The role of antibody responses against glycans in bioprosthetic heart valve calcification and deterioration.

Bioprosthetic heart valves (BHVs) are commonly used to replace severely diseased heart valves but their susceptibility to structural valve degeneration (SVD) limits their use in young patients. We hypothesized that antibodies against immunogenic glycans present on BHVs, particularly antibodies against the xenoantigens galactose-α1,3-galactose (αGal) and N-glycolylneuraminic acid (Neu5Gc), could mediate their deterioration through calcification. We established a large longitudinal prospective international cohort of patients (n = 1668, 34 ± 43 months of follow-up (0.1-182); 4,998 blood samples) to investigate the hemodynamics and immune responses associated with BHVs up to 15 years after aortic valve replacement. Early signs of SVD appeared in <5% of BHV recipients within 2 years. The levels of both anti-αGal and anti-Neu5Gc IgGs significantly increased one month after BHV implantation. The levels of these IgGs declined thereafter but anti-αGal IgG levels declined significantly faster in control patients compared to BHV recipients. Neu5Gc, anti-Neu5Gc IgG and complement deposition were found in calcified BHVs at much higher levels than in calcified native aortic valves. Moreover, in mice, anti-Neu5Gc antibodies were unable to promote calcium deposition on subcutaneously implanted BHV tissue engineered to lack αGal and Neu5Gc antigens. These results indicate that BHVs manufactured using donor tissues deficient in αGal and Neu5Gc could be less prone to immune-mediated deterioration and have improved durability.

Specific Detection of Neu5Gc in Animal Tissues by Immunohistochemistry.

One of the major obstacles in xenotransplantation is tissue expression of the non-human mammalian carbohydrate N-glycolylneuraminic acid (Neu5Gc). This 9-carbon backbone acidic sugar is the hydroxylated form of N-acetylneuraminic acid (Neu5Ac), and both constitute the two most common sialic acid types in mammals. Loss of CMP-Neu5Ac hydroxylase encoding gene in humans dictates the immunogenic nature of Neu5Gc-containing xenografts. Here we describe an immunohistochemistry method for the detection of Neu5Gc in mammalian-derived tissues using affinity-purified chicken anti-Neu5Gc IgY. Specificity is further demonstrated by competitive inhibition with free Neu5Gc or Neu5Gc-glycoproteins, but not with Neu5Ac or Neu5Ac-glycoproteins. This method can be used to evaluate potential Neu5Gc-immunogenicity of xenografts.

Biomimetic Glyconanoparticle Vaccine for Cancer Immunotherapy.

Cancer immunotherapy aims to harness the immune system to combat malignant processes. Transformed cells harbor diverse modifications that lead to formation of neoantigens, including aberrantly expressed cell surface carbohydrates. Targeting tumor-associated carbohydrate antigens (TACA) hold great potential for cancer immunotherapy. N-glycolylneuraminic acid (Neu5Gc) is a dietary non-human immunogenic carbohydrate that accumulates on human cancer cells, thereby generating neoantigens. In mice, passive immunotherapy with anti-Neu5Gc antibodies inhibits growth of Neu5Gc-positive tumors. Here, we designed an active cancer vaccine immunotherapy strategy to target Neu5Gc-positive tumors. We generated biomimetic glyconanoparticles using engineered αGal knockout porcine red blood cells to form nanoghosts (NGs) that either express (NGpos) or lack expression (NGneg) of Neu5Gc-glycoconjugates in their natural context. We demonstrated that optimized immunization of "human-like" Neu5Gc-deficient Cmah-/- mice with NGpos glyconanoparticles induce a strong, diverse and persistent anti-Neu5Gc IgG immune response. The resulting anti-Neu5Gc IgG antibodies were also detected within Neu5Gc-positive tumors and inhibited tumor growth in vivo. Using detailed glycan microarray analysis, we further demonstrate that the kinetics and quality of the immune responses influence the efficacy of the vaccine. These findings reinforce the potential of TACA neoantigens and the dietary non-human sialic acid Neu5Gc, in particular, as immunotherapy targets.

Novel non-invasive early detection of lung cancer using liquid immunobiopsy metabolic activity profiles.

