Impact of surgical strategies on the survival of gallbladder cancer patients: analysis of 715 cases.

OBJECTIVE: The aim of the study is to evaluate the impact of application of surgical strategies at different cancer stages on the survival of gallbladder cancer (GBC) patients. METHODS: The patients with GBC were divided into 3 groups according to their received surgical strategies: simple resection (full-thickness cholecystectomy for removal of primary tumor site), radical resection (gallbladder bed removal combined with partial hepatectomy), and palliative surgery (treatment at advanced stages). The overall survival (OS) of GBC patients who were received different surgical strategies was compared. RESULTS: Survival analysis showed that radical resection had a best OS at clinical stage II, and simple resection had a best OS at tumor clinical stage IV. Cox hazard proportional regression analysis showed that more advanced tumor stages, tumor location of gallbladder body or neck, and CA199 ≥ 27 U/mL were the major risk factors for the OS of GBC. CONCLUSIONS: At tumor stage II, radical resection should be the most effective surgical therapy for GBC. However, the effect of radical resection at advanced stages could be restricted. The utilization of radical resection should be increased at tumor stage II for a better long-term survival outcome.

Hemodynamic study of the ICA aneurysm evolution to attain the cerebral aneurysm rupture risk.

The influence of the aneurysm evolution on the hemodynamic characteristic of the blood flow inside the sac region is comprehensively investigated. By using the computational method, the blood flow through the vessel and aneurysm of the sac region is examined to find the role of aneurysm evolution on the wall shear stress, pressure, and risk of aneurysm rupture. Three different models of ICA aneurysms are chosen for the investigation of the aneurysm evolution at risk of rupture. Obtained data shows that the evolution of the aneurysm decreases the wall shear stress and pressure on the sac surface while an oscillatory index of blood increases on the aneurysm wall.

Sequence variation in promoter of Ica1 gene, which encodes protein implicated in type 1 diabetes, causes transcription factor autoimmune regulator (AIRE) to increase its binding and down-regulate expression.

ICA69 (islet cell autoantigen 69 kDa) is a protein implicated in type 1 diabetes mellitus in both the non-obese diabetic (NOD) mouse model and humans. ICA69 is encoded by the Ica1 gene on mouse chromosome 6 A1-A2. We previously reported reduced ICA69 expression in the thymus of NOD mice compared with thymus of several non-diabetic mouse strains. We propose that reduced thymic ICA69 expression could result from variations in transcriptional regulation of the gene and that polymorphisms within the Ica1 core promoter may partially determine this transcriptional variability. We characterized the functional promoter of Ica1 in NOD mice and compared it with the corresponding portions of Ica1 in non-diabetic C57BL/6 mice. Luciferase reporter constructs demonstrated that the NOD Ica1 promoter region exhibited markedly reduced luciferase expression in transiently transfected medullary thymus epithelial (mTEC(+)) and B-cell (M12)-derived cell lines. However, in a non-diabetic strain, C57BL/6, the Ica1 promoter region was transcriptionally active when transiently transfected into the same cell lines. We concomitantly identified five single nucleotide polymorphisms within the NOD Ica1 promoter. One of these single nucleotide polymorphisms increases the binding affinity for the transcription factor AIRE (autoimmune regulator), which is highly expressed in thymic epithelial cells, where it is known to play a key role regulating self-antigen expression. We conclude that polymorphisms within the NOD Ica1 core promoter may determine AIRE-mediated down-regulation of ICA69 expression in medullary thymic epithelial cells, thus providing a novel mechanistic explanation for the loss of immunologic tolerance to this self-antigen in autoimmunity.

An oncogenic gene, SNRPA1, regulates PIK3R1, VEGFC, MKI67, CDK1 and other genes in colorectal cancer.

