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1.  PITX2C loss-of-function mutations responsible for idiopathic atrial fibrillation 
Clinics  2014;69(1):15-22.
OBJECTIVE:
This study aimed to identify novel PITX2c mutations responsible for idiopathic atrial fibrillation.
METHODS:
A cohort of 210 unrelated patients with idiopathic atrial fibrillation and 200 unrelated, ethnically matched healthy individuals used as controls were recruited. The whole coding exons and splice junctions of the PITX2c gene, which encodes a paired-like homeobox transcription factor required for normal cardiovascular morphogenesis, were sequenced in 210 patients and 200 control subjects. The causative potentials of the identified mutations were automatically predicted by MutationTaster and PolyPhen-2. The functional characteristics of the PITX2c mutations were explored using a dual-luciferase reporter assay system.
RESULTS:
Two novel heterozygous PITX2c mutations (p.Q105L and p.R122C) were identified in 2 of the 210 unrelated patients with idiopathic atrial fibrillation. These missense mutations were absent in the 400 control chromosomes and were both predicted to be pathogenic. Multiple alignments of PITX2c protein sequences across various species showed that the altered amino acids were highly evolutionarily conserved. A functional analysis demonstrated that the mutant PITX2c proteins were both associated with significantly reduced transcriptional activity compared with their wild-type counterparts.
CONCLUSION:
The findings of this study associate PITX2c loss-of-function mutations with atrial fibrillation, supporting the hypothesis that dysfunctional PITX2c confers enhanced susceptibility to atrial fibrillation and suggesting potential implications for early prophylaxis and allele-specific therapy for this common arrhythmia.
doi:10.6061/clinics/2014(01)03
PMCID: PMC3870307  PMID: 24473555
Atrial Fibrillation; Transcriptional Factor; PITX2c; Genetics; Reporter Gene
2.  Mutation spectrum of GATA4 associated with congenital atrial septal defects 
Introduction
Congenital atrial septal defect (ASD) is the second commonest form of cardiac developmental anomaly, responsible for substantial morbidity and mortality in affected individuals. Previous studies have implicated genetic defects in the pathogenesis of ASD. However, ASD is largely a genetically heterogeneous disease and the genetic determinants for ASD in the majority of patients remain to be identified.
Material and methods
The entire coding region of GATA4, a gene encoding a zinc-finger transcription factor essential for normal cardiac morphogenesis, was sequenced in 220 unrelated patients with ASD. The available relatives of the patients harboring the identified mutations and 200 unrelated ethnicity-matched control individuals were genotyped.
Results
Four heterozygous missense GATA4 mutations, p.P36S, p.H190R, p.S262A, and p.V399G, were identified in four unrelated patients with ASD, respectively. These mutations were neither detected in 200 control individuals nor described in the human SNP database. Alignment of multiple GATA4 protein sequences across species indicated that the affected amino acids were highly conserved evolutionarily. Genetic analysis of the available relatives of the mutation carriers showed that in each family the mutation co-segregated with ASD.
Conclusions
The findings expand the spectrum of mutations in GATA4 linked to ASD and provide new insight into the molecular etiology associated with ASD, suggesting the potential implications for the genetic diagnosis and gene-specific therapy for this prevalent cardiovascular abnormality in humans.
doi:10.5114/aoms.2013.39788
PMCID: PMC3902718  PMID: 24482639
atrial septal defect; transcription factor; genetics
3.  p38 MAPK in myeloma cells regulates osteoclast and osteoblast activity and induces bone destruction 
Cancer research  2012;72(24):6393-6402.
p38 MAPK which is constitutively activated in human myeloma has been implicated in bone destruction by this cancer, but the processes it recruits are obscure. In this study, we demonstrate that p38 activity in myeloma inhibits osteoblast differentiation and bone formation but also enhances osteoclast maturation and bone resorption. p38 regulated the expression and secretion of the Wnt pathway antagonist DKK-1 and the monocyte chemoattractant MCP-1. Attenuating p38, DKK-1 or MCP-1 were each sufficient to reduce bone lesions in vivo. Although it is well known that DKK-1 inhibits osteoblast differentiation, we found that together with MCP-1 it could also promote osteoclast differentiation and bone resorption. The latter effects were mediated by enhancing expression of RANK in osteoclast progenitor cells and by upregulating secretion of its ligand RANKL from stromal cells and mature osteoblasts. In summary, our study defined the mechanisms by which p38 signaling in myeloma cells regulates osteoblastogenesis, osteoclastogenesis, and bone destruction. Our findings, which may have implications for bone invasion by other cancers where p38 is elevated, strongly suggests that targeting p38 for inhibition might offer an effective therapeutic approach to treat osteolytic bone lesions in myeloma patients.
doi:10.1158/0008-5472.CAN-12-2664
PMCID: PMC3525770  PMID: 23066034
4.  Bone Marrow Stromal Cells Derived MCP-1 Reverses the Inhibitory Effects of Multiple Myeloma Cells on Osteoclastogenesis by Upregulating the RANK Expression 
PLoS ONE  2013;8(12):e82453.
