Human cancer: is it linked to dysfunctional lipid metabolism?

BACKGROUND: Lipid metabolism dysfunction leading to excess fat deposits (obesity) may cause tumor (cancer) development. Both obesity and cancer are the epicenter of important medical issues. Lipid metabolism and cell death/proliferation are controlled by biochemical and molecular pathways involving many proteins, and organelles; alteration in these pathways leads to fat accumulation or tumor growth. Mammalian Krüppel-like factors, KLFs play key roles in both lipid metabolism and tumor development. SCOPE OF REVIEW: Substantial epidemiological and clinical studies have established strong association of obesity with a number of human cancers. However, we need more experimental verification to determine the exact role of this metabolic alteration in the context of tumor development. A clear understanding of molecules, pathways and the mechanisms involved in lipid metabolism and cell death/proliferation will have important implications in pathogenesis, and prevention of these diseases. MAJOR CONCLUSION: The regulatory role of KLFs, in both cell death/proliferation and lipid metabolism suggests a common regulation of both processes. This provides an excellent model for delivering a precise understanding of the mechanisms linking altered expression of KLFs to obesity and tumor development. GENERAL SIGNIFICANCE: Currently, mouse and rats are the models of choice for investigating disease mechanisms and pharmacological therapies but a genetic model is needed for a thorough examination of KLF function in vivo during the development of an organism. The worm Caenorhabditis elegans is an ideal model to study the connectivity between lipid metabolism and cell death/proliferation.

IL-12 immunotherapy of Braf(V600E)-induced papillary thyroid cancer in a mouse model.

Papillary thyroid carcinoma (PTC) accounts for >80% thyroid malignancies, and BRAF(V600E) mutation is frequently found in >40% PTC. Interleukin-12 (IL-12) is a proinflammatory heterodimeric cytokine with strong antitumor activity. It is not known whether IL-12 immunotherapy is effective against Braf(V600E)-induced PTC. In the present study, we investigated the effectiveness of IL-12 immunotherapy against Braf(V600E)-induced PTC in LSL-Braf(V600E)/TPO-Cre mice. LSL-Braf(V600E)/TPO-Cre mice were created for thyroid-specific expression of Braf(V600E) under the endogenous Braf promoter, and spontaneous PTC developed at about 5 weeks of age. The mice were subjected to two treatment regimens: (1) weekly intramuscular injection of 50 µg plasmid DNA expressing a single-chain IL-12 fusion protein (scIL-12/CMVpDNA), (2) daily intraperitoneal injection of mouse recombinant IL-12 protein (mrIL-12, 100 ng per day). The role of T cells, natural killer (NK) cells, and transforming growth factor-ß (TGF-ß) in IL-12-mediated antitumor effects was determined by a (51)Cr-release cytotoxicity assay. Tumor size and weight were significantly reduced by either weekly intramuscular injection of scIL-12/CMVpDNA or daily intraperitoneal injection of mrIL-12, and tumor became more localized. Survival was significantly increased when treatment started at 1 week of age as compared with that at the 6 weeks of age. Both NK and CD8(+) T cells were involved in the cytotoxicity against tumor cells and their antitumor activity was significantly reduced in tumor-bearing mice. TGF-ß also inhibited the antitumor activity of NK and CD8(+) T cells. The immune suppression was completely reversed by IL-12 treatment and partially recovered by anti-TGF-ß antibody. We conclude that both IL-12 gene therapy and recombinant protein therapy are effective against PTC. Given that the immune response is significantly suppressed in tumor-bearing mice and can be restored by IL-12, the current study raises a possibility of the application of IL-12 as an adjuvant therapy for thyroid cancer.

KRAS(G12D)-mediated oncogenic transformation of thyroid follicular cells requires long-term TSH stimulation and is regulated by SPRY1.

