[Lys40(Ahx-DTPA-111In)NH2]-Exendin-4 is a highly efficient radiotherapeutic for glucagon-like peptide-1 receptor-targeted therapy for insulinoma

PURPOSE: Although metabolic changes make diagnosis of insulinoma relatively easy, surgical removal is hampered by difficulties in locating it, and there is no efficient treatment for malignant insulinoma. We have previously shown that the high density of glucagon-like peptide-1 receptors (GLP-1R) in human insulinoma cells provides an attractive target for molecular imaging and internal radiotherapy. In this study, we investigated the therapeutic potential of [Lys(40)(Ahx-DTPA-(111)In)NH(2)]-Exendin-4, an (111)In-labeled agonist of GLP-1, in a transgenic mouse model of human insulinoma. EXPERIMENTAL DESIGN: [Lys(40)(Ahx-DTPA-(111)In)NH(2)]-Exendin-4 was assessed in the Rip1Tag2 mouse model of pancreatic beta-cell carcinogenesis, which exhibits a GLP-1R expression comparable with human insulinoma. Mice were injected with 1.1, 5.6, or 28 MBq of the radiopeptide and sacrificed 7 days after injection. Tumor uptake and response, the mechanism of action of the radiopeptide, and therapy toxicity were investigated. RESULTS: Tumor uptake was >200% injected activity per gram, with a dose deposition of 3 Gy/MBq at 40 pmol [Lys(40)(Ahx-DTPA-(111)In)NH(2)]-Exendin-4. Other GLP-1R-positive organs showed > or =30 times lower dose deposition. A single injection of [Lys(40)(Ahx-DTPA-(111)In)NH(2)]-Exendin-4 resulted in a reduction of the tumor volume by up to 94% in a dose-dependent manner without significant acute organ toxicity. The therapeutic effect was due to increased tumor cell apoptosis and necrosis and decreased proliferation. CONCLUSIONS: The results suggest that [Lys(40)(Ahx-DTPA-(111)In)NH(2)]-Exendin-4 is a promising radiopeptide capable of selectively targeting insulinoma. Furthermore, Auger-emitting radiopharmaceuticals such as (111)In are able to produce a marked therapeutic effect if a high tumor uptake is achieved.

GLP-1 receptor expression in human tumors and human normal tissues: potential for in vivo targeting

Peptide hormone receptors overexpressed in human tumors, such as somatostatin receptors, can be used for in vivo targeting for diagnostic and therapeutic purposes. A novel promising candidate in this field is the GLP-1 receptor, which was recently shown to be massively overexpressed in gut and lung neuroendocrine tumors--in particular, in insulinomas. Anticipating a major development of GLP-1 receptor targeting in nuclear medicine, our aim was to evaluate in vitro the GLP-1 receptor expression in a large variety of other tumors and to compare it with that in nonneoplastic tissues. METHODS: The GLP-1 receptor protein expression was qualitatively and quantitatively investigated in a broad spectrum of human tumors (n=419) and nonneoplastic human tissues (n=209) with receptor autoradiography using (125)I-GLP-1(7-36)amide. Pharmacologic competition experiments were performed to provide proof of specificity of the procedure. RESULTS: GLP-1 receptors were expressed in various endocrine tumors, with particularly high amounts in pheochromocytomas, as well as in brain tumors and embryonic tumors but not in carcinomas or lymphomas. In nonneoplastic tissues, GLP-1 receptors were present in generally low amounts in specific tissue compartments of several organs--namely, pancreas, intestine, lung, kidney, breast, and brain; no receptors were identified in lymph nodes, spleen, liver, or the adrenal gland. The rank order of potencies for receptor binding--namely, GLP-1(7-36)amide = exendin-4 >> GLP-2 = glucagon(1-29)--provided proof of specific GLP-1 receptor identification. CONCLUSION: The GLP-1 receptors may represent a novel molecular target for in vivo scintigraphy and targeted radiotherapy for a variety of GLP-1 receptor-expressing tumors. For GLP-1 receptor scintigraphy, a low-background signal can be expected, on the basis of the low receptor expression in the normal tissues surrounding tumors.