Multiple myeloma (MM) cells are responsible for aberrant osteoclast (OC) activation. However, when cocultured monocytes, but not OC precursors, with MM cells, we made a novel observation that MM cells inhibited receptor activator of nuclear factor κB ligand (RANKL)-induced increase of OC differentiation, OC gene expression, signaling pathways and bone resorption activity. Our results showed that MM cells produced multiple inhibitory cytokines of osteoclastogenesis, such as IL-10, which activated STAT3 signaling and induce OC inhibition. However, cocultures of bone marrow stromal cells (BMSCs) reversed MM-induced OC inhibition. We found that MM cells increased production of MCP-1 from BMSCs and BMSC-derived MCP-1 enhanced OC formation. Mechanistic studies showed that IL-10 downregulated RANK expression in monocytes and thus, inhibited RANKL-induced OC formation. In contrast, MCP-1 upregulated RANK expression and thus, enhanced OC formation. Overall, our studies for the first time demonstrated that MM cell have inhibitory effects on osteoclastogenesis by producing inhibitory cytokines. Our results further indicate that activation of osteoclastogenesis in bone marrow requests the crosstalk of MM cells, BMSCs and their produced cytokines. Thus, our studies provide evidences that targeting bone marrow microenvironmental cells and/or cytokines may be a new approach to treating MM bone destruction.
doi:10.1371/journal.pone.0082453
PMCID: PMC3858321  PMID: 24340030
5.  Gallic Acid Induces the Apoptosis of Human Osteosarcoma Cells In Vitro and In Vivo via the Regulation of Mitogen-Activated Protein Kinase Pathways 
Abstract
To examine the antitumor effects of gallic acid (GA) on osteosarcoma, two human osteosarcoma cell lines U-2OS and MNNG/HOS were treated by GA and subjected to cell proliferation and apoptosis assays. In addition, MNNG/HOS xenograft tumors were established in nude BALB/c mice to evaluate the anticancer capacity of GA in vivo. The results showed that GA inhibited the proliferation and induced the apoptosis of osteosarcoma cells, accompanied by the upregulation of p-38 activation and the downregulation of c-Jun N-terminal kinase (JNK) and extracellular signal regulated kinase (ERK1/2) activation. Additionally, p38 MAPK inhibitor abrogated GA-induced growth inhibition of osteosarcoma cells, whereas JNK or ERK1/2 inhibitors sensitized osteosarcoma cells to GA-induced growth inhibition. In vivo studies further showed that GA administration decreased xenograft tumor growth in a dose-dependent manner. Immunohistochemistry analysis demonstrated the downregulation of PCNA and CD31 expression and upregulation of apoptosis in MNNG/HOS tumor tissues following GA treatment. This study demonstrates the antitumor efficacy of GA for osteosarcoma that is mediated by the modulation of cell proliferation, apoptosis, and angiogenesis. Our findings suggest that GA could be a potent agent for osteosarcoma intervention.
doi:10.1089/cbr.2012.1245
PMCID: PMC3516425  PMID: 22849560
Apoptosis; Caspase; Gallic acid; MAPK kinases; Osteosarcoma
6.  Novel GATA6 Mutations Associated with Congenital Ventricular Septal Defect or Tetralogy of Fallot 
DNA and Cell Biology  2012;31(11):1610-1617.
Congenital heart disease (CHD) is the most common form of developmental malformation and is the leading noninfectious cause of infant mortality. Emerging evidence indicates that genetic defects are involved in the pathogenesis of CHD. Nevertheless, CHD is genetically heterogeneous, and the molecular basis for CHD in a majority of patients remains unknown. In this study, the whole coding region of GATA6, a gene encoding a zinc-finger transcription factor crucial for normal cardiogenesis, was sequenced in 380 unrelated patients with CHD. The relatives of the index patients harboring the identified mutations and 200 unrelated control individuals were subsequently genotyped. The functional effect of the mutations was characterized using a luciferase reporter assay system. As a result, two novel heterozygous GATA6 mutations, p.D404Y and p.E460X, were identified in two families with ventricular septal defect and tetralogy of Fallot, respectively. The mutations co-segregated with CHD in the families with complete penetrance, and were absent in 400 control chromosomes. Functional analysis demonstrated that the mutated GATA6 proteins were associated with significantly decreased transactivational activity in comparison with their wild-type counterpart. These findings provide novel insight into the molecular mechanism implicated in CHD, suggesting potential implications for the early prophylaxis and personalized treatment of CHD.