PURPOSE: Colorectal cancer (CRC) caused more than 65,000 mortalities worldwide per year. It is a result of one or a combination of chromosomal instability, CpG island methylator phenotype, and microsatellite instability. SNRPA1 (small nuclear ribonucleoprotein polypeptide A) is a subunit of spliceosome complex that is involved in the RNA processing. Overexpression of SNRPA1 has been implicated in a variety of cancers including CRC. Besides from its role in mediating the RNA processing, the other aspects regarding its function in the progression of colorectal cancer have not been revealed. METHODS: Herein, we combined regular gene overexpression or knock down in vitro and in vivo and microarray gene profiling analysis to decipher the unknow regulatory role of SNRPA1 in CRC. RESULTS: We found SNRPA1 widely expression in many representative CRC cell lines. Knocking down expression of SNRPA1 by shRNA lentivirus inhibited the cell proliferation in vitro and impaired tumor formation from implanted CRC cells transduced with SNRPA1 silencing shRNA lentivirus in nude mice. It also promoted the cell apoptosis by upregulating the caspase 3/7 activity. Additional microarray gene profiling analysis uncovered the gene interaction network of SNRPA1, special focus was placed on its association with tumor suppressor or oncogenes. CONCLUSIONS: According to the results of gene interaction network as well as qRT-PCR verification, it revealed that SNPRA1 regulates PIK3R1, VEGFC, MKI67, CDK1 in CRC. These novel findings identified new roles played by SNRPA1 in the progression of CRC and it may become a potential therapeutic target in the treatment of CRC.

IDH mutant gliomas escape natural killer cell immune surveillance by downregulation of NKG2D ligand expression.

BACKGROUND: Diffuse gliomas are poorly immunogenic, fatal brain tumors. The basis for insufficient antitumor immunity in diffuse gliomas is unknown. Gain-of-function mutations in isocitrate dehydrogenases (IDH1 and IDH2) promote diffuse glioma formation through epigenetic reprogramming of a number of genes, including immune-related genes. Here, we identify epigenetic dysregulation of natural killer (NK) cell ligand genes as significant contributors to immune escape in glioma. METHODS: We analyzed the database of The Cancer Genome Atlas for immune gene expression patterns in IDH mutant or wild-type gliomas and identified differentially expressed immune genes. NKG2D ligand expression levels and NK cell-mediated lysis were measured in IDH mutant and wild-type patient-derived glioma stem cells and genetically engineered astrocytes. Finally, we assessed the impact of hypomethylating agent 5-aza-2'deoxycytodine (decitabine) as a potential NK cell sensitizing agent in IDH mutant cells. RESULTS: IDH mutant glioma stemlike cell lines exhibited significantly lower expression of NKG2D ligands compared with IDH wild-type cells. Consistent with these findings, IDH mutant glioma cells and astrocytes are resistant to NK cell-mediated lysis. Decitabine increases NKG2D ligand expression and restores NK-mediated lysis of IDH mutant cells in an NKG2D-dependent manner. CONCLUSIONS: IDH mutant glioma cells acquire resistance to NK cells through epigenetic silencing of NKG2D ligands ULBP1 and ULBP3. Decitabine-mediated hypomethylation restores ULBP1 and ULBP3 expression in IDH mutant glioma cells and may provide a clinically useful method to sensitize IDH mutant gliomas to NK cell-mediated immune surveillance in patients with IDH mutated diffuse gliomas.

Phospho-BAD BH3 mimicry protects ß cells and restores functional ß cell mass in diabetes.

Strategies that simultaneously enhance the survival and glucose responsiveness of insulin-producing ß cells will greatly augment ß cell replacement therapies in type 1 diabetes (T1D). We show that genetic and pharmacologic mimetics of the phosphorylated BCL-2 homology 3 (BH3) domain of BAD impart ß-cell-autonomous protective effects in the face of stress stimuli relevant to ß cell demise in T1D. Importantly, these benefits translate into improved engraftment of donor islets in transplanted diabetic mice, increased ß cell viability in islet grafts, restoration of insulin release, and diabetes reversal. Survival of ß cells in this setting is not merely due to the inability of phospho-BAD to suppress prosurvival BCL-2 proteins but requires its activation of the glucose-metabolizing enzyme glucokinase. Thus, BAD phospho-BH3 mimetics may prove useful in the restoration of functional ß cell mass in diabetes.

