OBJECTIVE: To explore the predictors of hematologic responses of non-transfusion-dependent β-thalassemia (NTDT) to thalidomide. METHODS: 33 patients with NTDT who treated with thalidomide in the 923rd Hospital of the Joint Logistics Support Force of the People's Liberation Army from May 2016 to June 2019 were included in the study. The basic data, hematological indexes, degree of treatment response and genetic background of the patients were analyzed. RESULTS: The baseline fetal hemoglobin (HbF) level of main responders (MaR) was significantly higher than that of minor responders (MiR) and no responders (NR) (P=0.001). And the baseline HbF level was positively correlated with hemoglobin increment after treatment (r=0.601). Genetic background analysis showed that the frequencies of the genotype CT of HBG2 rs7482144 (P=0.031), the genotypes CT/CC (P=0.030) and the minor allele C (P=0.015) of HBS1L-MYB rs9399137, the genotypes AT/TT (P=0.030) and the minor allele T (P=0.028) of HBS1L-MYB rs4895440, the genotypes AG/GG (P=0.030) and the minor allele G (P=0.028) of HBS1L-MYB rs4895441 (P=0.030) in MaR group were significantly higher than those in MiR and NR groups. Comparing the area under the ROC curve (AUC) of the above indicators to predict the main response, the results demonstrated that the predictive value of baseline HbF level was significantly better than rs7482144 (0.91 vs 0.72, P=0.003), rs9399137 (0.91 vs 0.74, P=0.022), rs4895440 (0.91 vs 0.74, P=0.023) and rs4895441 (0.91 vs 0.74, P=0.023), but there was no significant difference in the predictive value between combined single nucleotide polymorphisms (SNPs) (0.91 vs 0.88, P=0.658）and baseline HbF combined SNPs (0.91 vs 0.97, P=0.132). The AUC value of baseline HbF predicting the efficacy of thalidomide as the main response was 0.91, the cut-off value was 27.4%, the sensitivity was 100%, and the specificity was 58.3% (P=0.001). CONCLUSION The hematologic response of NTDT to thalidomide is variable and complex. Compared to genetic background, baseline HbF may be a simpler and more efficient tool to predict efficacy response.
Fetal Hemoglobin/genetics* ; Humans ; MicroRNAs ; Polymorphism, Single Nucleotide ; Repressor Proteins/genetics* ; Thalidomide/therapeutic use* ; beta-Thalassemia/genetics*

Sickle cell disease (SCD) is a single gene genetic disease, which seriously threatens the life span and quality of patients. On the basis of the pathogenesis of SCD and the alternative therapy based on fetal hemoglobin F (HbF), the research progress of transcription factors involved in the regulation of HbF gene expression, such as BCL11A, ZBTB7A, KLF-1, c-MYB and SOX6, as well as the application of CRISPR / Cas9, TALEN, zinc finger nuclease and other gene editing technologies in this field has been made, providing a solid theoretical basis for the exploration of new treatment schemes for β- like hemoglobin diseases, such as sickle cell disease and β- thalassemia.
Anemia, Sickle Cell/therapy* ; Cell Line, Tumor ; DNA-Binding Proteins ; Fetal Hemoglobin/genetics* ; Genetic Therapy ; Humans ; Repressor Proteins/genetics* ; Transcription Factors

