GHRH-SST-GH-IGF axis regulates crosstalk between growth and immunity in rainbow trout (Oncorhynchus mykiss) infected with Vibrio anguillarum.

An energy trade-off is existed between immunological competence and growth. The axis of growth hormone releasing hormone, somatostatin, growth hormone, insulin-like growth factor (GHRH-SST-GH-IGF axis) regulates growth performances and immune competences in rainbow trout (Oncorhynchus mykiss). The salmonid-specific whole genome duplication event is known to result in duplicated copies of several key genes in GHRH-SST-GH-IGF axis. In this study, we evaluated the physiological functions of GHRH-SST-GH-IGF axis in regulating crosstalk between growth and immunity. Based on principal components analysis (PCA), we observed the overall expression profiles of GHRH-SST-GH-IGF axis were significantly altered by Vibrio anguillarum infection. Trout challenged with Vibrio anguillarum showed down-regulated igf1s subtypes and up-regulated igfbp1a1. The brain sst genes (sst1a, sst1b, sst3b and sst5) and igfpbs genes (igfbp4s and igfbp5b2) were significantly affected by V. anguillarum infection, while the igfbp4s, igfbp5s, igfbp6s and igf2bps genes showed significant changes in peripheral immune tissues in response to V. anguillarum infection. Gene enrichment analyses showed functional and signaling pathways associated with apoptosis (such as p53, HIF-1 or FoxO signaling) were activated. We further proposed a possible model that describes the IGF and IGFBPs-regulated interaction between cell growth and programmed death. Our study provided new insights into the physiological functions and potentially regulatory mechanisms of the GHRH-SST-GH-IGF axis, indicating the pleiotropic effects of GHRH-SST-GH-IGF axis in regulating crosstalk between growth and immunity in trout.

Development of IGF signaling antibody arrays for the identification of hepatocellular carcinoma biomarkers.

PURPOSE: Our objective was to develop a system to simultaneously and quantitatively measure the expression levels of the insulin-like growth factor (IGF) family proteins in numerous samples and to apply this approach to profile the IGF family proteins levels in cancer and adjacent tissues from patients with hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: Antibodies against ten IGF family proteins (IGF-1, IGF-1R, IGF-2, IGF-2R, IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4, IGFBP-6, and Insulin) were immobilized on the surface of a glass slide in an array format to create an IGF signaling antibody array. Tissue lysates prepared from patient's liver cancer tissues and adjacent tissues were then applied to the arrays. The proteins captured by antibodies on the arrays were then incubated with a cocktail of biotinylated detection antibodies and visualized with a fluorescence detection system. By comparison with standard protein amount, the exact protein concentrations in the samples can be determined. The expression levels of the ten IGF family proteins in 25 pairs of HCC and adjacent tissues were quantitatively measured using this novel antibody array technology. The differential expression levels between cancer tissues and adjacent tissues were statistically analyzed. RESULTS: A novel IGF signaling antibody array was developed which allows the researcher to simultaneously detect ten proteins involved in IGF signal pathway with high sensitivity and specificity. Using this approach, we found that the levels of IGF-2R and IGFBP-2 in HCC tissues were higher than those in adjacent tissues. CONCLUSION: Our IGF signaling antibody array which can detect the expression of ten IGF family members with high sensitivity and specificity will undoubtedly prove a powerful tool for drug and biomarker discovery.

Insulin-like growth factor: current concepts and new developments in cancer therapy.

The insulin-like growth factor (IGF) family and the IGF-1 receptor (IGF-1R) play an important role in cancer. This intricate and complex signaling pathway provides many opportunities for therapeutic intervention, and several novel therapeutics aimed at the IGF-1R, particularly monoclonal antibodies and small molecule tyrosine kinase inhibitors, are under clinical investigation. This article provides a patent overview of the IGF signaling pathway and its complexity, addresses the justification for the use of IGF-1R-targeted therapy, and reviews the results of in vivo and in vitro novel therapeutics. Over the past year, the completion of several phase I, II, and III trials have provided interesting new information about the clinical activity of these novel compounds, particularly CP-751,871, IMC-A12, R1507, AMG-479, AVE-1642, MK-0646, XL-228, OSI-906, and BMS-754807. We review the important preliminary results from clinical trials with these compounds and conclude with a discussion about future therapeutic efforts.

Specific allowance for antibody bivalence in the determination of dissociation constants by kinetic exclusion assay.