Immunohistochemical demonstration of interleukin-1 beta induced changes in acute-phase proteins and albumin in rat liver

Interleukin-1 beta is a potent mediator of the acute-phase response. However, the effects of interleukin-1 beta administration on the topic in vivo production of acute-phase proteins and albumin are so far not well understood. Overnight fasted rats were subcutaneously injected with 0.2 mL 0.9% NaCl (control group) or 6.25 micrograms recombinant human interleukin-1 beta, and rectal temperature was measured at intervals up to 48 h. Livers were perfused-fixed in vivo prior to injection (base-line), and at 9, 24, and 48 h following the interleukin-1 beta injection. Fibrinogen, orosomucoid (alpha 1-acid glycoprotein) and albumin were immunostained using a streptavidin-biotin-immunoperoxidase technique. Rectal temperature peaked 5 h after the single interleukin-1 beta injection, and fell gradually to base-line values by 24 h. Prior to injection only a few hepatocytes, randomly scattered throughout the liver lobule, stained positive for fibrinogen and orosomucoid. In contrast, all hepatocytes stained uniformly positive for fibrinogen and orosomucoid 9 h after interleukin-1 beta injection, whereas at 24 h a predominant centrilobular staining pattern occurred. Due to fasting, albumin positive hepatocytes were already reduced at base-line in both groups. Interleukin-1 beta induced a further significant loss of albumin positive cells in the periportal zone (35 +/- 21%) at 9 h when compared with controls (58 +/- 11%, p = 0.037). In conclusion, subcutaneous interleukin-1 beta (probably by stimulation of interleukin-6) strongly induces fibrinogen and orosomucoid expression in rat liver, and suppresses immunohistochemically stainable albumin in a heterogenous way, mainly in the periportal zone.

The Protein-tyrosine-phosphatase SHP2 is phosphorylated on serine residues 576 and 591 by protein kinase C isoforms alpha, beta 1, beta 2, and eta

To study whether protein kinase C (PKC) isoforms can interact with protein-tyrosine-phosphatases (PTPs) which are connected to the insulin signaling pathway, we co-overexpressed PKC isoforms together with insulin receptor, docking proteins, and the PTPs SHP1 and SHP2 in human embryonic kidney (HEK) 293 cells. After phorbol ester induced activation of PKC isoforms alpha, beta 1, beta 2, and eta, we could show a defined gel mobility shift of SHP2, indicating phosphorylation on serine/threonine residues. This phosphorylation was not dependent on insulin receptor or insulin receptor substrate-1 (IRS-1) overexpression and did not occur for the closely related phosphatase SHP1. Furthermore, PKC phosphorylation of SHP2 was completely blocked by the PKC inhibitor bisindolylmaleimide and was not detectable when SHP2 was co-overexpressed with kinase negative mutants of PKC beta 1 and -beta 2. The phosphorylation also occurred on endogenous SHP2 in Chinese hamster ovary (CHO) cells stably overexpressing PKC beta 2. Using point mutants of SHP2, we identified serine residues 576 and 591 as phosphorylation sites for PKC. However, no change of phosphatase activity by TPA treatment was detected in an in vitro assay. In summary, SHP2 is phosphorylated on serine residues 576 and 591 by PKC isoforms alpha, beta 1, beta 2, and eta.

Effects of Toll-like receptor 2 agonist Pam(3)CysSK(4) on inflammation and brain damage in experimental pneumococcal meningitis

TLR2 signaling participates in the pathogenesis of pneumococcal meningitis. In infant rats, the TLR2 agonist Pam(3)CysSK(4) was applied intracisternally (0.5 microg in 10 microl saline) alone or after induction of pneumococcal meningitis to investigate the effect of TLR2 activation on cerebrospinal fluid (CSF) inflammation and hippocampal apoptosis. A dose effect of Pam(3)CysSK(4) on apoptosis was investigated by intracisternal application of 0.5 microg in 10 microl saline and 40 microg in 20 microl saline. Pam(3)CysSK(4) neither induced apoptosis in sham-operated mice nor aggravated apoptosis in acute infection. However, Pam(3)CysSK(4) induced pleocytosis, TNF-alpha and MMP-9 in CSF in sham-infection but not during acute meningitis. We conclude that TLR2 signaling triggered by Pam(3)CysSK(4) at a dosage capable to induce a neuroinflammatory response does not induce hippocampal apoptosis in the infant rat model of experimental pneumococcal meningitis.