KRAS(G12D) can cause lung cancer rapidly, but is not sufficient to induce thyroid cancer. It is not clear whether long-term serum thyroid stimulating hormone (TSH) stimulation can promote KRAS(G12D)-mediated thyroid follicular cell transformation. In the present study, we investigated the effect of long-term TSH stimulation in KRAS(G12D) knock-in mice and the role of Sprouty1 (SPRY1) in KRAS(G12D)-mediated signaling. We used TPO-KRAS(G12D) mice for thyroid-specific expression of KRAS(G12D) under the endogenous KRAS promoter. Twenty TPO-KRAS(G12D) mice were given anti-thyroid drug propylthiouracil (PTU, 0.1% w/v) in drinking water to induce serum TSH and 20 mice were without PTU treatment. Equal number of wild-type littermates (TPO-KRAS(WT)) was given the same treatment. The expression of SPRY1, a negative regulator of receptor tyrosine kinase (RTK) signaling, was analyzed in both KRAS(G12D)-and BRAF(V600E)-induced thyroid cancers. Without PTU treatment, only mild thyroid enlargement and hyperplasia were observed in TPO-KRAS(G12D) mice. With PTU treatment, significant thyroid enlargement and hyperplasia occurred in both TPO-KRAS(G12D) and TPO-KRAS(WT) littermates. Thyroids from TPO-KRAS(G12D) mice were six times larger than TPO-KRAS(WT) littermates. Distinct thyroid histology was found between TPO-KRAS(G12D) and TPO-KRAS(WT) mice: thyroid from TPO-KRAS(G12D) mice showed hyperplasia with well-maintained follicular architecture whereas in TPO-KRAS(WT) mice this structure was replaced by papillary hyperplasia. Among 10 TPO-KRAS(G12D) mice monitored for 14 months, two developed follicular thyroid cancer (FTC), one with pulmonary metastasis. Differential SPRY1 expression was demonstrated: increased in FTC and reduced in papillary thyroid cancer (PTC). The increased SPRY1 expression in FTC promoted TSH-RAS signaling through PI3K/AKT pathway whereas downregulation of SPRY1 by BRAF(V600E) in PTC resulted in both MAPK and PI3K/AKT activation. We conclude that chronic TSH stimulation can enhance KRAS(G12D)-mediated oncogenesis, leading to FTC. SPRY1 may function as a molecular switch to control MAPK signaling and its downregulation by BRAF(V600E) favors PTC development.

Identification of the tetraspanin CD82 as a new barrier to xenotransplantation.

Significant immunological obstacles are to be negotiated before xenotransplantation becomes a clinical reality. An initial rejection of transplanted vascularized xenograft is attributed to Galα1,3Galß1,4GlcNAc-R (Galα1,3-Gal)-dependent and -independent mechanisms. Hitherto, no receptor molecule has been identified that could account for Galα1,3-Gal-independent rejection. In this study, we identify the tetraspanin CD82 as a receptor molecule for the Galα1,3-Gal-independent mechanism. We demonstrate that, in contrast to human undifferentiated myeloid cell lines, differentiated cell lines are capable of recognizing xenogeneic porcine aortic endothelial cells in a calcium-dependent manner. Transcriptome-wide analysis to identify the differentially expressed transcripts in these cells revealed that the most likely candidate of the Galα1,3-Gal-independent recognition moiety is the tetraspanin CD82. Abs to CD82 inhibited the calcium response and the subsequent activation invoked by xenogeneic encounter. Our data identify CD82 on innate immune cells as a major "xenogenicity sensor" and open new avenues of intervention to making xenotransplantation a clinical reality.

Partners in diabetes epidemic: A global perspective.

There is a recent increase in the worldwide prevalence of both obesity and diabetes. In this review we assessed insulin signaling, genetics, environment, lipid metabolism dysfunction and mitochondria as the major determinants in diabetes and to identify the potential mechanism of gut microbiota in diabetes diseases. We searched relevant articles, which have key information from laboratory experiments, epidemiological evidence, clinical trials, experimental models, meta-analysis and review articles, in PubMed, MEDLINE, EMBASE, Google scholars and Cochrane Controlled Trial Database. We selected 144 full-length articles that met our inclusion and exclusion criteria for complete assessment. We have briefly discussed these associations, challenges, and the need for further research to manage and treat diabetes more efficiently. Diabetes involves the complex network of physiological dysfunction that can be attributed to insulin signaling, genetics, environment, obesity, mitochondria and stress. In recent years, there are intriguing findings regarding gut microbiome as the important regulator of diabetes. Valid approaches are necessary for speeding medical advances but we should find a solution sooner given the burden of the metabolic disorder - What we need is a collaborative venture that may involve laboratories both in academia and industries for the scientific progress and its application for the diabetes control.