Theory that takes rigorous account of antibody bivalence in the characterization of immunospecific reactions by kinetic exclusion assay is presented. In addition to reinforcing the basic correctness of quantitative expressions currently being used for the determination of dissociation constants (K(d)) by this method, the current study highlights a requirement for conformity of the system with critical assumptions/approximations therein. Published results for the interaction between the extracellular domain of human insulin-like growth factor (hIGFR) and anti-hIGFR are used to illustrate aspects of the theoretical predictions for a system to which those assumptions/approximations may well apply; and those for a cadmium-ethylenediaminetetraacetic acid (Cd-EDTA) antibody interaction to emphasize the consequences of adopting the same analytical procedure in a situation where one of those assumptions does not apply. The major weakness of current protocols for the characterization of antigen-antibody interactions by kinetic exclusion assay is an absence of any check on the likely magnitude of the probability of antibody capture by the affinity beads--a parameter that needs to be 5% or lower for validity of the quantitative expression on which the analysis is based.

Mapping of the vitronectin-binding site on the urokinase receptor: involvement of a coherent receptor interface consisting of residues from both domain I and the flanking interdomain linker region.

The urokinase-type plasminogen activator receptor (uPAR) has been implicated as a modulator of several biochemical processes that are active during tumor invasion and metastasis, e.g. extracellular proteolysis, cell adhesion, and cell motility. The structural basis for the high affinity interaction between the urokinase-type plasminogen activator (uPA) and uPAR, which focuses cell surface-associated plasminogen activation in vivo, is now thoroughly characterized by site-directed mutagenesis studies and x-ray crystallography. In contrast, the structural basis for the interaction between uPAR and the extracellular matrix protein vitronectin, which is involved in the regulation of cell adhesion and motility, remains to be clarified. In this study, we have identified the functional epitope on uPAR that is responsible for its interaction with the full-length, extended form of vitronectin by using a comprehensive alanine-scanning library of purified single-site uPAR mutants (244 positions tested). Interestingly, the five residues identified as "hot spots" for vitronectin binding form a contiguous epitope consisting of two exposed loops connecting the central fourstranded beta-sheet in uPAR domain I (Trp(32), Arg(58), and Ile(63)) as well as a proximal region of the flexible linker peptide connecting uPAR domains I and II (Arg(91) and Tyr(92)). This binding topology provides the molecular basis for the observation that uPAR can form a ternary complex with uPA and vitronectin. Furthermore, it raises the intriguing possibility that the canonical receptor and inhibitor for uPA (uPAR and PAI-1) may have reached a convergent solution for binding to the somatomedin B domain of vitronectin.

FQPD, a novel immunomodulatory drug, has significant in vitro activity in multiple myeloma.

Multiple myeloma (MM) is a plasma cell malignancy that claims thousands of lives each year and has considerable morbidity. The disease remains incurable despite recent advances in the understanding of the disease biology and the introduction of more effective drugs is needed. This study evaluated the anti-MM activity of 3-(7-fluoro-4H-quinazolin-3-yl)-piperidine-2,6-dione, hydrochloride (FQPD), a novel immunomodulatory drug. FQPD inhibited the proliferation of multiple MM cell lines, including those resistant to conventional treatments, such as dexamethasone. It induced apoptosis in MM cell lines, as well as freshly isolated patient MM cells, without cytotoxicity on normal human lymphocytes. Moreover, it induced apoptosis in MM cells adherent to bone marrow (BM) stromal cells or in the presence of cytokines, such as interleukin-6 and vascular endothelial growth factor, confirming its ability to overcome the protective effects of the BM milieu. Apoptosis in the MM cells was mediated via poly-ADP ribose polymerase cleavage as well as cleavage of caspase 8 and caspase 9. Our studies therefore demonstrated in vitro anti-MM activity of FQPD and provide the rationale for its in vivo evaluation in animal models and derived clinical trials.

Inhibition of bone-derived insulin-like growth factors by a ligand-specific antibody suppresses the growth of human multiple myeloma in the human adult bone explanted in NOD/SCID mouse.

Multiple myeloma (MM) is a fatal disease that affects plasma cells. Patients with MM have 1 or more osteolytic lesions in their bone tissues, where insulin-like growth factors (IGFs; IGF-I and IGF-II) are mainly stored. The role of bone-derived IGFs in the development of MM has not been extensively studied because reliable animal models are lacking. We established an animal model using a human MM cell line, RPMI8226, in nonobese diabetic/severe-combined immunodeficient (NOD/SCID) mice implanted with human adult bone (HAB) fragments. Treatment with an anti-human IGF-neutralizing monoclonal antibody, KM1468, inhibited the IGF-I-stimulated phosphorylation of type-I IGF receptors (IGF-IR) in RPMI8226 cells and the activation of the downstream PI3-K/Akt signaling pathway in vitro. KM1468 inhibited IGF-I-mediated RPMI8226 cell growth in a dose-dependent manner. In the NOD/SCID-HAB model, treatment with KM1468 significantly inhibited the growth of RPMI8226 cells (p<0.02). These results indicated that the growth of MM cells was predominantly stimulated not by serum-derived IGFs, but by bone-derived IGFs. Furthermore, the targeting of bone-derived IGFs, using a neutralizing antibody, may offer a new therapeutic strategy for MM.

