Pharmacokinetics and lung distribution of a humanized anti-RAGE antibody in wild-type and RAGE-/- mice

A neutralizing antibody to the receptor for the advanced glycation end products (anti-RAGE Ab) was developed as a potential treatment of acute and chronic inflammatory conditions. Previous pharmacology studies demonstrated efficacy of the anti-RAGE antibody in the mouse model of sepsis. We examined pharmacokinetics and lung distribution of [¹²⁵I]anti-RAGE Ab in RAGE-/- and wild-type (129S5) mice following single IV administration. Serum pharmacokinetics of [¹²⁵I]anti-RAGE Ab was similar in RAGE-/- and 129S5 mice, with the total body clearance of 0.3 mL/hr/kg and the elimination half-life of 11-12 days, suggesting the target expression had limited impact on overall elimination of [¹²⁵I]anti-RAGE Ab from mice. [¹²⁵I]Anti-RAGE Ab accumulated in the lung of 129S5 mice, with ~4% of total dose retained in the lung at days 6-27 and the lung AUC0-∞ of ~300% of that in serum. The SDS-PAGE analysis suggested that most of retained lung radioactivity was attributed to intact antibody. No accumulation of radioactivity was observed in the lung of RAGE-/- mice, indicating that lung uptake of [¹²⁵I]anti-RAGE Ab was target-dependent in wild-type mice. These data suggest that the anti-RAGE Ab was able to localize to the site of RAGE expression, the lung, and support the findings in the previous pharmacology studies.     

Receptor for Advanced Glycation End Products (RAGE) Serves a Protective Role during Klebsiella pneumoniae - Induced Pneumonia

Klebsiella species is the second most commonly isolated gram-negative organism in sepsis and a frequent causative pathogen in pneumonia. The receptor for advanced glycation end products (RAGE) is expressed on different cell types and plays a key role in diverse inflammatory responses. We here aimed to investigate the role of RAGE in the host response to Klebsiella (K.) pneumoniae pneumonia and intransally inoculated rage gene deficient (RAGE^-/-) and normal wild-type (Wt) mice with K. pneumoniae. Klebsiella pneumonia resulted in an increased pulmonary expression of RAGE. Furthermore, the high-affinity RAGE ligand high mobility group box-1 was upregulated during K. pneumoniae pneumonia. RAGE deficiency impaired host defense as reflected by a worsened survival, increased bacterial outgrowth and dissemination in RAGE^-/- mice. RAGE^-/- neutrophils showed a diminished phagocytosing capacity of live K. pneumoniae in vitro. Relative to Wt mice, RAGE^-/- mice demonstrated similar lung inflammation, and slightly elevated—if any—cytokine and chemokine levels and unchanged hepatocellular injury. In addition, RAGE^-/- mice displayed an unaltered response to intranasally instilled Klebsiella lipopolysaccharide (LPS) with respect to pulmonary cell recruitment and local release of cytokines and chemokines. These data suggest that (endogenous) RAGE protects against K. pneumoniae pneumonia. Also, they demonstrate that RAGE contributes to an effective antibacterial defense during K. pneumoniae pneumonia, at least partly via its participation in the phagocytic properties of professional granulocytes. Additionally, our results indicate that RAGE is not essential for the induction of a local and systemic inflammatory response to either intact Klebsiella or Klebsiella LPS.     

RAGE mediates vascular injury and inflammation after global cerebral ischemia

The receptor for advanced glycation end products (RAGE) is a multi-ligand receptor involved in a diverse range of pathological conditions. To analyze the roles of RAGE and its decoy receptor, endogenous secretory RAGE (esRAGE), in the global cerebral ischemia, three different mouse cohorts, wild-type, RAGE^−/−, and esRAGE transgenic (Tg) mice were subjected to bilateral common carotid artery occlusion (BCCAO). RT-PCR and immunohistochemical analysis revealed that expression of RAGE was induced in the vascular cells at 12 h, and then in the neurons and glia from 3 to 7 days in the hippocampus after BCCAO. The numbers of surviving neurons in the hippocampal CA1 region were significantly higher in RAGE^−/− and esRAGE Tg mice than those in wild-type mice in the periods between 24 h and 7 days after BCCAO. Lower levels of 3-nitrotyrosine (3-NT) and higher levels of endothelial nitric oxide synthase (eNOS), together with enlarged vascular areas were observed in RAGE^−/− and esRAGE Tg mice at 12 h after BCCAO. In the later periods, expressions of glia-derived inflammatory mediators TNFα and inducible nitric oxide synthase (iNOS) were reduced in RAGE^−/− and esRAGE Tg mice. These results suggest that RAGE may contribute to delayed neuronal death after global cerebral ischemia by enhancing vascular injury and deleterious glia-mediated inflammation.     