The transcription factor GATA6 is a zinc finger DNA-binding protein that has been shown to be essential for cardiac development. In this article, two novel heterologous polymorphisms were associated with two cardiac malformations in families.
doi:10.1089/dna.2012.1814
PMCID: PMC3482375  PMID: 23020118
7.  Preparation and characterization of tetrandrine-phospholipid complex loaded lipid nanocapsules as potential oral carriers 
Background
Tetrandrine is an active constituent that is extracted from the root tuber of the Chinese herb Stephania tetrandra S. Moore. It has shown various pharmacological effects, such as antitumor activity, multidrug resistance reversal, and hepatic fibrosis resistance. In clinical applications, it has been used to treat hypertension, pneumosilicosis, and lung cancer. However, the poor water solubility of tetrandrine has limited its application. In this study, a newly emerging oral drug carrier of phospholipid complex loaded lipid nanocapsules was developed to improve the oral bioavailability of tetrandrine.
Methods
The phospholipid complex was prepared with the solvent-evaporation method to enhance the liposolubility of tetrandrine. The formation of the phospholipid complex was confirmed with a solubility study, infrared spectroscopy, and a differential scanning calorimetry (DSC) analysis. The tetrandrine-phospholipid complex loaded lipid nanocapsules (TPC-LNCs) were prepared using the phase inversion method. Lyophilization was performed with mannitol (10%) as a cryoprotectant. TPC-LNCs were characterized according to their particle size, zeta potential, encapsulation efficiency, morphology by transmission electron microscopy, and crystallinity by DSC. In addition, the in vitro release of tetrandrine from TPC-LNCs was examined to potentially illustrate the in vivo release behavior. The in vivo bioavailability of TPC-LNCs was studied and compared to tetrandrine tablets in rats.
Results
The liposolubility of tetrandrine in n-octanol improved from 8.34 μg/mL to 35.64 μg/mL in the tetrandrine-phospholipid complex. The prepared TPC-LNCs were spherical-shaped particles with a small size of 40 nm and a high encapsulation efficiency of 93.9%. DSC measurements revealed that the crystalline state was less ordered in lipid nanocapsules. The in vitro release study demonstrated a fast release of approximately 25% in the first 1 hour, which was followed by a sustained release of 70% over 12 hours. The relative bioavailability of TPC-LNCs compared to that of tablets was 208%, indicating a significant improvement in the oral absorption of tetrandrine.
Conclusion
The TPC-LNCs system developed in this study is a promising carrier that improves the oral bioavailability of tetrandrine in rats. The phospholipid complex loaded lipid nanocapsules have great potential for use as an oral drug delivery system for moderately lipophilic drugs that are encapsulated in the lipid nanocapsules.
doi:10.2147/IJN.S50557
PMCID: PMC3817030  PMID: 24204145
tetrandrine; phospholipid complex; lipid nanocapsules; oral bioavailability; enhanced liposolubility
8.  CpG or IFN-α are more potent adjuvants than GM-CSF to promote anti-tumor immunity following idiotype vaccine in multiple myeloma 
Cancer immunology, immunotherapy : CII  2011;61(4):10.1007/s00262-011-1123-2.
Idiotype (Id) protein in combination with GM-CSF has been used as vaccines for immunotherapy of patients with myeloma and B-cell tumors and the results have been disappointing. To search for better immune adjuvants to improve the efficacy of Id-based immunotherapy in myeloma, we evaluated and compared the efficacy of vaccination of Id protein in combination with CpG or IFN-α, or GM-CSF as a control, in the 5TGM1 myeloma mouse model. Our results showed that Id vaccine combined with CpG or IFN-α, but not GM-CSF, not only efficiently protected mice from developing myeloma but also eradicated established myeloma. The therapeutic responses were associated with an induction of strong humoral immune responses including anti-Id antibodies, and cellular immune responses including Id- and myeloma-specific CD8+ cytotoxic T lymphocytes (CTLs), CD4+ type-1 T-helper (Th1) cells and memory T cells in mice receiving Id vaccine combined with CpG or IFN-α. Furthermore, Id vaccine combined with CpG or IFN-α induced Id- and tumor-specific memory immune responses that protected surviving mice from tumor rechallenge. Thus, our study clearly shows that CpG or IFN-α are better immune adjuvants than GM-CSF. This information will be important for improving the strategies of Id-based immunotherapy for patients with myeloma and other B-cell tumors.