BACKGROUND/AIMS: For the clinical application of stem cell therapy, functional enhancement is needed to increase the survival rate and the engraftment rate. The purpose of this study was to investigate functional enhancement of the paracrine effect using stem cells and hepatocyte-like cells and to minimize stem cell homing by using a scaffold system in a liver disease model. METHODS: A microporator was used to overexpress Foxa2 in adipose tissue-derived stem cells (ADSCs), which were cultured in a poly(lactic-co-glycolic acid) (PLGA) scaffold. Later, the ADSCs were cultured in hepatic differentiation medium for 2 weeks by a 3-step method. For in vivo experiments, Foxa2-overexpressing ADSCs were loaded in the scaffold, cultured in hepatic differentiation medium and later were implanted in the dorsa of nude mice subjected to acute liver injury (thioacetamide intraperitoneal injection). RESULTS: Foxa2-overexpressing ADSCs showed greater increases in hepatocyte-specific gene markers (alpha fetoprotein [AFP], cytokeratin 18 [CK18], and albumin), cytoplasmic glycogen storage, and cytochrome P450 expression than cells that underwent the conventional differentiation method. In vivo experiments using the nude mouse model showed that 2 weeks after scaffold implantation, the mRNA expression of AFP, CK18, dipeptidyl peptidase 4 (CD26), and connexin 32 (CX32) was higher in the Foxa2-overexpressing ADSCs group than in the ADSCs group. The Foxa2-overexpressing ADSCs scaffold treatment group showed attenuated liver injury without stem cell homing in the thioacetamide-induced acute liver injury model. CONCLUSIONS: Foxa2-overexpressing ADSCs applied in a scaffold system enhanced hepatocyte-like differentiation and attenuated acute liver damage in an acute liver injury model without homing effects.
Animals ; Cytochrome P-450 Enzyme System ; Cytoplasm ; Dipeptidyl Peptidase 4 ; Fetal Proteins ; Glycogen ; Keratin-18 ; Liver Diseases ; Liver Failure, Acute ; Liver ; Mesenchymal Stromal Cells ; Methods ; Mice ; Mice, Nude ; RNA, Messenger ; Stem Cells ; Survival Rate

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a rare autosomal recessive disorder caused by a defect in the immunoglobulin mu binding protein 2 (IGHMBP2) gene, leading to motor neuron degeneration. We identified an infant with SMARD1 by targeted exome sequencing from a consanguineous Syrian family having a history of recurrent infant deaths. The patient initially presented intrauterine growth retardation, poor sucking, failure to thrive, and respiratory failure at the age of two months, and an inborn error of metabolism was suspected at first. Over a period of one month, the infant showed rapid progression of distal muscular weakness with hand and foot contractures, which were suggestive of neuromuscular disease. Using targeted exome sequencing, the mutation in IGHMBP2 was confirmed, although the first report was normal. Targeted exome sequencing enabled identification of the genetic cause of recurrent mysterious deaths in the consanguineous family. Additionally, it is suggested that a detailed phenotypic description and communication between bioinformaticians and clinicians is important to reduce false negative results in exome sequencing.
Carrier Proteins ; Contracture ; Exome ; Failure to Thrive ; Fetal Growth Retardation ; Foot ; Hand ; Humans ; Immunoglobulins ; Infant Death ; Infant ; Metabolism ; Motor Neurons ; Muscle Weakness ; Muscular Atrophy, Spinal ; Neuromuscular Diseases ; Respiratory Insufficiency

Human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) are used in tissue repair and regeneration; however, the mechanisms involved are not well understood. We investigated the hair growth-promoting effects of hUCB-MSCs treatment to determine whether hUCB-MSCs enhance the promotion of hair growth. Furthermore, we attempted to identify the factors responsible for hair growth. The effects of hUCB-MSCs on hair growth were investigated in vivo, and hUCB-MSCs advanced anagen onset and hair follicle neogeneration. We found that hUCB-MSCs co-culture increased the viability and up-regulated hair induction-related proteins of human dermal papilla cells (hDPCs) in vitro. A growth factor antibody array revealed that secretory factors from hUCB-MSCs are related to hair growth. Insulin-like growth factor binding protein-1 (IGFBP-1) and vascular endothelial growth factor (VEGF) were increased in co-culture medium. Finally, we found that IGFBP-1, through the co-localization of an IGF-1 and IGFBP-1, had positive effects on cell viability; VEGF secretion; expression of alkaline phosphatase (ALP), CD133, and β-catenin; and formation of hDPCs 3D spheroids. Taken together, these data suggest that hUCB-MSCs promote hair growth via a paracrine mechanism.
Alkaline Phosphatase ; Alopecia ; Cell Survival ; Coculture Techniques ; Fetal Blood* ; Hair Follicle ; Hair* ; Humans* ; In Vitro Techniques ; Insulin-Like Growth Factor Binding Protein 1 ; Insulin-Like Growth Factor I ; Intercellular Signaling Peptides and Proteins* ; Mesenchymal Stromal Cells ; Regeneration ; Stem Cells* ; Umbilical Cord* ; Vascular Endothelial Growth Factor A