ICAM-1 Targeted Nanogels Loaded with Dexamethasone Alleviate Pulmonary Inflammation

Lysozyme dextran nanogels (NG) have great potential in vitro as a drug delivery platform, combining simple chemistry with rapid uptake and cargo release in target cells with “stealth” properties and low toxicity. In this work, we study for the first time the potential of targeted NG as a drug delivery platform in vivo to alleviate acute pulmonary inflammation in animal model of LPS-induced lung injury. NG are targeted to the endothelium via conjugation with an antibody (Ab) directed to Intercellular Adhesion Molecule-1(ICAM-NG), whereas IgG conjugated NG (IgG-NG) are used for control formulations. The amount of Ab conjugated to the NG and distribution in the body after intravenous (IV) injection have been quantitatively analyzed using a tracer isotope-labeled [¹²⁵I]IgG. As a proof of concept, Ab-NG are loaded with dexamethasone, an anti-inflammatory therapeutic, and the drug uptake and release kinetics are measured by HPLC. In vivo studies in mice showed that: i) ICAM-NG accumulates in mouse lungs (∼120% ID/g vs ∼15% ID/g of IgG-NG); and, ii) DEX encapsulated in ICAM-NG, but not in IgG-NG practically blocks LPS-induced overexpression of pro-inflammatory cell adhesion molecules including ICAM-1 in the pulmonary inflammation.     

Knockout of receptor for advanced glycation end‐products attenuates age‐related renal lesions

Pro‐aging effects of endogenous advanced glycation end‐products (AGEs) have been reported, and there is increasing interest in the pro‐inflammatory and ‐fibrotic effects of their binding to RAGE (the main AGE receptor). The role of dietary AGEs in aging remains ill‐defined, but the predominantly renal accumulation of dietary carboxymethyllysine (CML) suggests the kidneys may be particularly affected. We studied the impact of RAGE invalidation and a CML‐enriched diet on renal aging. Two‐month‐old male, wild‐type (WT) and RAGE^?/? C57Bl/6 mice were fed a control or a CML‐enriched diet (200 μg CML/g_food) for 18 months. Compared to controls, we observed higher CML levels in the kidneys of both CML WT and CML RAGE^?/? mice, with a predominantly tubular localization. The CML‐rich diet had no significant impact on the studied renal parameters, whereby only a trend to worsening glomerular sclerosis was detected. Irrespective of diet, RAGE^?/? mice were significantly protected against nephrosclerosis lesions (hyalinosis, tubular atrophy, fibrosis and glomerular sclerosis) and renal senile apolipoprotein A‐II (ApoA‐II) amyloidosis (p < 0.001). A positive linear correlation between sclerosis score and ApoA‐II amyloidosis score (r = 0.92) was observed. Compared with old WT mice, old RAGE^?/? mice exhibited lower expression of inflammation markers and activation of AKT, and greater expression of Sod2 and SIRT1. Overall, nephrosclerosis lesions and senile amyloidosis were significantly reduced in RAGE^?/? mice, indicating a protective effect of RAGE deletion with respect to renal aging. This could be due to reduced inflammation and oxidative stress in RAGE^?/? mice, suggesting RAGE is an important receptor in so‐called inflamm‐aging.     