doi:10.1007/s00262-011-1123-2
PMCID: PMC3810206  PMID: 22002243
Multiple myeloma; Idiotype; Adjuvants; Vaccination; Immunotherapy
9.  Association of angiotensin-converting enzyme gene I/D and CYP11B2 gene −344T/C polymorphisms with lone atrial fibrillation and its recurrence after catheter ablation 
The renin-angiotensin-aldosterone system (RAAS) plays a key role in atrial structural and electrical remodeling. The aim of this study was to investigate the potential associations of angiotensin-converting enzyme (ACE) gene insertion/ deletion (I/D) and aldosterone synthase (CYP11B2) gene −344T/C polymorphisms with the risk and recurrence of lone atrial fibrillation (AF). One hundred and ninety-three patients who underwent successful catheter ablation for lone AF were recruited. Two hundred and ninety-seven sinus rhythm subjects without a history of arrhythmia served as controls. The subjects were genotyped for ACE gene I/D and CYP11B2 gene −344T/C polymorphisms. Results showed that the ACE gene DD genotype and D allele were associated with a greater prevalence of lone AF (both P<0.01). In addition, the ACE gene DD genotype had a significantly larger left atrial dimension (LAD; 41.6±5.7 mm vs. 39.6±5.2 mm; P=0.043) and higher risk of AF recurrence [44.7% vs. 23.2%; odds ratio (OR), 2.68; 95% confidence interval (CI), 1.28–5.61; P=0.008] compared with the II+ID genotype in lone AF patients. After adjustment for a variety of risk factors, the ACE gene DD genotype had a 1.97-fold increased risk for lone AF (OR, 1.97; 95% CI, 1.15–3.37; P= 0.013) and 2.35-fold increased risk for AF recurrence (RR, 2.35; 95% CI, 1.10–5.04; P=0.028) compared with the ACE gene II+ID genotype. However, no correlation between the CYP11B2 gene −344T/C polymorphism and lone AF or its recurrence was observed in this cohort. In conclusion, the ACE gene DD genotype was associated with an increased incidence of lone AF and its recurrence following ablation, which was partly mediated by LAD.
doi:10.3892/etm.2012.650
PMCID: PMC3501435  PMID: 23170137
angiotensin-converting enzyme gene; CYP11B2; lone atrial fibrillation; recurrence; polymorphisms
10.  Rs12218 In SAA1 gene was associated with serum lipid levels 
Background
Serum amyloid A (SAA) is a kind of apolipoprotein. Several studies indicated that SAA genetic polymorphism rs12218 was associated with carotid atherosclerosis, peripheral arterial disease, and serum uric acid levels. However, the relation between rs12218 and lipid levels remains unclear. This study assessed the correlation between SAA1 gene rs12218 polymorphism and lipid levels in a Chinese population.
Methods
A total of 823 participants were selected from the subjects for health check in Shanghai Huashan hospital from Jan. 2013 to Mach. 2013. Correlations between rs12218 polymorphism and lipid levels were investigated through the identification of rs12218 genotypes using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).
Results
We found that the SNP rs12218 was associated with triglyceride (TG), total cholesterol (TC), and low-density lipoprotein (LDL-C) levels by analyses of a dominant model (P<0.001, P=0.002, P=0.003, respectively), a recessive model (P <0.001, P=0.001, P=0.005, respectively) and an additive model (P < 0.001, P=0.001, P=0.002, respectively), and the difference remained significant after the adjustment of sex, age, alcohol intake, and smoking (All P < 0.01).
Conclusion
Our results indicated that the rs12218 in the SAA1gene was associated with lipid levels in a Chinese population.
doi:10.1186/1476-511X-12-116
PMCID: PMC3765124  PMID: 23898911
SAA1; Genetic polymorphism; Triglyceride; Total cholesterol; Low-density lipoprotein
11.  Clinical and experimental studies regarding the expression and diagnostic value of carcinoembryonic antigen-related cell adhesion molecule 1 in non-small-cell lung cancer 
BMC Cancer  2013;13:359.
Background
Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a multifunctional Ig-like cell adhesion molecule that has a wide range of biological functions. According to previous reports, serum CEACAM1 is dysregulated in different malignant tumours and associated with tumour progression. However, the serum CEACAM1 expression in non-small-cell lung carcinomas (NSCLC) is unclear. The different expression ratio of CEACAM1-S and CEACAM1-L isoform has seldom been investigated in NSCLC. This research is intended to study the serum CEACAM1 and the ratio of CEACAM1-S/L isoforms in NSCLC.
Methods
The expression of the serum CEACAM1 was determined by enzyme-linked immunosorbent assay. The protein expression and the location of CEACAM1 in tumours were observed by immunohistochemical staining. The CEACAM1 mRNA levels in tumour and normal adjacent tissues were measured using quantitative real-time PCR, and the expression patterns and the rate of CEACAM1-S and CEACAM1-L were analysed by reverse transcription-PCR.
Results
Serum CEACAM1 levels were significantly higher in NSCLC patients compared with that from normal healthy controls (P <0.0001). 17 patients (81%) among 21 showed high expression of CEACAM1 by immunohistochemical staining. Although no significant differences were found between tumour and normal tissues on mRNA expression levels of CEACAM1 (P >0.05), the CEACAM1-S and the CEACAM1-S/L (S: L) ratios were significantly higher in tumour than normal tissues (P <0.05).