Glucagon-like peptide-1 receptor imaging for the localisation of insulinomas: a prospective multicentre imaging study

BACKGROUND Small benign insulinomas are hard to localise, leading to difficulties in planning of surgical interventions. We aimed to prospectively assess the insulinoma detection rate of single-photon emission CT in combination with CT (SPECT/CT) with a glucagon-like peptide-1 receptor avid radiotracer, and compare detection rates with conventional CT/MRI techniques. METHODS In our prospective imaging study, we enrolled adults aged 25-81 years at centres in Germany, Switzerland, and the UK. Eligible patients had proven clinical and biochemical endogenous hyperinsulinaemic hypoglycaemia and no evidence for metastatic disease on conventional imaging. CT/MRI imaging was done at referring centres according to standard protocols. At three tertiary nuclear medicine centres, we used whole body planar images and SPECT/CT of the abdomen up to 168 h after injection of (111)In-[Lys40(Ahx-DTPA-(111)In)NH2]-exendin-4 ((111)In-DTPA-exendin-4) to identify insulinomas. Consenting patients underwent surgery and imaging findings were confirmed histologically. FINDINGS Between Oct 1, 2008, and Dec 31, 2011, we recruited 30 patients. All patients underwent (111)In-DTPA-exendin-4 imaging, 25 patients underwent surgery (with histological analysis), and 27 patients were assessed with CT/MRI. (111)In-DTPA-exendin-4 SPECT/CT correctly detected 19 insulinomas and four additional positive lesions (two islet-cell hyperplasia and two uncharacterised lesions) resulting in a positive predictive value of 83% (95% CI 62-94). One true negative (islet-cell hyperplasia) and one false negative (malignant insulinoma) result was identified in separate patients by (111)In-DTPA-exendin-4 SPECT/CT. Seven patients (23%) were referred to surgery on the basis of (111)In-DTPA-exendin-4 imaging alone. For 23 assessable patients, (111)In-DTPA-exendin-4 SPECT/CT had a higher sensitivity (95% [95% CI 74-100]) than did CT/MRI (47% [27-68]; p=0·011). INTERPRETATION (111)In-DTPA-exendin-4 SPECT/CT could provide a good second-line imaging strategy for patients with negative results on initial imaging with CT/MRI. FUNDING Oncosuisse, the Swiss National Science Foundation, and UK Department of Health.

Janus kinases 1 and 2 regulate chemokine-mediated integrin activation and naïve T-cell homing.

Janus kinases (JAKs) are central signaling molecules in cytokine receptor cascades. Although they have also been implicated in chemokine receptor signaling, this function continues to be debated. To address this issue, we established a nucleofection model in primary, nonactivated mouse T lymphocytes to silence JAK expression and to evaluate the ability of these cells to home to lymph nodes. Reduced JAK1 and JAK2 expression impaired naïve T-cell migration in response to gradients of the chemokines CXCL12 and CCL21. In vivo homing of JAK1/JAK2-deficient cells to lymph nodes decreased, whereas intranodal localization and motility were unaffected. JAK1 and JAK2 defects altered CXCL12- and CCL21-triggered ezrin/radixin/moesin (ERM) dephosphorylation and F-actin polymerization, as well as activation of lymphocyte function-associated Ag-1 and very late Ag-4 integrins. As a result, the cells did not adhere firmly to integrin substrates in response to these chemokines. The results demonstrate that JAK1/JAK2 participate in chemokine-induced integrin activation and might be considered a target for modulation of immune cell extravasation and therefore, control of inflammatory reactions.

{\it RAS\/} is required for the expression of interleukin-1$\beta$ in two diverse leukemic cell lines