Comparison of distinct protein isoforms of the receptor for advanced glycation end-products expressed in murine tissues and cell lines

The receptor for advanced glycation end-products (RAGE) is thought to be expressed ubiquitously as various protein isoforms. Our objective was to use Northern blotting, immunoblotting, and sensitivity to N-glycanase digestion to survey RAGE isoforms expressed in cell lines and mouse tissues in order to obtain a more comprehensive view of the RAGE expressome. Pulmonary RAGE mRNA (1.4 kb) was smaller than cell-line and tissue RAGE mRNA (6 kb-10 kb). Three anti-RAGE antibodies that recognized three distinct RAGE epitopes were used for protein studies (N-16, H-300, and αES). Lung expressed three predominant protein isoforms with apparent molecular masses of 45.1, 52.6, and 57.4 kDa (N-16/H-300) and four isoforms at 25.0, 46.9, 52.5, and 54.2 kDa (αES). These isoforms were expressed exclusively in lung. Heart, ileum, and kidney expressed a 44.0-kDa isoform (N-16), whereas aorta and pancreas expressed a 53.3-kDa isoform (αES). Each of these isoforms were absent in tissue extracts prepared from RAGE^−/− mice. Cell lines expressed a 70.0-kDa isoform, and a subset expressed a 30.0-kDa isoform (αES). Lung RAGE appeared to contain two N-linked glycans. Tissue and cell-line RAGE isoforms were completely insensitive to PNGase F digestion. Thus, numerous RAGE protein isoforms are detectable in tissues and cell lines. Canonical transmembrane and soluble RAGE appear to be expressed solely in lung (N-16/H-300). Non-pulmonary tissues and cell lines, regardless of the source tissue, both express distinct RAGE protein isoforms containing the N-terminal N-16 epitope or the αES RAGE epitope encoded by alternate exon 9, but lacking the H-300 epitope. This work was supported by NIH grants R01 GM37631 and GM68481.     

RAGE,Receptor of Advanced Glycation Endoproducts,Negatively Regulates Chondrocytes Differentiation

RAGE, receptor for advanced glycation endoproducts (AGE), has been characterized as an activator of osteoclastgenesis. However, whether RAGE directly regulates chondrocyte proliferation and differentiation is unclear. Here, we show that RAGE has an inhibitory role in chondrocyte differentiation. RAGE expression was observed in chondrocytes from the prehypertrophic to hypertrophic regions. In cultured cells, overexpression of RAGE or dominant-negative-RAGE (DN-RAGE) demonstrated that RAGE inhibited cartilaginous matrix production, while DN-RAGE promoted production. Additionally, RAGE regulated Ihh and Col10a1 negatively but upregulated PTHrP receptor. Ihh promoter analysis and real-time PCR analysis suggested that downregulation of Cdxs was the key for RAGE-induced inhibition of chondrocyte differentiation. Overexpression of the NF-κB inhibitor I-κB-SR inhibited RAGE-induced NF-κB activation, but did not influence inhibition of cartilaginous matrix production by RAGE. The inhibitory action of RAGE was restored by the Rho family GTPases inhibitor Toxin B. Furthermore, inhibitory action on Ihh, Col10a1 and Cdxs was reproduced by constitutively active forms, L63RhoA, L61Rac, and L61Cdc42, but not by I-κB-SR. Cdx1 induced Ihh and Col10a1 expressions and directly interacted with Ihh promoter. Retinoic acid (RA) partially rescued the inhibitory action of RAGE. These data combined suggests that RAGE negatively regulates chondrocyte differentiation at the prehypertrophic stage by modulating NF-κB-independent and Rho family GTPases-dependent mechanisms.     

Diaphanous-1 affects the nanoscale clustering and lateral diffusion of receptor for advanced glycation endproducts (RAGE)

The interactions between the cytoplasmic protein diaphanous-1 (Diaph1) and the receptor for advanced glycation endproducts (RAGE) drive the negative consequences of RAGE signaling in several disease processes. Reported in this work is how Diaph1 affects the nanoscale clustering and diffusion of RAGE measured using super-resolution stochastic optical reconstruction microscopy (STORM) and single particle tracking (SPT). Altering the Diaph1 binding site has a different impact on RAGE diffusion compared to when Diaph1 expression is reduced in HEK293 cells. In cells with reduced Diaph1 expression (RAGE-Diaph1^?/?), the average RAGE diffusion coefficient is increased by 35%. RAGE diffusion is known to be influenced by the dynamics of the actin cytoskeleton. Actin labeling shows that a reduced Diaph1 expression leads to cells with reduced filopodia density and length. In contrast, when two RAGE amino acids that interact with Diaph1 are mutated (RAGE_RQ/AA), the average RAGE diffusion coefficient is decreased by 16%. Since RAGE diffusion is slowed when the interaction between Diaph1 and RAGE is disrupted, the interaction of the two proteins results in faster RAGE diffusion. In both RAGE_RQ/AA and RAGE-Diaph1^?/? cells the number and size of RAGE clusters are decreased compared to cells expressing RAGE and native concentrations of Diaph1. This work shows that Diaph1 has a role in affecting RAGE clusters and diffusion.     