The immunoglobulin A isotype of the Arabian camel (Camelus dromedarius) preserves the dualistic structure of unconventional single-domain and canonical heavy chains.

Introduction: The evolution of adaptive immunity in Camelidae resulted in the concurrent expression of classic heterotetrameric and unconventional homodimeric heavy chain-only IgG antibodies. Heavy chain-only IgG bears a single variable domain and lacks the constant heavy (CH) γ1 domain required for pairing with the light chain. It has not been reported whether this distinctive feature of IgG is also observed in the IgA isotype. Methods: Gene-specific primers were used to generate an IgA heavy chain cDNA library derived from RNA extracted from the dromedary's third eyelid where isolated lymphoid follicles and plasma cells abound at inductive and effector sites, respectively. Results: Majority of the cDNA clones revealed hallmarks of heavy chain-only antibodies, i.e. camelid-specific amino acid substitutions in framework region 1 and 2, broad length distribution of complementarity determining region 3, and the absence of the CHα1 domain. In a few clones, however, the cDNA of the canonical IgA heavy chain was amplified which included the CHα1 domain, analogous to CHγ1 domain in IgG1 subclass. Moreover, we noticed a short, proline-rich hinge, and, at the N-terminal end of the CHα3 domain, a unique, camelid-specific pentapeptide of undetermined function, designated as the inter-α region. Immunoblots using rabbit anti-camel IgA antibodies raised against CHα2 and CHα3 domains as well as the inter-α region revealed the expression of a ~52 kDa and a ~60 kDa IgA species, corresponding to unconventional and canonical IgA heavy chain, respectively, in the third eyelid, trachea, small and large intestine. In contrast, the leporine anti-CHα1 antibody detected canonical, but not unconventional IgA heavy chain, in all the examined tissues, milk, and serum, in addition to another hitherto unexplored species of ~45 kDa in milk and serum. Immunohistology using anti-CHα domain antibodies confirmed the expression of both variants of IgA heavy chains in plasma cells in the third eyelid's lacrimal gland, conjunctiva, tracheal and intestinal mucosa. Conclusion: We found that in the dromedary, the IgA isotype has expanded the immunoglobulin repertoire by co-expressing unconventional and canonical IgA heavy chains, comparable to the IgG class, thus underscoring the crucial role of heavy chain-only antibodies not only in circulation but also at the mucosal frontiers.

Molecular characterization of a novel p.R118C mutation in the insulin receptor gene from patients with severe insulin resistance.

CONTEXT: Mutations of the insulin receptor gene (INSR) can cause genetic syndromes associated with severe insulin resistance. OBJECTIVES: We aimed to analyse INSR mutations in Saudi patients with severe insulin resistance. DESIGN: Ten patients with Type A insulin resistance syndrome from five unrelated Saudi families were investigated. The entire coding region of INSR was sequenced. The founder effect was assessed by microsatellite haplotype analysis. The functional effect of the mutation was investigated by in vitro functional assays. RESULTS: A novel biallelic c.433 C>T (p.R118C) mutation was detected in all patients. The c.433 C>T (p.R118C) sequence variation was not found in 100 population controls. The arginine residue at position 118 is located in the ligand-binding domain of INSR and is highly conserved across species. Microsatellite haplotype analysis of these patients indicated that p.R118C was a founder mutation created approximately 2900 years ago. The wild-type and mutant (R118C) INSR were cloned and expressed in CHO cells for functional analysis. Specific insulin binding to the mutant receptor was reduced by 83% as compared to wild-type (WT), although the mutant receptor was processed and expressed on the cell surface. Insulin-mediated receptor autophosphorylation was also significantly reduced in CHO(R118C) cells. CONCLUSIONS: Biallelic c.433 C>T (p.R118C) mutation of INSR causes significant damage to insulin binding and insulin-mediated signal transduction. p.R118C is a founder mutation frequently present in the Saudi patients with severe insulin resistance.