Background: Valproic acid (VPA) exposure during pregnancy has been proven to contribute to congenital heart disease (CHD). Our previous findings implied that disruption of planar cell polarity (PCP) signaling pathway in cardiomyocytes might be a factor for the cardiac teratogenesis of VPA. In addition, the teratogenic ability of VPA is positively correlated to its histone deacetylase (HDAC) inhibition activity. This study aimed to investigate the effect of the VPA on cardiac morphogenesis, HDAC1/2/3, and PCP key genes (Vangl2/Scrib/Rac1), subsequently screening out the specific HDACs regulating PCP pathway. Methods: VPA was administered to pregnant C57BL mice at 700 mg/kg intraperitoneally on embryonic day 10.5. Dams were sacrificed on E15.5, and death/absorption rates of embryos were evaluated. Embryonic hearts were observed by hematoxylin-eosin staining to identify cardiac abnormalities. H9C2 cells (undifferentiated rat cardiomyoblasts) were transfected with Hdac1/2/3 specific small interfering RNA (siRNA). Based on the results of siRNA transfection, cells were transfected with Hdac3 expression plasmid and subsequently mock-treated or treated with 8.0 mmol/L VPA. Hdac1/2/3 as well as Vangl2/Scrib/Rac1 mRNA and protein levels were determined by real-time quantitative polymerase chain reaction and Western blotting, respectively. Total HDAC activity was detected by colorimetric assay. Results: VPA could induce CHD (P < 0.001) and inhibit mRNA or protein expression of Hdac1/2/3 as well as Vangl2/Scrib in fetal hearts, in association with total Hdac activity repression (all P < 0.05). In vitro, Hdac3 inhibition could significantly decrease Vangl2/Scrib expression (P < 0.01), while knockdown of Hdac1/2 had no influence (P > 0.05); VPA exposure dramatically decreased the expression of Vanlg2/Scrib together with Hdac activity (P < 0.01), while overexpression of Hdac3 could rescue the VPA-induced inhibition (P > 0.05). Conclusion VPA could inhibit Hdac1/2/3, Vangl2/Scrib, or total Hdac activity both in vitro and in vivo and Hdac3 might participate in the process of VPA-induced cardiac developmental anomalies.
Animals ; Cell Polarity ; Enzyme Inhibitors ; adverse effects ; Female ; Fetal Heart ; embryology ; Heart Defects, Congenital ; chemically induced ; physiopathology ; Histone Deacetylase Inhibitors ; Histone Deacetylases ; drug effects ; physiology ; Mice ; Mice, Inbred C57BL ; Nerve Tissue Proteins ; Pregnancy ; Rats ; Transfection ; Valproic Acid ; adverse effects

OBJECTIVE: To investigate the effect of Piwil2-induced cancer stem-like cell (Piwil2-iCSC)-derived exosomes on the proliferation,migration and invasion of human umbilical cord blood-derived mesenchymal stem cells (hucMSCs). METHODS: Piwil2-iCSC-derived exosomes were isolated by ultracentrifugation and identified using transmission electron microscopy,nanoparticle tracking analysis and Western blotting.Exosome uptake assay was used to identify the pathway that Piwil2-iCSCderived exosomes utilized.HucMSCs were divided into control group,PBS intervention group and exosome intervention group,and CCK-8 assay,wound healing assay,Transwell assay,Western blotting and cell karyotype analysis were used to observe the proliferation,migration,invasion,expression levels of MMP2 and MMP9 proteins,and chromosome structure of hucMSCs. RESULTS: The diameter of Piwil2-iCSC-derived exosomes ranged from 50 nm to 100 nm,and most of them were oval or spherical capsules rich in CD9,CD63 and Piwil2 proteins.Exosomal uptake assay showed that the exosomes executed theirs functions after entering the cells.Compared with the control cells and PBS-treated cells,hucMSCs treated with the exosomes showed significantly increased number of proliferating cells (<0.05) with accelerated healing rate (<0.05 at 24 h;<0.01 at 48 h),increased invasive cells (<0.01),enhanced protein expressions of MMP2(<0.05 PBS group;<0.01 control group) and MMP9(<0.05),but their karyotype still remained 46XY without any abnormalities. CONCLUSIONS Piwil2-iCSC-derived exosomes can promote the proliferation,migration and invasion but does not cause cancer-like heterogeneity changes in hucMSCs.
Argonaute Proteins ; Cell Movement ; physiology ; Cell Proliferation ; physiology ; Exosomes ; physiology ; Fetal Blood ; cytology ; Humans ; Karyotyping ; Mesenchymal Stem Cells ; pathology ; Neoplasm Invasiveness ; Neoplastic Stem Cells ; Umbilical Cord ; Wound Healing