The receptor for advanced glycation end products influences the expression of its S100 protein ligands in melanoma tumors

Recent studies have suggested that the receptor for advanced glycation end products (RAGE) participates in melanoma progression by promoting tumor growth. However, the mechanisms of RAGE activation in melanoma tumors are not clearly understood. To get deeper insights into these mechanisms, we transfected a melanoma cell line, which was established from a human melanoma primary tumor, with RAGE, and studied the effect of RAGE overexpression on cell proliferation and migration in vitro. We observed that overexpression of RAGE in these cells not only resulted in significantly increased migration rates compared to control cells, but also in decreased proliferation rates (Meghnani et al., 2014).In the present study, we compared the growth of xenograft tumors established from RAGE overexpressing WM115 cells, to that of control cells. We observed that when implanted in mice, RAGE overexpressing cells generated tumors faster than control cells. Analysis of protein tumor extracts showed increased levels of the RAGE ligands S100B, S100A2, S100A4, S100A6 and S100A10 in RAGE overexpressing tumors compared to control tumors. We show that the tumor growth was significantly reduced when the mice were treated with anti-RAGE antibodies, suggesting that RAGE, and probably several S100 proteins, were involved in tumor growth. We further demonstrate that the anti-RAGE antibody treatment significantly enhanced the efficacy of the alkylating drug dacarbazine in reducing the growth rate of RAGE overexpressing tumors.     

Diesterified Derivatives of 5-Iodo-2′-Deoxyuridine as Cerebral Tumor Tracers

With the aim to develop beneficial tracers for cerebral tumors, we tested two novel 5-iodo-2′-deoxyuridine (IUdR) derivatives, diesterified at the deoxyribose residue. The substances were designed to enhance the uptake into brain tumor tissue and to prolong the availability in the organism. We synthesized carrier added 5-[¹²⁵I]iodo-3′,5′-di-O-acetyl-2′-deoxyuridine (Ac₂[¹²⁵I]IUdR), 5-[¹²⁵I]iodo-3′,5′-di-O-pivaloyl-2′-deoxyuridine (Piv₂[¹²⁵I]IUdR) and their respective precursor molecules for the first time. HPLC was used for purification and to determine the specific activities. The iodonucleoside tracer were tested for their stability against human thymidine phosphorylase. DNA integration of each tracer was determined in 2 glioma cell lines (Gl261, CRL2397) and in PC12 cells in vitro. In mice, we measured the relative biodistribution and the tracer uptake in grafted brain tumors. Ac₂[¹²⁵I]IUdR, Piv₂[¹²⁵I]IUdR and [¹²⁵I]IUdR (control) were prepared with labeling yields of 31–47% and radiochemical purities of >99% (HPLC). Both diesterified iodonucleoside tracers showed a nearly 100% resistance against degradation by thymidine phosphorylase. Ac₂[¹²⁵I]IUdR and Piv₂[¹²⁵I]IUdR were specifically integrated into the DNA of all tested tumor cell lines but to a less extend than the control [¹²⁵I]IUdR. In mice, 24 h after i.p. injection, brain radioactivity uptakes were in the following order Piv₂[¹²⁵I]IUdR>Ac₂[¹²⁵I]IUdR>[¹²⁵I]IUdR. For Ac₂[¹²⁵I]IUdR we detected lower amounts of radioactivities in the thyroid and stomach, suggesting a higher stability toward deiodination. In mice bearing unilateral graft-induced brain tumors, the uptake ratios of tumor-bearing to healthy hemisphere were 51, 68 and 6 for [¹²⁵I]IUdR, Ac₂[¹²⁵I]IUdR and Piv₂[¹²⁵I]IUdR, respectively. Esterifications of both deoxyribosyl hydroxyl groups of the tumor tracer IUdR lead to advantageous properties regarding uptake into brain tumor tissue and metabolic stability.     