Lung cancer is the leading cause of cancer death worldwide. Survival is largely dependent on the stage of diagnosis: the localized disease has a 5-year survival greater than 55%, whereas, for spread tumors, this rate is only 4%. Therefore, the early detection of lung cancer is key for improving prognosis. In this study, we present an innovative, non-invasive, cancer detection approach based on measurements of the metabolic activity profiles of immune system cells. For each Liquid ImmunoBiopsy test, a 384 multi-well plate is loaded with freshly separated PBMCs, and each well contains 1 of the 16 selected stimulants in several increasing concentrations. The extracellular acidity is measured in both air-open and hermetically-sealed states, using a commercial fluorescence plate reader, for approximately 1.5 h. Both states enable the measurement of real-time accumulation of 'soluble' versus 'volatile' metabolic products, thereby differentiating between oxidative phosphorylation and aerobic glycolysis. The metabolic activity profiles are analyzed for cancer diagnosis by machine-learning tools. We present a diagnostic accuracy study, using a multivariable prediction model to differentiate between lung cancer and control blood samples. The model was developed and tested using a cohort of 200 subjects (100 lung cancer and 100 control subjects), yielding 91% sensitivity and 80% specificity in a 20-fold cross-validation. Our results clearly indicate that the proposed clinical model is suitable for non-invasive early lung cancer diagnosis, and is indifferent to lung cancer stage and histological type.

Vitamin D inhibits development of liver fibrosis in an animal model but cannot ameliorate established cirrhosis.

1,25(OH)2D3, the active form of vitamin D, has an antiproliferative and antifibrotic effect on hepatic stellate cells. Our aim was to investigate the potential of 1,25(OH)2D3 to inhibit the development of liver fibrosis and to ameliorate established fibrosis in vivo. The antifibrotic effect of 1,25(OH)2D3 was investigated in a thioacetamide (TAA) model (as a preventive treatment and as a remedial treatment) and in a bile duct ligation model. In the preventive model, rats received simultaneously intraperitoneum injection of TAA and/or 1,25(OH)2D3 for 10 wk. In the remedial model, rats were treated with TAA for 10 wk and then received 1,25(OH)2D3 or saline for 8 wk. Fibrotic score was determined by Masson staining. Collagen I, α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase-1 (TIMP1), platelet-derived growth factor (PDGF), and transforming growth factor-ß (TGF-ß) expression were measured by Western blot analysis and real-time PCR. Hypercalemia was detected by chemistry measurements. Preventive treatment of 1,25(OH)2D3 significantly suppressed liver fibrosis both macroscopically and microscopically and significantly lowered the fibrotic score of the TAA + 1,25(OH)2D3 group compared with the TAA group. 1,25(OH)2D3 significantly inhibited expression of PDGF and TGF-ß by ∼50% and suppressed the expression of collagen Iα1, TIMP1, and α-SMA by approximately three-, two-, and threefold, respectively. In contrast, 1,25(OH)2D3 was inefficient in amelioration of established liver fibrosis. Administration of 1,25(OH)2D3 to bile duct ligation rats led to a high mortality rate probably caused by hypercalcemia. We conclude that 1,25(OH)2D3 may be considered as a potential preventive treatment in an in vivo model but failed to ameliorate established cirrhosis.

Vitamin D and S-farnesylthiosalicylic acid have a synergistic effect on hepatic stellate cells proliferation.

BACKGROUND: Hepatic stellate cells (HSCs) have a key role in the formation of hepatic fibrosis. The active form of vitamin D, 1,25(OH)2D3, has been found to have antiproliferative and antifibrotic effects in various tissues including liver. Farnesylthiosalicylic acid (FTS), a novel Ras antagonist, was also found to inhibit hepatic fibrosis. AIMS: The purpose of this study was to examine the antiproliferative and antifibrotic effects of the combined treatment of 1,25(OH)2D3 and FTS on primary cultured HSCs. METHODS: Primary HSCs, isolated from rat's livers, were treated with 1,25(OH)2D3, FTS or a combination of both. Proliferation was assessed by bromodeoxyuridine. Expression of p-ERK, ERK, Ras-GTP, total-Ras, CyclinD1 and fibrotic markers was measured by western blotting analysis and real-time PCR. Cytotoxicity was assessed by lactate dehydrogenase method. RESULTS: The combined treatment inhibited HSCs proliferation by threefold. The effect was synergistic and non-cytotoxic. In concordance, the combined treatment suppressed CyclinD1 expression by ~2-fold, whereas 1,25(OH)2D3 or FTS alone showed a significantly lower inhibitory effect. The effect of the combined treatment on CyclinD1 expression was mediated via Ras-GTP and p-ERK signal transduction pathway. The effect on fibrotic markers showed that 1,25(OH)2D3 decreased collagen Iα1 expression by ~40%, FTS by ~50% and the combined treatment by ~60%. 1,25(OH)2D3 inhibited tissue inhibitor of metalloproteinases-1 (TIMP-1) expression by 20%. FTS alone or 1,25(OH)2D3 + FTS inhibited TIMP-1 expression by 60%. FTS inhibited transforming growth factor-ß (TGF-ß) expression by 25%, while 1,25(OH)2D3 had no effect. CONCLUSION: Although the combination of 1,25(OH)2D3 and FTS did not demonstrate an additive antifibrotic effect, it showed a synergistic antiproliferative effect on primary HSCs. Therefore, the combined treatment may have a potential therapeutic value in the initiation of fibrotic process.