Transcriptomal Insights of Heart Failure from Normality to Recovery.

Current management of heart failure (HF) is centred on modulating the progression of symptoms and severity of left ventricular dysfunction. However, specific understandings of genetic and molecular targets are needed for more precise treatments. To attain a clearer picture of this, we studied transcriptome changes in a chronic progressive HF model. Fifteen sheep (Ovis aries) underwent supracoronary aortic banding using an inflatable cuff. Controlled and progressive induction of pressure overload in the LV was monitored by echocardiography. Endomyocardial biopsies were collected throughout the development of LV failure (LVF) and during the stage of recovery. RNA-seq data were analysed using the PANTHER database, Metascape, and DisGeNET to annotate the gene expression for functional ontologies. Echocardiography revealed distinct clinical differences between the progressive stages of hypertrophy, dilatation, and failure. A unique set of transcript expressions in each stage was identified, despite an overlap of gene expression. The removal of pressure overload allowed the LV to recover functionally. Compared to the control stage, there were a total of 256 genes significantly changed in their expression in failure, 210 genes in hypertrophy, and 73 genes in dilatation. Gene expression in the recovery stage was comparable with the control stage with a well-noted improvement in LV function. RNA-seq revealed the expression of genes in each stage that are not reported in cardiovascular pathology. We identified genes that may be potentially involved in the aetiology of progressive stages of HF, and that may provide future targets for its management.

Toward allogenizing a xenograft: Xenogeneic cardiac scaffolds recellularized with human-induced pluripotent stem cells do not activate human naïve neutrophils.

The limited availability of human donor organs suitable for transplantation has resulted in ever-increasing patient waiting lists globally. Xenotransplantation is considered a potential option, but is yet to reach clinical practice. Although remarkable progress has been made in overcoming immunological rejection, issues with functionality are still to be resolved. Bioengineering approaches have been used to create cardiac tissues with optimized functions. The use of decellularized xenogeneic cardiac tissues seeded with donor-derived cardiac cells may prove to be a viable strategy as supporting structures of the native tissue such as vasculature can be utilized. Here we used sequential perfusion to decellularize adult rat hearts. The acellular scaffolds were reseeded with human endothelial cells, human fibroblasts, human mesenchymal stem cells, and cardiac cells derived from human-induced pluripotent stem cells. The ability of the resultant recellularized rat scaffolds to activate human naïve neutrophils in vitro was investigated to measure xenogeneic recognition. Our results demonstrate that in contrast to cadaveric xenogeneic hearts, acellular and recellularized xenogeneic scaffolds did not activate human naïve neutrophils and suggest that decellularization removes the xenogeneic antigens that lead to human naïve neutrophil activation thus allowing human cells to populate the now "allogenized" xenogeneic scaffolds.

Recombinant Ov-ASP-1, a Th1-biased protein adjuvant derived from the helminth Onchocerca volvulus, can directly bind and activate antigen-presenting cells.

We previously reported that rOv-ASP-1, a recombinant Onchocerca volvulus activation associated protein-1, was a potent adjuvant for recombinant protein or synthetic peptide-based Ags. In this study, we further evaluated the adjuvanticity of rOv-ASP-1 and explored its mechanism of action. Consistently, recombinant full-length spike protein of SARS-CoV or its receptor-binding domain in the presence of rOv-ASP-1 could effectively induce a mixed but Th1-skewed immune response in immunized mice. It appears that rOv-ASP-1 primarily bound to the APCs among human PBMCs and triggered Th1-biased proinflammatory cytokine production probably via the activation of monocyte-derived dendritic cells and the TLR, TLR2, and TLR4, thus suggesting that rOv-ASP-1 is a novel potent innate adjuvant.

Intracellular calcium and NF-kB regulate hypoxia-induced leptin, VEGF, IL-6 and adiponectin secretion in human adipocytes.