Genetically-determined hyperfunction of the S100B/RAGE axis is a risk factor for aspergillosis in stem cell transplant recipients

Invasive aspergillosis (IA) is a major threat to the successful outcome of hematopoietic stem cell transplantation (HSCT), although individual risk varies considerably. Recent evidence has established a pivotal role for a danger sensing mechanism implicating the S100B/receptor for advanced glycation end products (RAGE) axis in antifungal immunity. The association of selected genetic variants in the S100B/RAGE axis with susceptibility to IA was investigated in 223 consecutive patients undergoing HSCT. Furthermore, studies addressing the functional consequences of these variants were performed. Susceptibility to IA was significantly associated with two distinct polymorphisms in RAGE (-374T/A) and S100B (+427C/T) genes, the relative contribution of each depended on their presence in both transplantation counterparts [patient SNP_RAGE, adjusted hazard ratio (HR), 1.97; P = 0.042 and donor SNP_RAGE, HR, 2.03; P = 0.047] or in donors (SNP_S100B, HR, 3.15; P = 7.8e-⁴) only, respectively. Functional assays demonstrated a gain-of-function phenotype of both variants, as shown by the enhanced expression of inflammatory cytokines in RAGE polymorphic cells and increased S100B secretion in vitro and in vivo in the presence of the S100B polymorphism. These findings point to a relevant role of the danger sensing signaling in human antifungal immunity and highlight a possible contribution of a genetically-determined hyperfunction of the S100B/RAGE axis to susceptibility to IA in the HSCT setting.     

Advanced glycation end products increase endothelial permeability through the RAGE/Rho signaling pathway

Although increased vascular permeability is known to be a major characteristic of diabetic vasculopathy, the precise mechanisms and relevance of advanced glycation end products (AGE) to hyperpermeability of vessels remains unclear. Here, we studied changes in cytoskeletal configuration and the signaling mechanism induced by AGE in human endothelial cells. AGE-BSA stimulation induced Rho activation, intercellular gap formation, prominent actin stress fiber and cell contraction without changing VE-cadherin, and subsequently transendothelial diffusion of FITC-labeled dextran. These processes induced by AGE-BSA were inhibited by either Rho-kinase inhibitor Y27632 or anti-RAGE antibody. We also showed that RhoA and RAGE spontaneously formed a complex. These findings suggest that activation of RAGE/Rho is involved in AGE-BSA-induced hyperpermeability through gap formation and actin reorganization in diabetes.

Structured summary

MINT-7301170: Rhotekin (uniprotkb:Q9BST9) physically interacts (MI:0915) with RhoA (uniprotkb:P61586) by pull down (MI:0096)MINT-7301204, MINT-7301186: RhoA (uniprotkb:P61586) physically interacts (MI:0915) with RAGE (uniprotkb:Q15109) by anti bait coimmunoprecipitation (MI:0006)     

Radioiodinated sunitinib as a potential radiotracer for imaging angiogenesis-radiosynthesis and first radiopharmacological evaluation of 5-[125I]Iodo-sunitinib

Sunitinib® (SU11248) is a highly potent tyrosine kinase inhibitor targeting vascular endothelial growth factor receptor (VEGFR). Radiolabeled inhibitors of RTKs might be useful tools for monitoring RTKs levels in tumour tissue giving valuable information for anti-angiogenic therapy. We report here the synthesis of a ¹²⁵I-labeled derivative of sunitinib® and its first radiopharmaceutical characterization.The non-radioactive reference compound 5-iodo-sunitinib 4 was prepared by Knoevenagel condensation of 5-iodo-oxindole with the corresponding substituted 5-formyl-1H-pyrrole. In a competition binding assay against VEGFR-2 a binding constant (K_d) of 16 nM for 4 was found. The ability of 4 to inhibit tyrosine kinase activity was demonstrated on RTK expressing cells suggesting this radiotracer as a useful tool for monitoring VEGFR expression. 5-[¹²⁵I]lodo-sunitinib, [¹²⁵I]-4 was obtained via destannylation of the corresponding tributylstannyl precursor with [¹²⁵I]NaI in the presence of H₂O₂ in high radiochemical yield (>95%) and radiochemical purity (<98%) after HPLC purification. Determination of human plasma protein binding at time intervals of 0; 1; 2; 4 and 24 h suggested a low non-specific binding of 5-10%. Preliminary biodistribution studies of [¹²⁵I]-4 in healthy CD-1 mice showed a relatively high uptake in VEGFR-2 rich tissues like kidney and lung followed by rapid washout (9.6 and 9.7; 4.5 and 3.8% ID/g of kidney and lung at 1 and 4 h, respectively).     

