Screening and identification of immune-related genes for immunotherapy and prognostic assessment in colorectal cancer patients.

BACKGROUND: Increasing evidence indicates that the immune microenvironment plays a key role in the genesis and progression of colorectal cancer (CRC). This study aimed to establish an immune-related gene (IRG) signature and determine its clinical prognostic value in patients with CRC. METHODS: The RNA sequencing and associated clinical data of CRC were downloaded from The Cancer Genome Atlas (TCGA) database. We then screened for differentially expressed IRGs by intersecting with IRGs obtained from the Immunology Database and Analysis Portal. Functional enrichment analyses were carried out to determine the potential biological functions and pathways of the IRGs. We also explored the specific molecular mechanisms of the IRGs by constructing regulatory networks. Prognostic IRGs were obtained by LASSO regression analysis, and subsequently, gene models were constructed in the TCGA dataset to confirm the predictive capacity of these IRGs. Finally, we used the TIMER tool to assess the immune properties of prognostic IRGs and correlate them with immune cells. RESULTS: We identified 409 differentially expressed IRGs in patients with CRC. Kyoto Encyclopaedia of Genes and Genomes and Gene Ontology enrichment analyses suggested that these differentially expressed IRGs were significantly related to 102 cancer signalling pathways and various biological functions. Based on the prediction and interaction results, we obtained 59 TF-IRG, 48 miRNA-IRG, and 214 drug-IRG interaction networks for CRC. Four prognostic genes (POMC, TNFRSF19, FGF2, and SCG2) were developed by integrating 47 survival-related IRGs and 42 characteristic CRC genes. The results of gene model showed that patients in the low risk group had better survival outcomes compared to those in the high risk group. The expression of POMC, TNFRSF19, FGF2, and SCG2 was significantly correlated with immune cells. CONCLUSION: This study identified some valid IRGs, and these findings can provide strong evidence for precision immunotherapy in patients with CRC.

Long-term follow-up and safety assessment of angiogenic gene therapy trial VIF-CAD: Transcatheter intramyocardial administration of a bicistronic plasmid expressing VEGF-A165/bFGF cDNA for the treatment of refractory coronary artery disease.

There have been a number of angiogenic gene therapy trials, yielding mixed results as to efficacy, but demonstrating uniform short-term treatment safety. Data regarding long-term safety of angiogenic gene therapy are limited. Double-blind VIF-CAD trial (NCT00620217) assessed myocardial perfusion and clinical data in 52 refractory coronary artery disease (CAD) patients randomized into treatment (VIF; n = 33) and Placebo (n = 19) arms. VIF group received electromechanical system NOGA-guided intramyocardial injections of VEGF-A165/bFGF plasmid (VIF) into ischemic regions, while the Placebo group-placebo plasmid injections. Full 1-year follow-up data have been published. This study presents the results of over 10-year (median 133 months, range 95-149) safety follow-up of VIF-CAD patients. Overall, 12 (36.4%) patients died in VIF and 8 (42.1%) in Placebo group (P = .68). Cardiovascular mortality was 12/33 (36.4%) in the VIF group and 6/19 (31.6%) in Placebo group (P = .73). Two Placebo patients died due to malignancies, but no VIF patients (P = .17). The Kaplan-Meier curves of combined endpoint: cardiovascular mortality, myocardial infarction and stroke were similar for both patient groups (P = .71). Odds ratio of Placebo group increasing (reaching a worse) their CCS class versus VIF was non-significant (OR 1.28, 95% CI = 0.66-2.45; P = .47). However, CCS class improved in time irrespectively of treatment-OR of reaching a less favorable CCS class per each year of follow-up was 0.74 (95% CI 0.685-0.792; P < .0001, pooled data). There were no differences in readmission rates. Intramyocardial VEGF-A165/bFGF plasmid administration appears safe, with no evidence of an increase in the incidence of death, malignancy, myocardial infarction or stroke during 10-year follow-up in this limited patient population.

Expression and Purification of Recombinant Human Basic Fibroblast Growth Factor Fusion Proteins and Their Uses in Human Stem Cell Culture.

To reduce the cost of cytokines and growth factors in stem cell research, a simple method for the production of soluble and biological active human basic fibroblast growth factor (hbFGF) fusion protein in Escherichia coli was established. Under optimal conditions, approximately 60-80 mg of >95% pure hbFGF fusion proteins (Trx-6xHis-hbFGF and 6xHis-hbFGF) were obtained from 1 liter of culture broth. The purified hbFGF proteins, both with and without the fusion tags, were biologically active, which was confirmed by their ability to stimulate proliferation of NIH3T3 cells. The fusion proteins also have the ability to support several culture passages of undifferentiated human embryonic stem cells and induce pluripotent stem cells. This paper describes a low-cost and uncomplicated method for the production and purification of biologically active hbFGF fusion proteins.

The noninvasive, quantitative, in vivo assessment of adenoviral-mediated gene delivery in skin wound biomaterials.