AIMS: Hypoxia-induced adipokine release has been attributed mainly to HIF-1α. Here we investigate the role of intracellular calcium and NF-kB in the hypoxia-dependent release of leptin, VEGF, IL-6 and the hypoxia-induced inhibition of adiponectin release in human adipocytes. MAIN METHODS: We used intracellular calcium imaging to compare calcium status in preadipocytes and in adipocytes. We subjected both cell types to hypoxic conditions and measured the release of adipokines induced by hypoxia in the presence and absence of HIF-1α inhibitor YC-1, NF-κB inhibitor SN50 and intracellular calcium chelator BAPTA-AM. KEY FINDINGS: We demonstrate reduced intracellular calcium oscillations and increased oxidative stress as the cells transitioned from preadipocytes to adipocytes. We show that differentiation of preadipocytes to adipocytes is associated with distinct morphological changes in the mitochondria. We also show that hypoxia-induced secretion of leptin, VEGF, IL-6 and hypoxia-induced inhibition of adiponectin secretion are independent of HIF-1α expression. The hypoxia-induced leptin, VEGF and IL-6 release are [Ca++]i dependent whereas adiponectin is NF-kB dependent. SIGNIFICANCE: Our work suggests a major role for [Ca++]i in preadipocyte differentiation to adipocytes and that changes in mitochondrial morphology in the adipocytes might underlie the reduced calcium oscillations observed in the adipocytes. It also demonstrates that multiple signaling pathways are associated with the hypoxia-induced adipokine secretion.

Data on hypoxia-induced VEGF, leptin and NF-kB p65 expression.

The data set presented here is associated with the article "Intracellular calcium and NF-kB regulate hypoxia-induced leptin, VEGF, IL-6 and adiponectin secretion in human adipocytes" (Al-Anazi et al., 2018). Data illustrate hypoxia-induced VEGF and leptin expression in human adipocytes treated with the calcium chelator BAPTA-AM (1 µM). It also shows NF-κB p65 induced expression by hypoxia. Preadipocytes were differentiated for 14 days and then subjected to 0.5-1.5% oxygen in the presence and absence of BAPTA-AM or the NF-κB inhibitor SN50 for 48 h prior to RNA isolation and PCR analysis.

Cyp24a1 Attenuation Limits Progression of BrafV600E -Induced Papillary Thyroid Cancer Cells and Sensitizes Them to BRAFV600E Inhibitor PLX4720.

CYP24A1, the primary inactivating enzyme for vitamin D, is often overexpressed in human cancers, potentially neutralizing the antitumor effects of calcitriol, the active form of vitamin D. However, it is unclear whether CYP24A1 expression serves as a functional contributor versus only a biomarker for tumor progression. In this study, we investigated the role of CYP24A1 on malignant progression of a murine model of BrafV600E -induced papillary thyroid cancer (PTC). Mice harboring wild-type Cyp24a1 (BVECyp24a1-wt) developed PTC at 5 weeks of age. Mice harboring a homozygous deletion of Cyp24a1 (BVECyp24a1-null) exhibited a 4-fold reduction in tumor growth. Notably, we found the tumorigenic potential of BVECyp24a1-null-derived tumor cells to be nearly abolished in immunocompromised nude mice. This phenotype was associated with downregulation of the MAPK, PI3K/Akt, and TGFß signaling pathways and a loss of epithelial-mesenchymal transition (EMT) in BVECyp24a1-null cells, associated with downregulation of genes involved in EMT, tumor invasion, and metastasis. While calcitriol treatment did not decrease cell proliferation in BVECyp24a1-null cells, it strengthened antitumor responses to the BRAFV600E inhibitor PLX4720 in both BVECyp24a1-null and BVECyp24a1-wt cells. Our findings offer direct evidence that Cyp24a1 functions as an oncogene in PTC, where its overexpression activates multiple signaling cascades to promote malignant progression and resistance to PLX4720 treatment. Cancer Res; 77(8); 2161-72. ©2017 AACR.

Defective lipid metabolism associated with mutation in klf-2 and klf-3: important roles of essential dietary salts in fat storage.