Immunoinhibitory DNA vaccine protects against autoimmune diabetes through cDNA encoding a selective CTLA-4 (CD152) ligand.

Cytotoxic T lymphocyte antigen 4 (CTLA-4 or CD152) is a strong negative regulator of T cell activity. Like CD28 (a positive regulator) it binds to B7-1 and B7-2, and there is no known natural selective ligand. Monoclonal antibodies to CTLA-4 generally have a masking effect, enhancing rather than suppressing responses. However, a single amino acid substitution in B7-1 (W88 > A; denoted B7-1wa) abrogates binding to CD28 but not to CTLA-4. We constructed plasmids encoding B7-1 or B7-1wa, as cell-surface or Ig fusion proteins. In a bound state, B7-1-Ig enhanced CD3-mediated T cell activation, but B7-1wa-Ig was inhibitory, as expected of a CTLA-4 ligand. To alter immunity in vivo, we inoculated mice intramuscularly (i.m.) with a carcinoembryonic antigen (CEA) plasmid. Gene transfer was amplified by electroporation. Co-injection of a B7-1wa (membrane-bound form) plasmid blocked induction of anti-CEA immunity, whereas a B7-1 plasmid was stimulatory. We studied this DNA covaccination method in nonobese diabetic (NOD) mice with autoimmune diabetes. Delivery of either preproinsulin I (PPIns) or B7-1wa cDNA alone did not suppress the autoimmune anti-insulin response of spleen cells. However, co-delivery of B7-1wa and PPIns cDNA abrogated reactivity to insulin and ameliorated disease. Interferon-gamma and interleukin-4 were both depressed, arguing against a Th2 bias. Reactivity to glutamic acid decarboxylase 65, another major islet autoantigen, was not altered and suppressor cells were not identified, suggesting induction of tolerance to insulin by either T cell anergy or deletion. Selective engagement of CTLA-4 through gene transfer represents a novel and powerful way to block autoimmunity specifically.

Intramuscular gene transfer of soluble B7.1/IgG(1) fusion cDNA induces potent antitumor immunity as an adjuvant for DNA vaccination.

Soluble B7.1/IgG Fc fusion protein, which has costimulatory effects, is an effective molecular adjuvant in tumor immune therapy. Here, we describe a nonviral intramuscular (i.m.) gene transfer method to deliver this therapeutic protein. Gene transfer was greatly enhanced by electroporation and highly efficient production of this protein was achieved. Serum levels reached up to 1 microg/ml with considerable length of expression and without apparent systemic adverse effects. Lymphocytes from mice coinjected with soluble B7.1/IgG(1) and carcinoembryonic antigen (CEA)-encoding plasmids showed significantly elevated CEA-stimulated proliferation, cytokine production, and cytotoxic T-lymphocyte (CTL) activity. These mice gained significant protection against a CEA-positive transplanted tumor, in terms of reduced tumor incidence and growth. The effects were superior when soluble B7.1/IgG(1) was expressed as compared to membrane-bound wild-type B7.1. Notably, expression of soluble B7.1/IgG(1) alone did not induce any protection against tumor, confirming its primary role as a costimulatory molecule rather than a direct antitumor agent. The plasmid encoding B7.1/IgG(1) did not have to be injected at the same site as the antigen-encoding plasmid to exert its adjuvant effect, indicating that circulating protein was sufficient. Muscle histopathology revealed minimal damage to DNA-injected muscles. Importantly, we show that, after gene transfer, muscle tissue can produce this protein in large quantity to exert its immune costimulatory effect for cancer therapy and it would be otherwise difficult and expensive to maintain this high a level of recombinant protein.

Antidiabetic sulfonylurea stimulates insulin secretion independently of plasma membrane KATP channels.