OBJECTIVETo investigate the effect of histone acetylation/deacetylation imbalances on embryonic hearts of mice and its effect on key genes of planar cell polarity (PCP) pathway-Vangl2, Scrib and Rac1 in H9C2 cells.

METHODSForty pregnant C57/B6 mice were randomly assigned into three groups: blank group (n=10), vehicle group (n=10), and valproic acid (VPA)-treated group (n=20). In the VPA-treated group, VPA, a histone deacetylase (HDAC) inhibitor, was administered to each individual dam intraperitoneally at a single dose of 700 mg/kg on embryonic day 10.5 (E10.5). The vehicle and blank groups received equivalent saline or no interventions, respectively. Dams were sacrificed on E15.5, and death rates of embryos were evaluated. Subsequently, embryonic hearts of survival fetus were removed to observe cardiac abnormalities by hematoxylin-eosin (HE) staining. H9C2 cells were cultured and allotted to the blank, vehicle, and VPA-treated groups: the VPA treated group received VPA exposure at concentrations of 2.0, 4.0 and 8.0 mmol/L; the vehicle and blank groups received equivalent saline or no interventions, respectively. HDAC1-3 as well as Vangl2, Scrib and Rac1 mRNA and protein expression levels were determined by quantitative real-time PCR and Western blot, respectively. The total HDAC activity was analyzed by colorimetric assay.

RESULTSThe fetus mortality rate after VPA treatment was 31.7%, with a significantly higher rate of cardiac abnormalities in comparison with the controls (P<0.05). In comparison with the blank and vehicle groups, HDAC1 mRNA was significantly increased at various concentrations of VPA treatment at all time points of exposure (P<0.05), together with a reduction of protein level after 48 and 72 hours of exposure (P<0.05). The inhibition of HDAC2 mRNA after various concentrations of VPA incubation was pronounced at 24 hours of exposure (P<0.05), while the protein levels were reduced at all time points (P<0.05). HDAC3 mRNA was prominently induced by VPA (4.0 and 8.0 mmol/L) at all time points of treatment (P<0.05). In contrast, the protein level was inhibited after VPA treatment (P<0.05). In comparison with the blank and vehicle groups, Vangl2 mRNA as well as Scrib mRNA/protein expression levels were markedly reduced after 48 and 72 hours of VPA treatment (P<0.05), together with a reduction of protein level in Vangl2 at 72 hours (P<0.05). Compared with the blank and vehicle groups, a significant repression in the total HDAC activity was observed in the VPA-treated group at concentrations of 4.0 and 8.0 mmol/L after 24 hours of treatment (P<0.05), and the effect persisted up to 48 and 72 hours, exhibiting pronounced inhibition at all concentrations (P<0.05).

CONCLUSIONSVPA might result in acetylation/deacetylation imbalances by inhibiting HDAC1-3 protein expression and total HDAC activity, leading to the down-regulation of mRNA and protein expression of Vangl2 and Scrib. This could be one of the mechanisms contributing to congenital heart disease.