VPg unlinkase,the phosphodiesterase that hydrolyzes the bond between VPg and picornavirus RNA: a minimal nucleic moiety of the substrate

VPg unlinkase is an unusual eukaryotic enzyme that catalyzes hydrolysis of the phosphodiester bond between residues of the unique tyrosine of VPg (viral protein genome-linked) and the 5"-terminal uridylic acid of picornavirus RNA. Cellular targets of the VPg unlinking enzyme are yet unknown. To determine an essential nucleic part of the covalent linkage unit that is necessary for the VPg unlinkase reaction, the following derivatives of the encephalomyocarditis virus (EMCV) VPg–RNA complex were used: [¹²⁵I]Kp–pUpUpGp, [¹²⁵I]Kp–pUp, and [¹²⁵I]Kp–pU (Kp is residual peptides bound to RNA after proteinase K treatment of VPg–RNA). [¹²⁵I]K-peptides were unlinked from [¹²⁵I]Kp–pUpUpGp and [¹²⁵I]Kp–RNA with similar velocity, but [¹²⁵I]Kp–pUp was split much slower. Under the same conditions [¹²⁵I]Kp–pU was not dissociated at all. Thus, pUp is a minimal part of picornavirus RNA that is necessary for VPg unlinkase. We speculate that cellular substrates of the enzyme are phosphodiesters of oligo(poly)ribonucleotides and tyrosine or tyrosine peptides. In no case [¹²⁵I]VPg–pU, [¹²⁵I]VPg–pUp, and [¹²⁵I]VPg–pUpUpGp were hydrolyzed by VPg unlinkase, in contrast with [¹²⁵I]VPg–RNA and [¹²⁵I]VPg–pUpUpGpApApApGp. We conclude that the whole VPg, when bound to trinucleotide (but not to heptanucleotide), protects the inter-polymeric phosphodiester bond against hydrolysis of the covalent linkage unit. We speculate that VPg unlinkase might repair covalent complexes of RNA and topoisomerases and trigger degradation process of the picornavirus RNA.     

Disposition of125I-labeled recombinant human tumor necrosis factor in the tumor-bearing mouse

Disposition of [¹²⁵I]rHu-TNF was elucidated in BALB/c mice bearing Meth A fibrosarcoma 7 days after transplantation. Afteri.v. administration, [¹²⁵I]rHu-TNF measured by radioactivity and immunoreactivity biphasically decreased in plasma. Tumor level of [¹²⁵I]rHu-TNF was the maximum at 1 h, then decreased and finally remained essentially constant. After i.t. administration, plasma level reached the maximum at 1 h. Tumor level decreased quickly and then became essentially constant. [¹²⁵I]rHu-TNF was suggested to be degraded to small fragments in the tumor. Significant distribution of [¹²⁵I]rHu-TNF was found in the kidney, lung, liver and tumor. Most tissue levels decreased with time in parallel with plasma levels. [¹²⁵I]rHu-TNF radioactivity was found in proximal convoluted tubules of kidney and in those areas of tumor consisting of degenerating cells with pyknotic nuclei. Urine contained most of administered radioactivity, which being neither immunoreactive nor protein-bound.     