Understanding mechanisms by which glibenclamide stimulates insulin release is important, particularly given recent promising treatment by glibenclamide of permanent neonatal diabetic subjects. Antidiabetic sulfonylureas are thought to stimulate insulin secretion solely by inhibiting their high-affinity ATP-sensitive potassium (K(ATP)) channel receptors at the plasma membrane of beta-cells. This normally occurs during glucose stimulation, where ATP inhibition of plasmalemmal K(ATP) channels leads to voltage activation of L-type calcium channels for rapidly switching on and off calcium influx, governing the duration of insulin secretion. However, growing evidence indicates that sulfonylureas, including glibenclamide, have additional K(ATP) channel receptors within beta-cells at insulin granules. We tested nonpermeabilized beta-cells in mouse islets for glibenclamide-stimulated insulin secretion mediated by granule-localized K(ATP) channels by using conditions that bypass glibenclamide action on plasmalemmal K(ATP) channels. High-potassium stimulation evoked a sustained rise in beta-cell calcium level but a transient rise in insulin secretion. With continued high-potassium depolarization, addition of glibenclamide dramatically enhanced insulin secretion without affecting calcium. These findings support the hypothesis that glibenclamide, or an increased ATP/ADP ratio, stimulates insulin secretion in part by binding at granule-localized K(ATP) channels that functionally contribute to sustained second-phase insulin secretion.

Protective regulatory T cell generation in autoimmune diabetes by DNA covaccination with islet antigens and a selective CTLA-4 ligand.

DNA vaccination of autoimmune diabetes-prone NOD mice with unmodified target islet antigens, i.e., preproinsulin (PPIns) or glutamic acid decarboxylase 65 (GAD65), is poorly protective. However, in this study, we demonstrate protection against disease by covaccination with a mutant B7-1 molecule (B7-1wa) that binds the negative T cell regulator CTLA-4 (CD152), but not CD28. Codelivery of plasmids encoding a PPIns-GAD65 fusion construct and B7-1wa protected against both insulitis and diabetes. In vitro, the T cells of covaccinated mice had negative responses to both insulin and GAD65, and this was restored by adding blocking antibodies to transforming growth factor beta1 (TGF-beta1), suggesting a role for this cytokine. Adoptive transfer experiments revealed that DNA vaccination generated protective CD4(+) regulatory T cells (Tr) of either CD25(+) or CD25(-) phenotype. Furthermore, vaccinated mice had increased numbers of T cells with Tr-associated markers, such as CTLA-4, Foxp3, and membrane-bound TGF-beta1. Tr cells inhibited the responses of diabetogenic T cells to islet antigens, and depletion of T cells expressing membrane-bound TGF-beta1 abolished the suppressive effect. Thus, selective engagement of CTLA-4 during islet-antigen DNA vaccination induces Tr cells that protect against this autoimmune disease.

In vivo immune modulatory activity of hepatic stellate cells in mice.

Accumulating data suggest that hepatic tolerance, initially demonstrated by spontaneous acceptance of liver allografts in many species, results from an immune regulatory activity occurring in the liver. However, the responsible cellular and molecular components have not been completely understood. We have recently described profound T cell inhibitory activity of hepatic stellate cells (HSCs) in vitro. In this study, we demonstrate in vivo evidence of immune modulatory activity of HSCs in mice using an islet transplantation model. Co-transplanted HSCs effectively protected islet allografts from rejection, forming a multi-layered capsule, which reduced allograft immunocyte infiltrates by enhancement of apoptotic death. The immune modulation by HSCs appeared to be a local effect, and regulated by inducible expression of B7-H1, an inhibitory molecule of B7 family. This may reflect an intrinsic mechanism of immune inhibition mediated by liver-derived tissue cells. In conclusion, these results may lead to better understanding of liver immunobiology and development of new strategies for treatment of liver diseases.

A mutant B7-1/Ig fusion protein that selectively binds to CTLA-4 ameliorates anti-tumor DNA vaccination and counters regulatory T cell activity.