BACKGROUND: Dietary salts are important factors in metabolic disorders. They are vital components of enzymes, vitamins, hormones, and signal transduction that act synergistically to regulate lipid metabolism. Our previous studies have identified that Krüppel-like factor -3 (KLF-3) is an essential regulator of lipid metabolism. However, it is not known if KLF-2 also regulates lipid metabolism and whether KLF-2 and -3 mediate the effects of dietary salts on lipid metabolism. METHODS: In this study, we used klf mutants [homozygous klf-2 (ok1043) V and klf-3 (ok1975) II mutants] to investigate the role of dietary salts in lipid metabolism. All gene expression was quantified by qRT-PCR. Localization of KLF-2 was analyzed by the expression of klf-2::gfp (in pPD95.75 vector) using a fluorescent microscope. Fat storage was measured by Oil Red O staining. RESULTS: Klf-2 was identified to express in the intestine during all stages of Caenorhabditis elegans development with peak expression at L3 stage. Mutation of klf-2 increased fat accumulation. Under regular growth media free of Ca2+, the expression of both klf-2 and -3 was inhibited slightly; further their expression reduced significantly in WT worms fed on 10X Ca2+ diet. When klf-3 was mutated, the expression of klf-2 increased under 10X Ca2+ diet; but when klf-2 was mutated, the expression of klf-3 was not altered under 10X Ca2+ diet. Overall, Mg2+ and K+ were less effective on the gene expression of klfs. KLF target gene Ce-C/EBP-2 showed elevated expression in WT and klf-3 (ok1975) worms with changed Ca2+ concentrations but not in klf-2 (ok1043) worms. However, high Ca+2 diet exhibited inhibitory effect on Ce-SREBP expression in WT worms. CONCLUSION: Dietary Ca2+ is most effective on fat storage and klf-2 expression, wherein high Ca2+ diet decreased klf-2 expression and reduced fat buildup. Mechanistic study identified Ce-C/EBP (C48E7.3; lpd-2) and Ce-SREBP (Y47D3B.7; lpd-1) as the target genes of klf-2 and/or klf-3 to mediate lipid metabolism. This study identifies a new function of klf-2 in inhibiting fat buildup and reveals the interplay between dietary salts and klf-2 and klf-3 in lipid metabolism.

Rapid static adhesion of human naïve neutrophil to naïve xenoendothelium under physiologic flow is independent of Galalpha1,3-gal structures.

Adhesion interactions under flow have long been known to depend on applied wall shear stress. We investigated the ability of human naïve neutrophils to adhere to xenogeneic endothelial cells under static and flow conditions. We demonstrate that human naïve neutrophils bind to xenogeneic endothelial cells under flow conditions. This binding is dependent on the applied stress and is independent of Galalpha1,3-gal structures, ICAM-1, or its counter ligands LFA-1alpha and Mac-1. The binding was rapid and is characterized by stationary attachment with no obvious rolling or change in morphology. This binding leads to a transient increase in intracellular-free calcium levels in xenogeneic but not allogeneic-endothelial cells with occasional oscillations that persist long after the initial contact between the two cell types. Previous activation of xenoendothelium by autologous serum or human TNF-alpha augments binding of human naïve neutrophils to the endothelial cells. Our data suggest novel interaction sites between the xenogeneic endothelial cells and human naïve neutrophils.

IL-12-dependent nuclear factor-kappaB activation leads to de novo synthesis and release of IL-8 and TNF-alpha in human neutrophils.

The cytokine interleukin (IL)-12 plays a bridging role between innate and adaptive immunity. Here, we demonstrate that treatment of neutrophils with IL-12 leads to a transient increase in intracellular-free calcium [Ca(+)(+)](i) levels, which is necessary for the production of reactive oxygen metabolites (ROM). This production is associated with the activation and nuclear translocation of the transcription factor nuclear factor (NF)-kappaB and is inhibited in the presence of the intracellular calcium chelator 1,2-bis(O-amminophenoxy) ethane-N,N-N',N'-tetraacetic acid-acetoxymethyl ester and the ROM production inhibitor diphenyl iodonium. We show that IL-12 causes a significant increase in total mRNA levels, which appear dependent on the generated ROM. In addition IL-12 induces the de novo synthesis and production of IL-8 and tumor necrosis factor alpha (TNF-alpha) in a calcium- and ROM-dependent manner. Our data demonstrate a direct role for IL-12 in the activation of human neutrophils and suggest a ROM-dependent interplay between IL-12-induced [Ca(+)(+)](i) transient and the release of IL-8 and TNF-alpha through NF-kappaB activation.