Conclusions
Our data indicated that the serum levels of CEACAM1 could discriminate lung cancer patients from health donors and that CEACAM1 might be a useful marker in early diagnosis of NSCLC. Moreover, our results showed that the expression patterns of CEACAM1 isoforms could be changed during oncogenesis, even when total CEACAM1 in tumour tissues did not show significant changes. Our study suggested that the expression ratios of CEACAM1-S/CEACAM1-L might be a better diagnostic indicator in NSCLC than the quantitative changes of CEACAM1.
doi:10.1186/1471-2407-13-359
PMCID: PMC3728234  PMID: 23885995
Carcinoembryonic antigen-related cell adhesion molecule 1; Non-small-cell lung carcinomas; Enzyme-linked immunosorbent assay; Receiver operating characteristic curve
12.  Prevalence and spectrum of Nkx2.5 mutations associated with idiopathic atrial fibrillation 
Clinics  2013;68(6):777-784.
OBJECTIVE:
The aim of this study was to evaluate the prevalence and spectrum of Nkx2.5 mutations associated with idiopathic atrial fibrillation (AF).
METHODS:
A cohort of 136 unrelated patients with idiopathic atrial fibrillation and 200 unrelated, ethnically matched healthy controls were enrolled. The coding exons and splice junctions of the Nkx2.5 gene were sequenced in 136 atrial fibrillation patients, and the available relatives of mutation carriers and 200 controls were subsequently genotyped for the identified mutations. The functional characteristics of the mutated Nkx2.5 gene were analyzed using a dual-luciferase reporter assay system.
RESULTS:
Two novel heterozygous Nkx2.5 mutations (p.N19D and p.F186S) were identified in 2 of the 136 unrelated atrial fibrillation cases, with a mutational prevalence of approximately 1.47%. These missense mutations co-segregated with atrial fibrillation in the families and were absent in the 400 control chromosomes. Notably, 2 mutation carriers also had congenital atrial septal defects and atrioventricular block. Multiple alignments of the Nkx2.5 protein sequences across various species revealed that the altered amino acids were completely conserved evolutionarily. Functional analysis demonstrated that the mutant Nkx2.5 proteins were associated with significantly reduced transcriptional activity compared to their wild-type counterpart.
CONCLUSION:
These findings associate the Nkx2.5 loss-of-function mutation with atrial fibrillation and atrioventricular block and provide novel insights into the molecular mechanism involved in the pathogenesis of atrial fibrillation. These results also have potential implications for early prophylaxis and allele-specific therapy of this common arrhythmia.
doi:10.6061/clinics/2013(06)09
PMCID: PMC3674279  PMID: 23778487
Atrial Fibrillation; Transcriptional Factor; Nkx2.5; Genetics; Reporter Gene
13.  The endoglycosidase heparanase enters the nucleus of T lymphocytes and modulates H3 methylation at actively transcribed genes via the interplay with key chromatin modifying enzymes 
Transcription  2012;3(3):130-145.
The methylation of histones is a fundamental epigenetic process regulating gene expression programs in mammalian cells. Dysregulated patterns of histone methylation are directly implicated in malignant transformation. Here, we report the unexpected finding that the invasive extracellular matrix degrading endoglycosidase heparanase enters the nucleus of activated human T lymphocytes and regulates the transcription of a cohort of inducible immune response genes by controlling histone H3 methylation patterns. It was found that nuclear heparanase preferentially associates with euchromatin. Genome-wide ChIP-on-chip analyses showed that heparanase is recruited to both the promoter and transcribed regions of a distinct cohort of transcriptionally active genes. Knockdown and overexpression of the heparanase gene also showed that chromatin-bound heparanase is a prerequisite for the transcription of a subset of inducible immune response genes in activated T cells. Furthermore, the actions of heparanase seem to influence gene transcription by associating with the demethylase LSD1, preventing recruitment of the methylase MLL and thereby modifying histone H3 methylation patterns. These data indicate that heparanase belongs to an emerging class of proteins that play an important role in regulating transcription in addition to their well-recognized extra-nuclear functions.
doi:10.4161/trns.19998
PMCID: PMC3616083  PMID: 22771948
T lymphocytes; chromatin; heparanase; histone methylation; immune response genes; inducible genes; transcription
14.  Molecular mechanism of monodisperse colloidal tin-doped indium oxide nanocrystals by a hot-injection approach 
Nanoscale Research Letters  2013;8(1):153.