Amplified inhibition of stellate cell activation pathways by PPAR-γ, RAR and RXR agonists.

Peroxisome proliferator activator receptors (PPAR) ligands such as 15-Δ12,13-prostaglandin L(2) [PJ] and all trans retinoic acid (ATRA) have been shown to inhibit the development of liver fibrosis. The role of ligands of retinoic X receptor (RXR) and its ligand, 9-cis, is less clear. The purpose of this study was to investigate the effects of combined treatment of the three ligends, PJ, ATRA and 9-cis, on key events during liver fibrosis in rat primary hepatic stellate cells (HSCs). We found that the anti-proliferative effect of the combined treatment of PJ, ATRA and 9-cis on HSCs was additive. Further experiments revealed that this inhibition was due to cell cycle arrest at the G0/G1 phase as demonstrated by FACS analysis. In addition, the combined treatment reduced cyclin D1 expression and increased p21 and p27 protein levels. Furthermore, we found that the three ligands down regulated the phosphorylation of mTOR and p70(S6K). The activation of HSCs was also inhibited by the three ligands as shown by inhibition of vitamin A lipid droplets depletion from HSCs. Studies using real time PCR and western blot analysis showed marked inhibition of collagen Iα1 and αSMA by the combination of the three ligands. These findings suggest that the combined use of PJ, ATRA and 9-cis causes inhibition of cell proliferation by cell cycle arrest and down-regulation of fibrotic markers to a greater extent compared to each of the ligands alone.

Vitamin D inhibits proliferation and profibrotic marker expression in hepatic stellate cells and decreases thioacetamide-induced liver fibrosis in rats.

BACKGROUND AND AIM: Hepatic stellate cells (HSCs) are key participants in liver fibrosis development. 1,25(OH)(2)D(3), the active form of vitamin D, has antiproliferative properties and antifibrotic potential, as well as a role in extracellular matrix and matrix metalloproteinase (MMP) regulation in renal and lung fibrosis. Little is known about the role of 1,25(OH)(2)D(3) in liver and its involvement in liver fibrosis. Therefore, we investigated the antiproliferative and antifibrotic effects of 1,25(OH)(2)D(3) in primary cultured HSCs and in a rat model of liver fibrosis induced by thioacetamide (TAA). METHODS: Primary HSCs were isolated from rats' livers and treated with 1,25(OH)(2)D(3). Proliferation was examined by bromodeoxyuridine. Vitamin D receptor (VDR) expression and several fibrotic markers were detected by western blot analysis and real-time PCR. Collagen Iα1 and MMP-9 promoter activity were measured by luciferase assay. MMP-9 enzymatic activity was investigated by zymography. VDR silencing was performed by sh-RNA. An in vivo study was performed on TAA-induced liver fibrosis model in rats treated with or without 1,25(OH)(2)D(3). The fibrotic score and collagen deposition were determined by Masson and by Sirius red staining. RESULTS: While VDR was highly expressed in quiescent HSCs, its expression decreased up to 40% during activation. Addition of 1,25(OH)(2)D(3) to activated HSCs stimulated VDR expression. 1,25(OH)(2)D(3) suppressed HSC proliferation and cyclin D1 expression by ~50% and tissue inhibitor of metalloproteinase 1 (TIMP-1) by 60% and led to a 40% downregulation of collagen Iα1 expression. Moreover, 1,25(OH)(2)D(3) increased MMP-9 activity by 30%. Silencing VDR by sh-RNA demonstrated that suppression of cyclin D1 and collagen Iα1 protein expression was VDR dependent. Treatment with 1,25(OH)(2)D(3) significantly reduced extracellular matrix deposition and lowered the fibrotic score in TAA-induced liver fibrosis. CONCLUSION: 1,25(OH)(2)D(3) has antiproliferative and antifibrotic effects on liver fibrosis in in vitro and in vivo models and may be considered as having potential therapeutic value.

Transcriptional regulation of the insulin-like growth factor-I receptor gene in breast cancer.

