, than VP7.