Molecular mechanisms and precursor conversion pathways associated with the reactions that generate colloidal nanocrystals are crucial for the development of rational synthetic protocols. In this study, Fourier transform infrared spectroscopy technique was employed to explore the molecular mechanism associated with the formation of tin-doped indium oxide (ITO) nanocrystals. We found that the reaction pathways of the indium precursor were not consistent with simple ligand replacements proposed in the literature. The resulting understanding inspired us to design a hot-injection approach to separate the ligand replacements of indium acetate and the aminolysis processes, generating quality ITO nanocrystals with decent size distributions. The hot-injection approach was readily applied to the synthesis of ITO nanocrystals with a broad range of tin doping. Structural, chemical, and optical analyses revealed effective doping of Sn4+ ions into the host lattices, leading to characteristic and tunable near-infrared surface plasmon resonance peaks. The size control of ITO nanocrystals by multiple hot-injections of metal precursors was also demonstrated.
doi:10.1186/1556-276X-8-153
PMCID: PMC3626570  PMID: 23547801
ITO; Molecular mechanism; Synthesis; FTIR; Nanocrystals
15.  Constitutive activation of p38 MAPK in tumor cells contributes to osteolytic bone lesions in multiple myeloma 
Bone destruction is a hallmark of multiple myeloma and affects more than 80% of patients. However, current therapy is unable to completely cure and/or prevent bone lesions. Although it is accepted that myeloma cells mediate bone destruction by inhibition of osteoblasts and activation of osteoclasts, the underlying mechanism is still poorly understood. This study demonstrates that constitutive activation of p38 mitogen-activated protein kinase in myeloma cells is responsible for myeloma-induced osteolysis. Our results show that p38 is constitutively activated in most myeloma cell lines and primary myeloma cells from patients. Myeloma cells with high/detectable p38 activity, but not those with low/undetectable p38 activity, injected into SCID or SCID-hu mice caused bone destruction. Inhibition or knockdown of p38 in human myeloma reduced or prevented myeloma-induced osteolytic bone lesions without affecting tumor growth, survival, or homing to bone. Mechanistic studies showed that myeloma cell p38 activity inhibited osteoblastogenesis and bone formation and activated osteoclastogenesis and bone resorption in myeloma-bearing SCID mice. This study elucidates a novel molecular mechanism—sactivation of p38 signaling in myeloma cells—by which myeloma cells induce osteolytic bone lesions and indicates that targeting myeloma cell p38 may be a viable approach to treating or preventing myeloma bone disease.
doi:10.1038/leu.2012.71
PMCID: PMC3381862  PMID: 22425892
Myeloma; p38 MAPK; Osteolytic bone lesions; Osteoblastogenesis; Osteoclastogenesis
16.  Utilizing TH9 cells as a novel therapeutic strategy for malignancies 
Oncoimmunology  2013;2(3):e23084.
TH9 cells join the ever-growing list of CD4+ T helper subsets and primarily mediate anti-parasite immune responses. We have recently demonstrated that tumor-specific TH9 cells induce a CCL20-CCR6-dependent regulation of DCs while stimulating CD8+ T cell-mediated antitumor immunity. These findings offer a novel immunotherapeutic strategy against cancer.
doi:10.4161/onci.23084
PMCID: PMC3661147  PMID: 23802062
Th9 cell; TCLs; Ccl20; DCs; melanoma
17.  GATA5 loss-of-Function Mutations Underlie Tetralogy of Fallot 
Tetraology of Fallot (TOF) is the most common form of cyanotic congenital heart disease and is a major cause of significant morbidity and mortality. Emerging evidence demonstrates that genetic risk factors are involved in the pathogenesis of TOF. However, TOF is genetically heterogeneous and the genetic defects responsible for TOF remain largely unclear. In the present study, the whole coding region of the GATA5 gene, which encodes a zinc-finger transcription factor essential for cardiogenesis, was sequenced in 130 unrelated patients with TOF. The relatives of the index patients harboring the identified mutations and 200 unrelated control individuals were subsequently genotyped. The functional characteristics of the mutations were analyzed using a luciferase reporter assay system. As a result, 2 novel heterozygous GATA5 mutations, p.R187G and p.H207R, were identified in 2 families with autosomal dominantly inherited TOF, respectively. The variations were absent in 400 control alleles and the altered amino acids were completely conserved evolutionarily. Functional analysis showed that the GATA5 mutants were associated with significantly decreased transcriptional activation compared with their wild-type counterpart. To our knowledge, this is the first report on the association of GATA5 loss-of-function mutations with TOF, suggesting potential implications for the early prophylaxis and allele-specific therapy of human TOF.
doi:10.7150/ijms.5270
PMCID: PMC3534875  PMID: 23289003
Congenital heart disease; Tetralogy of Fallot; Genetics; Transcription factor; GATA5.