Because there are few reports using gene delivery in clinically-approved synthetic matrices, we examined the feasibility of using a noninvasive imaging system to study the kinetics of luciferase gene expression when delivered in an adenoviral vector. Using a mouse model of full thickness injury, we quantified the kinetics of gene expression, determined the optimal dose of particle delivery, and established the temporal importance of drug delivery in obtaining optimal gene expression. Specifically, we found that the ideal time to deliver adenovirus to a graft is during the early phase of graft wound closure (days 0-3 post-operatively) for a peak of gene expression to occur 7 days after delivery. Under these conditions, there is a saturating dose of 6 x 10(8) adenoviral particles per graft. In light of these findings, we examined whether the efficacy of delivery could be increased by modulating the composition of the grafts. When a collagen gene-activated matrix (GAM) containing basic fibroblast growth factor (FGF2) was compared to matrix alone, a significant increase in gene expression is observed when identical amounts of vector are delivered (p<0.05). Taken together, these results show how a noninvasive and quantitative assessment of gene expression can be used to optimize gene delivery and that the composition of matrices can dramatically influence gene expression in the wound bed.

Isolation, characterization, and functional assessment of oxidatively modified subfractions of circulating low-density lipoproteins.

OBJECTIVE: Current evidence suggests that oxidatively modified human plasma low-density lipoproteins (ox-LDLs) are proatherogenic and cytotoxic to endothelial and vascular smooth muscle cells. The present study describes a method using ion-exchange chromatography that is capable of large-scale subfractionation of LDL for adequate analyses of composition or bioactivities. METHODS AND RESULTS: LDLs from normolipidemic (N-LDL) and homozygous familial hypercholesterolemic (FH-LDL) subjects were separated into 5 subfractions (L1 through L5) by high-capacity ion-exchange chromatography. The most strongly retained fraction from FH subjects, FH-L5, suppressed DNA synthesis in cultured bovine aortic endothelial cells and stimulated mononuclear cell adhesion to cultured endothelial cells under flow conditions in vitro. L5, which represented 1.1+/-0.2% and 3.7+/-1.7% of the LDL from N-LDL and FH-LDL, respectively, was more triglyceride-rich (17% versus 5%) and cholesteryl ester-poor (23% versus 33%) than were L1 through L4. Electrophoretic mobilities on agarose gels increased from L1 to L5. According to SDS-PAGE, apolipoprotein B-100 in N-LDL fractions L1 through L5 appeared as a single approximately 500-kDa band. In contrast, the fractions isolated from FH-LDL showed substantial fragmentation of the apolipoprotein B-100, including bands between 200 and 116 kDa. Competitive ELISA analyses using a malondialdehyde-specific monoclonal antibody against Cu2+ ox-LDL suggest that FH-L5 is malondialdehyde-modified. CONCLUSIONS: Relative to N-LDL, FH-LDL contains higher concentrations of a fraction, L5, that exhibits distinctive physicochemical properties and biological activities that may contribute to initiation and progression of atherogenesis in vivo.

Assessment of the role of sphingosine 1-phosphate and its receptors in high-density lipoprotein-induced stimulation of astroglial cell function.

It has been suggested that lipoproteins in the central nervous system are involved in the regulation of several neural functions independent of cholesterol metabolism as well as those related to lipid metabolism. We recently demonstrated that lipoproteins are carriers for sphingosine 1-phosphate (S1P). This raised the possibility that S1P mediates the neural cell functions induced by lipoproteins. In the current study, we examined the effects of plasma high-density lipoprotein (HDL) on astroglial cell functions, focusing especially on the role of the lipoprotein-associated S1P. In rat type I astrocytes or C6 glioma cells, similar to S1P, HDL stimulated DNA synthesis and mRNA expression of fibroblast growth factor-2, a potent neurotrophic factor, which was associated with the activation of extracellular signal-regulated kinase (ERK) in a pertussis toxin-sensitive manner. The data from fractionation studies of HDL indicated that S1P may be a major component for the activation of ERK. In C6 glioma cells, HDL also induced phospholipase C-dependent intracellular Ca(2+) mobilization. Desensitization of the C6 glioma cells with S1P abolished these HDL-induced actions. Furthermore, overexpression of S1P receptors in C6 glioma cells led to a significant enhancement of HDL-induced ERK activation and Ca(2+) mobilization. Thus, at least some HDL-induced actions may be mediated by cell-surface S1P receptors in astroglial cells. These results imply that S1P might partially mediate lipoprotein-induced cholesterol metabolism-independent neural cell functions in the central nervous system.

Mapping four genes from human chromosome 4 to porcine chromosome 8 further develops the comparative map for an economically important chromosome of the swine genome.

Because porcine chromosome (SSC) 8 has become the focal point of many efforts aimed at identifying quantitative trait loci affecting ovulation rate, genes distributed across human chromosome (HSA) 4 were physically mapped in the pig. A more refined comparative map of this region for these two species was produced. In this study, four genes were selected based on their location in the human genome, the availability of nucleotide sequence and their genomic organization. The genes selected were fibroblast growth factor basic (FGF2; HSA 4q25-27), gonadotropin releasing hormone receptor (GNRHR; HSA 4q13), phosphodiesterase 6 B (PDE6B; HSA 4p16.3) and aminopeptidase S (PEPS; HSA 4p11-q12). Genomic libraries were screened via PCR and clones were physically assigned using fluorescence in situ hybridization (FISH). These four genes from HSA 4 were physically mapped to SSC 8p2.3 (PDE6B), 8p1.1 (PEPS), 8q1.1-1.2 (GNRHR) and 8q2.2-2.4 (FGF2). These assignments provide additional benchmarks for the comparative map and help define the level of gene order conserved between HSA 4 and SSC 8.