Under normal physiological conditions, cells of the hematopoietic system produce Interleukin-1$\beta$(IL-1$\beta)$ only when a stimulus is present. Leukemic cells, however, can constitutively produce this cytokine without an exogenous source of activation. In addition, IL-1$\beta$ can operate as an autocrine and/or paracrine growth factor for leukemic blasts. In order to study the cellular basis for this aberrant production, we analyzed two leukemic cell lines (B1 and W1) which express high levels of IL-1$\beta$ and use IL-1$\beta$ as an autocrine growth factor. Initial studies demonstrated: (1) lack of rearrangement and/or amplification in the IL-1$\beta$ gene and its promoter; and (2) intact responsiveness to regulators such as cycloheximide and dexamethasone, implying that the molecular defect was upstream. Analysis of the Ras inducible transcription factors by gel shift assay demonstrated constitutive transcription factor binding in the IL-1$\beta$ promoter. Furthermore, RAS mutations were found at codon 12 in the K-RAS and N-RAS genes in the B1 and W1 cells, respectively. To deduce the effects of activated Ras on IL-1$\beta$ expression, two classes of farnesyltransferase inhibitors and an adenoviral vector expressing antisense targeted to K-RAS were utilized. The farnesyltransferase inhibitors perillyl alcohol and B581 were able to reduce IL-1$\beta$ levels by 80% and 50% in the B1 cells, respectively. In W1 cells, IL-1$\beta$ was reduced by 60% with 1mM perillyl alcohol. Antisense RNA targeted to K-RAS confirmed the results demonstrating a 50% reduction in IL-1$\beta$ expression in the B1 cells. In addition, decreased binding at the crucial NF-IL6/CREB binding site correlated with decreased IL-1$\beta$ production and cellular proliferation implying that this site was a downstream effector of Ras signaling. Our data suggest that mutated RAS genes may be responsible for autocrine IL-1$\beta$ production in some leukemias by stimulating signal transduction pathways that activate the IL-1$\beta$ promoter. ^

Glucagon-like-peptide-1 receptor expression in normal and diseased human thyroid and pancreas.

Glucagon-like-peptide-1 (GLP1) analogs may induce thyroid or pancreatic diseases in animals, raising questions about their use in diabetic patients. There is, however, controversy regarding expression of GLP1 receptors (GLP1R) in human normal and diseased thyroid and pancreas. Here, 221 human thyroid and pancreas samples were analyzed for GLP1R immunohistochemistry and compared with quantitative in vitro GLP1R autoradiography. Neither normal nor hyperplastic human thyroids containing parafollicular C cells express GLP1R with either method. Papillary thyroid cancer do not, and medullary thyroid carcinomas rarely express GLP1R. Insulin- and somatostatin-producing cells in the normal pancreas express a high density of GLP1R, whereas acinar cells express them in low amounts. Ductal epithelial cells do not express GLP1R. All benign insulinomas express high densities of GLP1R, whereas malignant insulinomas rarely express them. All ductal pancreatic carcinomas are GLP1R negative, whereas 6/20 PanIN 1/2 and 0/12 PanIN 3 express GLP1R. Therefore, normal thyroid, including normal and hyperplastic C cells, or papillary thyroid cancer are not targets for GLP1 analogs in humans. Conversely, all pancreatic insulin- and somatostatin-producing cells are physiological GLP1 targets, as well as most acini. As normal ductal epithelial cells or PanIN 3 or ductal pancreatic carcinomas do not express GLP1R, it seems unlikely that GLP1R is related to neoplastic transformation in pancreas. GLP1R-positive medullary thyroid carcinomas and all benign insulinomas are candidates for in vivo GLP1R targeting.Modern Pathology advance online publication, 12 September 2014; doi:10.1038/modpathol.2014.113.

Effects of colostrum versus formula feeding on hepatic glucocorticoid and α1- and β2-adrenergic receptors in neonatal calves and their effect on glucose and lipid metabolism