Acetylation ; Animals ; Cell Polarity ; Cells, Cultured ; Fetal Heart ; drug effects ; metabolism ; Heart Defects, Congenital ; etiology ; Histone Deacetylase 1 ; genetics ; Histone Deacetylase 2 ; genetics ; Histones ; metabolism ; Mice ; Mice, Inbred C57BL ; Nerve Tissue Proteins ; genetics ; RNA, Messenger ; analysis ; Valproic Acid ; pharmacology

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) with portal vein tumor thrombosis (PVTT) exhibits poor prognosis. The aim of this study is to evaluate factors associated with survival of HCC patients with PVTT to suggest better therapeutic options. METHODS: Patients with HCC which were newly diagnosed at three tertiary hospitals between January 2004 and December 2012, were reviewed retrospectively. Among them, Barcelona Clinic of Liver Cancer stage C patients with PVTT were identified. Factors affecting overall survival (OS) were analyzed and efficacies of the treatment modalities were compared. RESULTS: Four hundred sixty five patients with HCC and PVTT were included. Liver function, tumor burden, presence of extrahepatic tumor, alfa fetoprotein, and treatment modalities were significant factors associated with OS. Treatment outcomes were different according to the initial modalities. OS of the patients who received hepatic resection, radiofrequency ablation (RFA), transarterial chemoembolization (TACE), hepatic arterial infusion chemotherapy (HAIC), sorafenib, systemic cytotoxic chemotherapy, radiation therapy (without combination), and supportive care were 27.8, 7.1, 6.7, 5.3, 2.5, 3.0, 1.8, and 0.9 months, respectively (P<0.001). Curative-intent treatments such as hepatic resection or RFA were superior to noncurativeintent treatments (P<0.001). TACE or HAIC was superior to sorafenib or systemic chemotherapy (P<0.001). Combining radiotherapy to TACE or HAIC did not provide additional benefit on OS (P=0.096). CONCLUSIONS: Treatment modalities as well as baseline factors significantly influenced on OS of HCC patients with PVTT. Whenever possible, curative intent treatments should be preferentially considered. If unable, locoregional therapy would be a better choice than systemic therapy in HCC patients with PVTT.
Carcinoma, Hepatocellular* ; Catheter Ablation ; Drug Therapy ; Fetal Proteins ; Humans ; Liver ; Liver Neoplasms ; Portal Vein* ; Prognosis* ; Radiotherapy ; Retrospective Studies ; Tertiary Care Centers ; Thrombosis* ; Tumor Burden

OBJECTIVETo delineate the phenotypic characteristics of 22q11.2 deletion syndrome and the role of CRKL gene in the pathogenesis of cardiac abnormalities.

METHODSG-banded karyotyping, single nucleotide polymorphism (SNP) array and fluorescence in situ hybridization (FISH) were performed on a fetus with tetralogy of Fallot detected by ultrasound. Correlation between the genotype and phenotype was explored after precise mapping of the breakpoints on chromosome 22q11.2. SNP array was also performed on peripheral blood samples from both parents to clarify its origin.

RESULTSThe fetus showed a normal karyotype of 46,XY. SNP array performed on fetal blood sample revealed a 749 kb deletion (chr22: 20 716 876-21 465 659) at 22q11.21, which encompassed the CRKL gene but not TBX1, HIRA, COMT and MAPK1. Precise mapping of the breakpoints suggested that the deleted region has overlapped with that of central 22q11.2 deletion syndrome. SNP array analysis of the parental blood samples suggested that the 22q11.21 deletion has a de novo origin. The presence of 22q11.21 deletion in the fetus was also confirmed by FISH analysis.

CONCLUSIONCentral 22q11.21 deletion probably accounts for the cardiac abnormalities in the fetus, for which the CRKL gene should be considered as an important candidate.

Adaptor Proteins, Signal Transducing ; genetics ; Adult ; Chromosome Deletion ; Chromosomes, Human, Pair 22 ; genetics ; DiGeorge Syndrome ; diagnosis ; embryology ; genetics ; Female ; Fetal Diseases ; diagnosis ; genetics ; Genotype ; Humans ; In Situ Hybridization, Fluorescence ; Male ; Nuclear Proteins ; genetics ; Phenotype ; Pregnancy ; Prenatal Diagnosis