RAGE-mediated neutrophil dysfunction is evoked by advanced glycation end products (AGEs).

The accumulation of advanced glycation end products (AGEs) in the tissue and serum of subjects with diabetes has been linked to the pathogenesis of vascular complications. Because diabetes may be also complicated by increased susceptibility to recurrent infection, we investigated the effects of AGEs on human neutrophils, because their burst of activity immediately upon engagement of pathogens or other inflammatory triggers is critical to host response. We demonstrate the presence of receptor for advanced glycation end products (RAGE) at the message and protein levels. We also demonstrate that AGE albumin (but not control albumin) binds with high affinity to human neutrophils (K(d) of 3.7 +/- 0.4 nM). The binding was blocked almost completely by excess soluble RAGE, anti-RAGE antibodies, or antibodies to CML-modified albumin. AGE albumin induced a dose-dependent increase in intracellular-free calcium as well as actin polymerization. Further, AGE albumin inhibited transendothelial migration and Staphylococcus aureus-induced but not fMLP-induced production of reactive oxygen metabolite. Moreover, although AGE albumin enhanced neutrophil phagocytosis of S. aureus, it inhibited bacterial killing. We conclude that functional RAGE is present on the plasma membrane of human neutrophils and is linked to Ca(2)(+) and actin polymerization, and engagement of RAGE impairs neutrophil functions.

Progression of matrixin and cardiokine expression patterns in an ovine model of heart failure and recovery.

BACKGROUND: The molecular mechanisms underlying the geometrical changes of the left ventricle during the progression to heart failure and recovery are not well defined. OBJECTIVE: Here we investigate the involvement of matrixins and cardiokines in an ovine model of pressure-induced left ventricular failure (LVF). METHODS: Fifteen sheep underwent supracoronary aortic banding with an inflatable cuff. A controlled and progressive increase of LV pressure was monitored echocardiographically. Endomyocardial biopsies were collected throughout the development of LVF and subsequent recovery after pressure unloading. RESULTS: Thirteen sheep developed LVF with a subsequent recovery. Peak left ventricular hypertrophy (LVH) and dilatation (LVD) occurred at 31.5 ± 1.6 weeks and 102.7 ± 2.2 weeks post-banding respectively, with an increase in LV internal diameter in diastole (LVIDd 5.11 ± 0.12 compared to the control 3.37 ± 0.07 cm, p<0.001), with preserved LV ejection fraction (LVEF). Reduced LVEF became evident 116.5 ± 2.7 weeks post-banding. Clinical and echocardiographic improvements were observed following deflation of the aortic banding cuff. By 138.1 ± 3.1 weeks cardiac performance recovered with restoration of LVEF. Significant changes in the expression of matrix metalloproteinases (MMP)-1, -2, -3, vascular endothelial cell growth factor (VEGF), fibroblast growth factor (FGF)-2, interferon (INF)-α-2 and soluble CD40 ligand (sCD40L) were observed throughout the progression to failure and recovery. CONCLUSIONS: We used an ovine model to study reversible LV remodelling without interruption and found significant changes in matrixin and cardiokine expression during LV progression to failure and recovery.

Gene therapy of anaplastic thyroid carcinoma with a single-chain interleukin-12 fusion protein.