The impact of receptor of advanced glycation end-products polymorphisms on prostate cancer progression and clinicopathological characteristics

The receptor for advanced glycation end products (RAGE) overexpression was suggested to be associated with prostate cancer development and poor prognosis. In this study, we focused on the correlations between the clinicopathological characteristics and susceptibility of prostate cancer and RAGE single-nucleotide polymorphisms (SNPs). In 579 prostate cancer patients, the RAGE SNPs rs1800625, rs1800624, rs2070600 and rs184003 in patients with or without grade group upgrade were analysed with real-time polymerase chain reaction. The results demonstrated that the prostate cancer patients who carried the RAGE SNPs rs2070600 ‘GA’ genotypic variants were significantly associated with lower risk to develop grade group upgrade. Moreover, patients with the RAGE rs1800625 ‘TC + CC’ genotypic variants were associated with higher risk of perineural invasion. In 343 prostate cancer patients who carried the RAGE rs1800625 ‘TC + CC’ genotype without grade group upgrade were correlated with higher risk of biochemical recurrence and perineural invasion. In the analysis of TCGA database, significant differences of the RAGE mRNA level were found between the normal controls and prostate cancer patients (p < 0.0001), and the pathologic stage N1 and N0 patients (p = 0.0027). The prostate cancer patients with high RAGE expression were associated with lower overall survival rate (p = 0.025). In conclusion, our results have revealed that the RAGE SNPs rs2070600 and rs1800625 were associated with the grade group upgrade of prostate cancer and clinical status. The RAGE polymorphisms may provide as a pivotal predictor to evaluate prostate cancer disease progression and prognosis.     

Phenolic ring deiodination in cultured rat hepatoma cells, and subcellular localization of deiodinases in cultured rat hepatoma, monkey hepatocarcinoma cells and normal rat liver homogenates

The cultured rat hepatoma cell (R117-21B) homogenates metabolized 3,[3′,5′-¹²⁵I]triiodothyronine by phenolic ring deiodination and produced radioactive iodide and 3,3′-diiodothyronine. Thyroxine (T₄) was converted to 3,3′,5-triidothyronine (T₃). The production of ¹²⁵I⁻ presented the deiodinase activity. The optimal pH for phenolic ring deiodination was observed to be pH 6.0–7.0. This enzyme reaction was accelerated by dithiothreitol. Propylthiouracil strongly inhibited the phenolic ring deiodination at 0.1 mM, whereas an effect of 20 mM methylmercaptoimidazol on the deiodination was very weak or absent.Excess unlabeled iodothyronines (T₄, T₃ and 3,5-diiodo-l-thyronine inhibited the phenolic ring deiodination of labeled 3,3′,5′-triiodothyronine, althought their inhibitory effect was slightly different. Triiodothyroacetic acid was a better inhibitor than T₃. Diiodotyrosine did not affect phenolic ring deiodination in cultured rat hepatoma cell homogenates.Phenolic and nonphenolic ring deiodinase activities of cultured monkey hepatocarcinoma cell and rat liver homogenates were also studied by the use of 3,[3′,5′-¹²⁵I]triiodothyronine and [3,5-¹²⁵I]thyroxine, respectively. Both deiodinase activities were observed in particulate fractions (mitochondrial and microsomal) of cultured cell and rat liver homogenates.     

Anti-RAGE (Receptor Advanced Glycation End products) Antibody Improves Diabetic Retinopathy in Rats via Hypoglycemic and Anti-inflammatory Mechanism

Background:Receptor advanced glycation end products (RAGE) activation plays an essential role in diabetic retinopathy (DR) progression. This study was aimed to explore the role of anti-RAGE antibodies (RAGE antagonists) in inhibiting DR progression through their hypoglycemic and anti-inflammatory mechanism in diabetic retinopathy induced rats.Methods:A total of 30 male Wistar rats were randomly divided into five group. The group was consisted of normal control group, DR group without treatment, DR group with anti-RAGE 1 ηg/kg BW, 10 ηg/kg BW, and 100 ηg/kg BW. To assess the diabetic retinopathy, fundus photographs were taken every week using a camera with 16x magnification placed in front of the rat''s eyes. Blood glucose was checked by the glucose oxidase-peroxidase method. Retinal TNF-α levels and VEGF were examined using an enzyme-linked immunosorbent assay (ELISA) kit.Results:The finding of this study showed that anti-RAGE treatment at dose of 10 and 100 ηg/kg BW, HbA1c levels were significantly higher (p< 0.05) compared to the normal control group but significantly lower (p< 0.05) than in the diabetes group. The mean blood vessel diameter in the DR+anti-RAGE 10 and 100 ηg/kg BW groups was significantly lower than in the diabetic retinopathy group (p< 0.05). The administration of anti-RAGE 10 and 100 ηg/kg BW showed the ability to significantly reduce VEGF levels compared to the DR group (p< 0.05).DiscussionThis study revealed at doses of 10 and 100 ηg/kg BW, anti-RAGE antibodies improved diabetic retinopathy in Wistar rats through hypoglycemic effects and anti-inflammatory mechanisms.Key Words: Anti-RAGE (Receptor Advanced Glycation End products), Diabetic Retinopathy, Glycated Hemoglobin A, Hypoglycemic Agents, Peroxidases, Vascular Endothelial Growth Factor A     