The insulin-like growth factor-I receptor (IGF-IR) has an important role in normal mammary gland growth and morphogenesis. In addition, the IGF-IR has been implicated in the initiation and progression of breast cancer. Previous studies have indicated that acquisition of the malignant phenotype in breast cancer is initially IGF-IR dependent. Most breast cancer-derived cell lines and primary tumors express high levels of IGF-IR mRNA and protein, whereas metastatic stages are usually associated with a decrease in IGF-IR levels. Transcription of the IGF-IR gene is controlled by complex interactions involving DNA-binding and non DNA-binding transcription factors. This review highlights selected examples of tumor suppressors, including BRCA1, p53, and WT1, whose mechanism of action involves regulation of IGF-IR gene expression.

The p53-family members p63 and p73 inhibit insulin-like growth factor-I receptor gene expression in colon cancer cells.

The insulin-like growth factor-I receptor (IGF-IR) has a critical role in malignant transformation. Consistent with its antiapoptotic role, the IGF-IR gene is overexpressed in most types of cancer, including colorectal tumors. The recently identified p53 homologues, p63 and p73, exhibit some of the biological properties of p53, including the ability to transactivate p53-responsive genes and to induce apoptosis. In the present study, we examined the hypothesis that p63/p73 proteins may contribute to colon cancer cell proliferation via mechanism/s that involve regulation of IGF-IR gene expression. Using transient co-expression assays in colon cancer-derived HCT116 cells, we showed that both proteins inhibit IGF-IR promoter activity and endogenous IGF-IR levels in a dose-dependent manner, whereas mutant proteins are significantly impaired in their ability to suppress IGF-IR gene expression. These results are compatible with the notion that disruption of p63/p73-mediated signal transduction pathways in colon cancer may lead to increased IGF-IR gene transcription. In summary, we have identified the IGF-IR gene as a novel downstream target for p63/p73 action.

The WT1 Wilms' tumor suppressor gene product interacts with estrogen receptor-alpha and regulates IGF-I receptor gene transcription in breast cancer cells.

The IGF-I receptor (IGF-IR) has an important role in breast cancer development and progression. Previous studies have suggested that the IGF-IR gene is negatively regulated by a number of transcription factors with tumor suppressor activity, including the Wilms' tumor protein WT1. The present study was aimed at evaluating the hypothesis that IGF-IR gene transcription in breast cancer cells is under inhibitory control by WT1 and, furthermore, that the mechanism of action of WT1 involves functional and physical interactions with estrogen receptor-alpha (ERalpha). Results of transient coexpression experiments showed that all four predominant isoforms of WT1 (including or lacking alternatively spliced exons 5 and 9) repressed IGF-IR promoter activity by 39-49%. To examine the potential interplay between WT1 and ERalpha in control of IGF-IR gene transcription we employed ER-depleted C4 cells that were generated by clonal selection of ER-positive MCF-7 cells that were maintained in estrogen-free conditions. IGF-IR levels in C4 cells were approximately 43% of the values in MCF-7 cells whereas WT1 levels in C4 cells were 4.25-fold higher than in MCF-7. Triple cotransfection experiments using an ERalpha expression vector in the absence or presence of WT1 expression vectors, along with an IGF-IR promoter reporter plasmid, revealed that ERalpha stimulated IGF-IR promoter activity whereas coexpression of WT1 abrogated the effect of ERalpha. In addition, co-immunoprecipitation experiments demonstrated a specific association between WT1 and ERalpha. Combined, our results suggest that WT1 suppresses IGF-IR gene transcription in breast cancer cells via a mechanism that involves protein-protein association with ERalpha. As a result of this interaction, the ability of ERalpha to transactivate the IGF-IR promoter is abrogated. These findings are consistent with a potential tumor suppressor role for WT1 in breast cancer and suggest that WT1 inactivation in tumoral cells may result in deregulated IGF-IR gene expression and enhanced mitogenic activation by locally produced and/or circulating IGFs.

BRCA1-Sp1 interactions in transcriptional regulation of the IGF-IR gene.

The insulin-like growth factor-I receptor (IGF-IR) plays a critical role in breast tumorigenesis and is overexpressed in most primary tumors. BRCA1 is a transcription factor involved in numerous cellular processes, including DNA damage repair, cell growth, and apoptosis. Consistent with its tumor suppressor role, we demonstrated that BRCA1 repressed the activity of co-transfected IGF-IR promoter reporter constructs in a number of breast cancer-derived cell lines. Results of electrophoretic mobility shift assay showed that BRCA1 did not exhibit any specific binding to the IGF-IR promoter, although it prevented binding of Sp1. Co-immunoprecipitation experiments demonstrated that BRCA1 action was associated with specific interaction with Sp1 protein. Furthermore, using a series of glutathione S-transferase-tagged BRCA1 fragments, we mapped the Sp1-binding domain to a segment located between aa 260 and 802. In summary, our data suggest that the IGF-IR gene is a novel downstream target for BRCA1 action.