Anaplastic thyroid carcinoma is the most aggressive type of thyroid malignancy with a mean survival time of less than 8 months. No effective therapeutic approach is currently available, making the development of novel treatments necessary. Interleukin (IL)-12 is a proinflammatory heterodimeric cytokine with strong antitumor activity. In the present study, we investigated the potential of IL-12 gene therapy for anaplastic thyroid carcinoma in BALB/c (nu/nu) nude mice. A single-chain IL-12 fusion protein construct was created to assure equal expression of its p35 and p40 subunits. Human anaplastic thyroid carcinoma cell line ARO was stably transfected with an IL-12 expression plasmid under the control of cytomegalovirus (CMV) promoter (scIL-12/CMVpDNA). High levels of functional IL-12 (26.78 +/- 4.11 ng/ml per 10(6) cells per 48 hr) were produced by scIL-12-transfected ARO cells (ARO/IL-12). Tumorigenicity in nude mice was completely lost in scIL-12-transfected ARO cells, as demonstrated by the lack of tumor formation after subcutaneous injection of 2 x 10(6) ARO/IL-12 cells, even though there was no difference in cell proliferation between ARO and ARO/IL-12 cells. Tumor growth was observed after challenge with ARO tumor cells, indicating that protective immunity had not developed against the parental cells. Furthermore, the growth rate of established subcutaneous ARO tumors was significantly reduced by either subcutaneous injection of 2 x 10(6) ARO/IL-12 cells weekly or intramuscular injection of 50 microg scIL-12/CMVpDNA twice weekly. The antineoplastic activity of ARO/IL-12 cells was, however, abrogated by intraperitoneal injection of anti-natural killer (NK) cell antibody. Moreover, significantly higher number of ARO/IL-12 cells and ARO cells were killed by splenocytes from nude mice previously treated with ARO/IL-12 compared to those treated with ARO cells (32% vs. 9% when ARO were used as target cells, 43% vs. 17% when ARO/IL12 were used as target cells; p < 0.01) in an in vitro cytotoxicity assay. Again, tumor cell killing was neutralized by the addition of anti-NK cell antibody in the assay. In conclusion, we have demonstrated successful gene therapy with a scIL-12 fusion protein against anaplastic thyroid carcinoma in an in vivo model. The immune response against ARO/IL-12 cells is mediated by NK cells. These results may set the stage for clinical application of IL-12 gene therapy for poorly differentiated thyroid carcinoma.

Microarray analysis of metastasis-associated gene expression profiling in a murine model of thyroid carcinoma pulmonary metastasis: identification of S100A4 (Mts1) gene overexpression as a poor prognostic marker for thyroid carcinoma.

Tumor cell invasion and metastasis are the hallmark of malignant neoplasm. Despite advances in the management of thyroid carcinoma and other solid tumors, metastasis continues to be the most significant cause in cancer mortality. To gain new insights into this complex process in thyroid carcinoma, we established a thyroid carcinoma cell line (ARO-met2) with high metastatic capacity to the lung by sequential passage of a human anaplastic thyroid cancer cell line (ARO) through the lung of a nude mouse. Global patterns of gene expression were analyzed in cells of the parental ARO and the ARO-met2, using Atlas human cancer 1.2 array with 1176 cancer-related genes. In total, 184 genes were differentially expressed more than 1.5 times, and 64 genes were differentially expressed over two times. Among those 64 genes, 43 were overexpressed, and 21 genes were underexpressed. Many genes whose increased expression was thought to be related to tumor progression were identified, such as c-Met, ezrin, integrin, motility-related protein-1, cadherin, and S100A4. The most highly expressed gene is the S100A4 (8-fold higher than control), which is a member of a small calcium binding protein family and is involved in the cell proliferation and cancer progression. The S100A4 overexpression in the ARO-met2 cells was later confirmed by Northern blot and real-time reverse transcriptase-PCR. Analysis of 49 thyroid tumor specimens by real-time reverse transcriptase-PCR (eight benign goiters, 36 papillary, and five anaplastic carcinomas) revealed that S100A4 overexpression was present in most advanced thyroid carcinomas and lymph node metastases, and was associated with poor prognosis. None of the benign goiters was found to have S100A4 overexpression. These data suggest that S100A4 could be used as a prognostic marker for thyroid carcinoma. Given that S100A4 is involved in tumor progression and metastasis, it may be a potential target for therapeutic intervention.