We have shown that a plasmid encoding a B7-1/Ig fusion protein enhanced DNA vaccination against human carcinoembryonic antigen (CEA) more effectively than the plasmid encoding membrane-bound B7-1. However, it was not known if B7-1/Ig acted only by binding CD28 (amplifying a stimulatory signal) or by blocking CTLA-4 on T cells (removing inhibitory signals). Here, we aimed to determine this using a plasmid encoding mutant B7-1/Ig (B7-1wa/Ig), which binds only to CTLA-4 but not to CD28. Our results showed that both the B7-1/Ig and B7-1wa/Ig plasmids, when co-administered with a CEA plasmid, enhanced tumor rejection and the in vitro anti-CEA response. Therefore, B7-1wa/Ig ameliorates DNA vaccination, presumably by binding to CTLA-4. This could result from a number of non-exclusive mechanisms, such as a reduced threshold for T-cell activation, or blockade of CTLA-4/B7-mediated tolerogenic signals in DCs or T cells. We found that, in vitro, a significant fraction of CD3/CD28-activated T cells (in the absence of DCs) expressed CTLA-4 and B7-1. Primed T cells of CTLA-4(+)B7-1(+/-) phenotype acted as regulatory T cells by inhibiting IFNgamma production by re-stimulated CTLA-4(-)B7-1(-) cells, and this was reversed by antibodies against IL-10 or TGF-beta1. Both B7-1wa/Ig and CTLA-4/Ig, which bind to CTLA-4 and B7-1/B7-2 respectively, enhanced IFNgamma production, but not the proliferation or IL-4 release in mixed T-cell populations containing these two cell types. In contrast, CTLA-4(-)B7-1(-) T cells produced IFNgamma which was not affected by B7-1wa/Ig or CTLA-4/Ig. These results suggest that blocking of CTLA-4/B7-1 binding in T cell/T cell interactions blocks negative regulatory signals. This might be the mechanism, at least in part, of the enhancement of anti-tumor immunity by the B7-1wa/Ig and B7-1/Ig plasmids.

Plasmids encoding membrane-bound IL-4 or IL-12 strongly costimulate DNA vaccination against carcinoembryonic antigen (CEA).

Vaccination with plasmids encoding an antigen of interest (DNA vaccination) is a new strategy to achieve effective immunization against many agents. DNA vaccination can be ameliorated by co-administration of plasmids encoding a cytokine. Thus far, only plasmids encoding soluble cytokines have been used for this purpose. However, these plasmids can induce release of cytokines into the circulation and could potentially cause many undesirable effects. We undertook this study to determine whether membrane-bound cytokines, which would restrict their localization at the site of administration, can act as immunoadjuvants. We and others have previously shown that plasmids encoding soluble IL-4 and IL-12 are effective adjuvants for DNA vaccination. In this study, we demonstrate that DNA co-vaccination with membrane-bound IL-4 (mbIL-4) or membrane-bound IL-12 (mbIL-12) both enhance anti-CEA immunity, as detected by in vitro and in vivo assays. Mice co-injected with plasmids encoding CEA and either type of membrane-bound cytokine rejected transplanted CEA-positive tumor cells strongly. Notably, unlike secreted IL-4, mbIL-4 was the most effective adjuvant for anti-tumor immunity. This study demonstrates that membrane-bound cytokines are suitable adjuvants for DNA vaccination.

Flexible management of enzymatic digestion improves human islet isolation outcome from sub-optimal donor pancreata.

Worldwide growing interest in reproducing the result of the Edmonton protocol in islet transplantation trials poses the problem of paucity of donors to supply sufficient amount of islets for clinical use. Improved outcomes include finding better ways to obtain higher yields from every donor organ processed and the possibility of extending islet isolation processing to glands of suboptimal quality. In order to optimize enzymatic digestion of marginal donor organs, we have modified the technique of tissue collection following enzymatic digestion of human pancreatic organs, allowing for reduced time of exposure of free islets to warm Liberase trade mark solution. Our results indicate that better controlled exposure to enzyme yields: (i) higher islet numbers; (ii) complete dissociation of all parts of pancreatic tissue; (iii) successful islet harvest from organs otherwise excluded. We also show that by limiting the exposure of free islets to enzyme solution, islet fragmentation and loss of insulin content are reduced. We further support evidence that enzymatic digestion may contribute to impairment of insulin secretory capacity of the islets in vitro during culture.