Neonatal energy metabolism in calves has to adapt to extrauterine life and depends on colostrum feeding. The adrenergic and glucocorticoid systems are involved in postnatal maturation of pathways related to energy metabolism and calves show elevated plasma concentrations of cortisol and catecholamines during perinatal life. We tested the hypothesis that hepatic glucocorticoid receptors (GR) and α₁- and β₂-adrenergic receptors (AR) in neonatal calves are involved in adaptation of postnatal energy metabolism and that respective binding capacities depend on colostrum feeding. Calves were fed colostrum (CF; n=7) or a milk-based formula (FF; n=7) with similar nutrient content up to d 4 of life. Blood samples were taken daily before feeding and 2h after feeding on d 4 of life to measure metabolites and hormones related to energy metabolism in blood plasma. Liver tissue was obtained 2 h after feeding on d 4 to measure hepatic fat content and binding capacity of AR and GR. Maximal binding capacity and binding affinity were calculated by saturation binding assays using [(3)H]-prazosin and [(3)H]-CGP-12177 for determination of α₁- and β₂-AR and [(3)H]-dexamethasone for determination of GR in liver. Additional liver samples were taken to measure mRNA abundance of AR and GR, and of key enzymes related to hepatic glucose and lipid metabolism. Plasma concentrations of albumin, triacylglycerides, insulin-like growth factor I, leptin, and thyroid hormones changed until d 4 and all these variables except leptin and thyroid hormones responded to feed intake on d 4. Diet effects were determined for albumin, insulin-like growth factor I, leptin, and thyroid hormones. Binding capacity for GR was greater and for α₁-AR tended to be greater in CF than in FF calves. Binding affinities were in the same range for each receptor type. Gene expression of α₁-AR (ADRA1) tended to be lower in CF than FF calves. Binding capacity of GR was related to parameters of glucose and lipid metabolism, whereas β₂-AR binding capacity was negatively associated with glucose metabolism. In conclusion, our results indicate a dependence of GR and α₁-AR on milk feeding immediately after birth and point to an involvement of hepatic GR and AR in postnatal adaptation of glucose and lipid metabolism in calves.

Direct adenovirus-mediated gene transfer of interleukin 1 and tumor necrosis factor α soluble receptors to rabbit knees with experimental arthritis has local and distal anti-arthritic effects

Adenoviral vectors were used to deliver genes encoding a soluble interleukin 1 (IL-1)-type I receptor-IgG fusion protein and/or a soluble type I tumor necrosis factor α (TNFα) receptor-IgG fusion protein directly to the knees of rabbits with antigen-induced arthritis. When tested individually, knees receiving the soluble IL-1 receptor had significantly reduced cartilage matrix degradation and white blood cell infiltration into the joint space. Delivery of the soluble TNFα receptor was less effective, having only a moderate effect on white blood cell infiltration and no effect on cartilage breakdown. When both soluble receptors were used together, there was a greater inhibition of white blood cell infiltration and cartilage breakdown with a considerable reduction of synovitis. Interestingly, anti-arthritic effects were also seen in contralateral control knees receiving only a marker gene, suggesting that sustained local inhibition of disease activity in one joint may confer an anti-arthritic effect on other joints. These results suggest that local intra-articular gene transfer could be used to treat systemic polyarticular arthritides.

Luminal trypsin may regulate enterocytes through proteinase-activated receptor 2

Proteinase-activated receptor 2 (PAR-2) is a recently characterized G-protein coupled receptor that is cleaved and activated by pancreatic trypsin. Trypsin is usually considered a digestive enzyme in the intestinal lumen. We examined the hypothesis that trypsin, at concentrations normally present in the lumen of the small intestine, is also a signaling molecule that specifically regulates enterocytes by activating PAR-2. PAR-2 mRNA was highly expressed in the mucosa of the small intestine and in an enterocyte cell line. Immunoreactive PAR-2 was detected at the apical membrane of enterocytes, where it could be cleaved by luminal trypsin. Physiological concentrations of pancreatic trypsin and a peptide corresponding to the tethered ligand of PAR-2, which is exposed by trypsin cleavage, stimulated generation of inositol 1,4,5-trisphosphate, arachidonic acid release, and secretion of prostaglandin E2 and F1α from enterocytes and a transfected cell line. Application of trypsin to the apical membrane of enterocytes and to the mucosal surface of everted sacs of jejunum also stimulated prostaglandin E2 secretion. Thus, luminal trypsin activates PAR-2 at the apical membrane of enterocytes to stimulate secretion of eicosanoids, which regulate multiple cell types in a paracrine and autocrine manner. We conclude that trypsin is a signaling molecule that specifically regulates enterocytes by triggering PAR-2.