Imaging Axl expression in pancreatic and prostate cancer xenografts

The receptor tyrosine kinase Axl is overexpressed in and leads to patient morbidity and mortality in a variety of cancers. Axl–Gas6 interactions are critical for tumor growth, angiogenesis and metastasis. The goal of this study was to investigate the feasibility of imaging graded levels of Axl expression in tumors using a radiolabeled antibody. We radiolabeled anti-human Axl (Axl mAb) and control IgG1 antibodies with ¹²⁵I with high specific radioactivity and radiochemical purity, resulting in an immunoreactive fraction suitable for in vivo studies. Radiolabeled antibodies were investigated in severe combined immunodeficient mice harboring subcutaneous CFPAC (Axl^high) and Panc1 (Axl^low) pancreatic cancer xenografts by ex vivo biodistribution and imaging. Based on these results, the specificity of [¹²⁵I]Axl mAb was also validated in mice harboring orthotopic Panc1 or CFPAC tumors and in mice harboring subcutaneous 22Rv1 (Axl^low) or DU145 (Axl^high) prostate tumors by ex vivo biodistribution and imaging studies at 72 h post-injection of the antibody. Both imaging and biodistribution studies demonstrated specific and persistent accumulation of [¹²⁵I]Axl mAb in Axl^high (CFPAC and DU145) expression tumors compared to the Axl^low (Panc1 and 22Rv1) expression tumors. Axl expression in these tumors was further confirmed by immunohistochemical studies. No difference in the uptake of radioactivity was observed between the control [¹²⁵I]IgG1 antibody in the Axl^high and Axl^low expression tumors. These data demonstrate the feasibility of imaging Axl expression in pancreatic and prostate tumor xenografts.     

Comparative Affinities of Adrenomedullin (AM) and Calcitonin Gene-Related Peptide (CGRP) for [125I] AM and [125I] CGRP Specific Binding Sites in Porcine Tissues

Abstract

We have investigated the binding characteristics of rat [¹²⁵I] adrenomedullin (AM) and human [¹²⁵I] calcitonin gene-related peptide (CGRP) to membranes prepared from a number of porcine tissues including atrium, ventricle, lung, spleen, liver, renal cortex and medulla. These membranes displayed specific, high affinity binding for [¹²⁵I] rat AM and [¹²⁵I] human CGRP. Porcine lung displayed the highest density of binding sites for radiolabeled AM and CGRP followed by porcine renal cortex. Competition experiments performed with [¹²⁵I] rat AM indicated that the rank order of potencies of various peptides for inhibiting [¹²⁵I] rat AM binding to various tissues were rat AM ≥ human AM ≥ human AM(22–52) > hαCGRP ≥ hαCGRP(8–37) <<<< sCT except spleen, atrium, renal cortex and renal medulla where rAM and hAM were 20–300 fold more potent than hAM(22–52). When the same experiments were performed using [¹²⁵I] hαCGRP as the radioligand, the rank order potencies for various peptides were rAM = hAM > hαCGRP > hαCGRP(8–37) in most of the tissues except in spleen and liver. where hαCGRP was the most potent ligand. In lung, hαCGRP was almost as potent as rAM and hAM in displacing [¹²⁵I] hαCGRP binding. These data suggest the existence of distinct CGRP and AM specific binding sites in contrast to previous reports that showed that both peptides interact differently in rat tissues.