18.  PITX2c Loss-of-Function Mutations Responsible for Congenital Atrial Septal Defects 
Congenital heart disease (CHD) is the most common form of developmental anomaly and is the leading non-infectious cause of infant mortality. A growing body of evidence demonstrates that genetic risk factors are involved in the pathogenesis of CHD. However, CHD is a genetically heterogeneous disease and the genetic determinants for CHD in most patients remain unclear. In the present study, the entire coding region and splice junction sites of the PITX2c gene, which encodes a homeobox transcription factor crucial for normal cardiovascular genesis, was sequenced in 150 unrelated patients with various CHDs. The 200 unrelated control individuals were subsequently genotyped. The functional characteristics of the mutations were explored using a dual-luciferase reporter assay system. As a result, two novel heterozygous PITX2c mutations, p.H98Q and p.M119T, were identified in 2 unrelated patients with atrial septal defects, respectively. The variations were absent in 400 control chromosomes and the affected amino acids were completely conserved evolutionarily. The two variants were both predicted to be disease-causing by MutationTaster and PolyPhen-2, and the functional analysis revealed that the PITX2c mutants were consistently associated with significantly reduced transcriptional activity compared with their wild-type counterpart. These findings firstly link PITX2c loss-of-function mutations to atrial septal defects in humans, which provide novel insight into the molecular mechanism responsible for CHD, suggesting potential implications for the early prophylaxis and allele-specific treatment of CHD.
doi:10.7150/ijms.6809
PMCID: PMC3753420  PMID: 23983605
Congenital heart disease; Atrial septal defect; Genetics; Transcription factor; PITX2c
19.  Immunotherapy for Multiple Myeloma 
doi:10.1155/2012/797165
PMCID: PMC3546469  PMID: 23346187
20.  The relationship between low pH in intervertebral discs and low back pain: a systematic review 
Introduction
To systematically review the relationship between low pH in intervertebral discs and low back pain.
Material and methods
Electronic database (PubMed, ISI Web of Science, Cochrane Library, CINAHL, AMED, and China National Knowledge Infrastructure) searches and hand searching of conference proceedings were conducted. Two authors independently evaluated the methodological quality and abstracted relevant data according to standard criteria. Then the experimental methods and samples employed in the finally retrieved articles were assessed.
Results
We first retrieved 136 articles regarding pain and pH, and only 16 of them were mainly about low back pain and pH. Finally, 7 articles met our expectation to focus on the pathogenesis of low back pain caused by pH. In these 7 studies the authors held three opinions to explain the pathogenesis of low back pain in relation to low pH. First, low pH caused by lactate stimulates the muscle and increases the muscle tension, which causes low back pain. Second, low pH stimulates the nerve roots and produces the feeling of pain. Third, low pH changes the matrix metabolism, leading to neuronal death and low back pain.
Conclusions
In this systematic review we propose a new hypothesis that low back pain may be caused by low pH based on the previous literature. Further experimental studies are necessary to verify our hypothesis. This hypothesis will promote our understanding of the pathogenesis of low back pain and the development of novel diagnostic and therapeutic approaches for low back pain.
doi:10.5114/aoms.2012.32401
PMCID: PMC3542485  PMID: 23319966
low back pain; pH; acidity; intervertebral disc; systematic review
21.  Novel GATA5 loss-of-function mutations underlie familial atrial fibrillation 
Clinics  2012;67(12):1393-1399.
OBJECTIVE:
This study aimed to identify novel GATA5 mutations that underlie familial atrial fibrillation.
METHODS:
A total of 110 unrelated patients with familial atrial fibrillation and 200 unrelated, ethnically matched healthy controls were recruited. The entire coding region of the GATA5 gene was sequenced in 110 atrial fibrillation probands. The available relatives of the mutation carriers and 200 controls were subsequently genotyped for the identified mutations. The functional effect of the mutated GATA5 was characterized using a luciferase reporter assay system.
RESULTS:
Two novel heterozygous GATA5 mutations (p.Y138F and p.C210G) were identified in two of the 110 unrelated atrial fibrillation families. These missense mutations cosegregated with AF in the families and were absent in the 400 control chromosomes. A cross-species alignment of GATA5 protein sequence showed that the altered amino acids were completely conserved evolutionarily. A functional analysis revealed that the mutant GATA5 proteins were associated with significantly decreased transcriptional activation when compared with their wild-type counterpart.
CONCLUSION:
The findings expand the spectrum of GATA5 mutations linked to AF and provide novel insights into the molecular mechanism involved in the pathogenesis of atrial fibrillation, suggesting potential implications for the early prophylaxis and personalized treatment of this common arrhythmia.
doi:10.6061/clinics/2012(12)08
PMCID: PMC3521801  PMID: 23295592
Atrial Fibrillation; Transcriptional Factor; GATA5; Genetics; Reporter Gene
22.  Th9 cells promote antitumor immune responses in vivo 
The Journal of Clinical Investigation  2012;122(11):4160-4171.