Severe reduction in leukocyte adhesion and monocyte extravasation in mice deficient in CC chemokine receptor 2

CC chemokine receptor 2 (CCR2) is a prominent receptor for the monocyte chemoattractant protein (MCP) group of CC chemokines. Mice generated by gene targeting to lack CCR2 exhibit normal leukocyte rolling but have a pronounced defect in MCP-1-induced leukocyte firm adhesion to microvascular endothelium and reduced leukocyte extravasation. Constitutive macrophage trafficking into the peritoneal cavity was not significantly different between CCR2-deficient and wild-type mice. However, after intraperitoneal thioglycollate injection, the number of peritoneal macrophages in CCR2-deficient mice did not rise above basal levels, whereas in wild-type mice the number of macrophages at 36 h was ≈3.5 times the basal level. The CCR2-deficient mice showed enhanced early accumulation and delayed clearance of neutrophils and eosinophils. However, by 5 days neutrophils and eosinophils in both CCR2-deficient and wild-type mice had returned to near basal levels, indicating that resolution of this inflammatory response can occur in the absence of macrophage influx and CCR2-mediated activation of the resident peritoneal macrophages. After intravenous injection with yeast β-glucan, wild-type mice formed numerous large, well-defined granulomas throughout the liver parenchyma, whereas CCR2-deficient mice had much fewer and smaller granulomas. These results demonstrate that CCR2 is a major regulator of induced macrophage trafficking in vivo.

A Role for Cadherin-5 in Regulation of Vascular Endothelial Growth Factor Receptor 2 Activity in Endothelial Cells

FLK-1/vascular endothelial growth factor receptor 2 (VEGFR-2) is one of the receptors for VEGF. In this study we examined the effect of cell density on activation of VEGFR-2. VEGF induces only very slight tyrosine phosphorylation of VEGFR-2 in confluent (95–100% confluent) pig aortic endothelial (PAE) cells. In contrast, robust VEGF-dependent tyrosine phosphorylation of VEGFR-2 was observed in cells plated in sparse culture conditions (60–65% confluent). A similar cell density-dependent phenomenon was observed in different endothelial cells but not in NIH-3T3 fibroblast cells expressing VEGFR-2. Stimulating cells with high concentrations of VEGF or replacing the extracellular domain of VEGFR-2 with that of the colony-stimulating factor 1 receptor did not alleviate the sensitivity of VEGFR-2 to cell density, indicating that the confluent cells were probably not secreting an antagonist to VEGF. Furthermore, in PAE cells, ectopically introduced platelet-derived growth factor α receptor could be activated at both high and low cell density conditions, indicating that the density effect was not universal for all receptor tyrosine kinases expressed in endothelial cells. In addition to lowering the density of cells, removing divalent cations from the medium of confluent cells potentiated VEGFR-2 phosphorylation in response to VEGF. These findings suggested that cell–cell contact may be playing a role in regulating the activation of VEGFR-2. To this end, pretreatment of confluent PAE cells with a neutralizing anti-cadherin-5 antibody potentiated the response of VEGFR-2 to VEGF. Our data demonstrate that endothelial cell density plays a critical role in regulating VEGFR-2 activity, and that the underlying mechanism appears to involve cadherin-5.

Chimeric Cytokine Receptor Can Transduce Expansion Signals in Interleukin 6 Receptor α (IL-6Rα)-, IL-11Rα-, and gp130-low to -negative Primitive Hematopoietic Progenitors

We generated transgenic mice expressing chimeric receptors, which comprise extracellular domains of the human granulocyte–macrophage colony-stimulating factor (hGM-CSF) receptor and transmembrane and cytoplasmic domains of the mouse leukemia inhibitory factor receptor. In suspension cultures of lineage-negative (Lin−), 5-fluorouracil-resistant bone marrow cells of the transgenic mice, a combination of hGM-CSF and stem cell factor (SCF) induced exponential expansions of mixed colony-forming unit. The combination of hGM-CSF and SCF was effective on enriched, Lin−Sca-1+c-kit+ progenitors and increased either mixed colony-forming unit or cobblestone area–forming cells. In case of stimulation with hGM-CSF and SCF, interleukin-6 (IL-6) and SCF, or IL-11 and SCF, the most efficient expansion was achieved with hGM-CSF and SCF. When Lin−Sca-1+c-kit+CD34− further enriched progenitors were clone sorted and individually incubated in the presence of SCF, hGM-CSF stimulated a larger number of cells than did IL-6, IL-6 and soluble IL-6 receptor (IL-6R), or IL-11. These data suggest the presence of IL-6Rα-, IL-11Rα-, and gp130-low to -negative primitive hematopoietic progenitors. Such primitive progenitors are equipped with signal transduction molecules and can expand when these chimeric receptors are genetically introduced into the cells and stimulated with hGM-CSF in the presence of SCF.