Th9 cells are a subset of CD4+ Th cells that produce the pleiotropic cytokine IL-9. IL-9/Th9 can function as both positive and negative regulators of immune response, but the role of IL-9/Th9 in tumor immunity is unknown. We examined the role of IL-9/Th9 in a model of pulmonary melanoma in mice. Lack of IL-9 enhanced tumor growth, while tumor-specific Th9 cell treatment promoted stronger antitumor responses in both prophylactic and therapeutic models. Th9 cells also elicited strong host antitumor CD8+ CTL responses by promoting Ccl20/Ccr6-dependent recruitment of DCs to the tumor tissues. Subsequent tumor antigen delivery to the draining LN resulted in CD8+ T cell priming. In agreement with this model, Ccr6 deficiency abrogated the Th9 cell–mediated antitumor response. Our data suggest a distinct role for tumor-specific Th9 cells in provoking CD8+ CTL-mediated antitumor immunity and indicate that Th9 cell–based cancer immunotherapy may be a promising therapeutic approach.
doi:10.1172/JCI65459
PMCID: PMC3484462  PMID: 23064366
23.  Targeting human B-cell malignancies through Ig light chain specific cytotoxic T lymphocytes 
Purpose
The variable regions of Ig (idiotype, Id) expressed by malignant B cells can be used as tumor-specific antigens that induce humoral and cellular immunity. However, epitopes derived from Id that stimulate human CD8+ T-cell immunity are incompletely characterized.
Experimental design
The clonal Ig VL of human myeloma cell line U266 and five primary B-cell tumors were sequenced and peptides corresponding to the Ig VL region were tested for their ability to stimulate cytotoxic T lymphocytes (CTLs) from ten HLA-A* 0201 positive normal donors. The CTLs thus generated were tested against peptide-pulsed T2 cells and autologous tumor cells.
Results
14 peptides derived from Ig light chain (VL) of U266 and primary B-cell tumors were used to generate 68 Cytotoxic T lymphocytes (CTLs) lines that specifically produced IFN-γ when co-cultured with peptide-pulsed T2 cells. These CTLs lysed peptide-pulsed T2 cell as well as U266 or autologous tumor targets in an HLA class I-dependent manner. Sequence analysis revealed shared VL T-cell epitopes in U266 and primary B-cell tumors, not previously reported within Ig heavy chain (VH) sequences.
Conclusion
This study thus identifies novel immunogenic CTLs epitopes from Id VL, suggests that they are naturally presented on the surface of B-cell malignancies, and supports their inclusion in next generation Id vaccines. The ability to prime T cells derived from normal HLA-matched donors, rather than patients, also may have direct application to current strategies, designed to generate allogeneic tumor-specific T cells for adoptive transfer.
doi:10.1158/1078-0432.CCR-11-0970
PMCID: PMC3176952  PMID: 21813633
myeloma; plasma cell leukemia; peptide; allogeneic T cells; immunotherapy; donor lymphocyte infusion; vaccine; stem cell transplantation
24.  Id2 regulates the proliferation of squamous cell carcinoma in vitro via the NF-κB/Cyclin D1 pathway 
Chinese Journal of Cancer  2012;31(9):430-439.
Squamous cell carcinoma (SCC) is a significant cause of cancer morbidity and mortality worldwide, with an incidence of up to 166 cases per 100 000 population. It arises in the skin, upper aerodigestive tract, lung, and cervix and affects more than 200 000 Americans each year. We report here that a microarray experiment comparing 41 SCC and 13 normal tissue specimens showed that Id2, a gene that controls the cell cycle, was significantly up-regulated in SCC. Enforced expression of Id2 in vitro stimulated the proliferation of SCC cells and up-regulated the transcription of nuclear factor kappa B (NF-κB) and cyclin D1. Enhancement of the NF-κB activity with p65 significantly increased the cell proliferation and the transcription of cyclin D1, whereas inhibition of the NF-κB activity with I kappa B alpha mutant (IκBα M) and pyrroline dithiocarbamate (PDTC) abrogated cell proliferation and transcription of cyclin D1. Furthermore, a mutated NF-κB binding site in the cyclin D1 promoter fully abrogated the Id2-induced transcription of cyclin D1. Taken together, these data indicate that Id2 induces SCC tumor growth and proliferation through the NF-κB/cyclin D1 pathway.
doi:10.5732/cjc.011.10454
PMCID: PMC3777501  PMID: 22835384
Id2; head and neck squamous cell carcinoma; NF-κB; cyclin D1; human
25.  DKK1 as a novel target for myeloma immunotherapy 
Oncoimmunology  2012;1(5):756-758.
Novel, potent tumor-associated antigens are needed to improve the efficacy of immunotherapy for myeloma. We demonstrated that active vaccination using the DKK1-DNA vaccine in the myeloma mouse model protected mice from developing myeloma and effectively treated established myeloma. Therefore, DKK1 could be developed as a novel vaccine for myeloma immunotherapy.
doi:10.4161/onci.19655
PMCID: PMC3429585  PMID: 22934273
multiple myeloma; Dickkopf-1; vaccine; immunotherapy; DNA

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