Regulation of adult olfactory neurogenesis by insulin-like growth factor-I.

Insulin-like growth factor-I (IGF-I) has multiple effects within the developing nervous system but its role in neurogenesis in the adult nervous system is less clear. The adult olfactory mucosa is a site of continuing neurogenesis that expresses IGF-I, its receptor and its binding proteins. The aim of the present study was to investigate the roles of IGF-I in regulating proliferation and differentiation in the olfactory mucosa. The action of IGF-I was assayed in serum-free culture combined with bromodeoxyuridine-labelling of proliferating cells and immunochemistry for specific cell types. IGF-I and its receptor were expressed by globose basal cells (the neuronal precursor) and by olfactory neurons. IGF-I reduced the numbers of proliferating neuronal precursors, induced their differentiation into neurons and promoted morphological differentiation of neurons. The evidence suggests that IGF-I is an autocrine and/or paracrine signal that induces neuronal precursors to differentiate into olfactory sensory neurons. These effects appear to be similar to the cellular effects of IGF-I in the developing nervous system.

Monitoring insulin-like growth factors in HIV infection and AIDS.

There is a close association between the growth hormone (GH)-insulin-like growth factor I (IGF-I) axis, infection and immunity. Infection with the human immunodeficiency virus (HIV) is often associated with a decrease of the concentrations of IGF-I, IGF-II, IGF-binding protein 3 (IGFBP-3) and an increase of IGFBP-1 and -2. Many investigators have studied the relationship between the GH-IGF-I system and some of the most common characteristics of disease progression, such as decreased CD4 cell counts, weight loss and fat redistribution. Although conditions for restoration of thymic function and lymphopoiesis with GH or IGF-I are still not well defined, many studies led to the development of clinical trials on the therapeutic use of GH, IGF-I and GHRH for the treatment of weight loss or fat redistribution, two problems which persist despite the introduction of highly active antiretroviral therapy. Monitoring IGF-I concentrations during treatment with GH and GHRH is likely to become an essential component of their therapeutic use. IGF-I levels are the first indicator of treatment efficacy and can be used to monitor compliance. High levels of IGF-I are a warning sign for the increased risk of potential adverse effects, such as acromegalic-like symptoms or malignancy. This could lead to a reduction of the therapeutic dose or the temporary interruption of treatment until IGF levels reach a safe range. IGF-I levels are also likely to increase with other hormones used in HIV patients, such as erythropoietin for the treatment of anemia or anabolic androgens in HIV-infected women.

Blockade of paracrine supply of insulin-like growth factors using neutralizing antibodies suppresses the liver metastasis of human colorectal cancers.

Environmental stimuli, such as organ-specific growth factors, can influence the metastatic potential of a tumor. The liver is the main source of insulin-like growth factors (IGFs). The importance of IGF signal in hepatic metastasis has been clarified mainly by IGF-I receptor targeting strategies. This study aims to confirm these precedent reports by novel tool, neutralizing antibodies against IGFs and to show that IGFs are promising therapeutic targets for hepatic metastasis in vivo. Hepatic metastasis was induced by intrasplenic injection of human colorectal cancer cell line, HT29. The antimetastatic effects of three antibodies (anti-mouse IGF-I, anti-mouse IGF-II, and anti-human/mouse IGF-II designated KM1468) were tested singly or in combination in the early phase of metastasis. The dose escalation effect of KM1468 and its survival benefit were examined in the early and late phases of metastasis. The mechanism of IGF neutralization was investigated with immunohistochemistry. Dual neutralization of paracrine IGF-I and IGF-II showed modest additive antimetastatic effects than single neutralization of IGF-I or IGF-II. In any phase of metastasis, neutralization led to significant tumor growth inhibition and longer survival. Dose escalation of KM1468 influenced survival only in the late phase of metastasis. Apoptosis increased significantly in the antibody-treated group compared with the control group (P = 0.0025) In conclusion, IGFs are promising therapeutic targets for hepatic metastases of colorectal cancers. However, the IGF dependency is probably variable in the metastatic process.

Effects of insulin-like growth factors and insulin-like growth factor binding protein-2 on the in vitro proliferation of peripheral blood mononuclear cells.

Increasing evidence has implicated that insulin-like growth factors (IGFs), polypeptides structurally related to proinsulin, are involved in the function and development of the immune system. To probe the relevance of IGF binding protein 2 (IGFBP-2) in T-cell activation and proliferation, we studied the role of IGFBP-2 in anti-CD3 monoclonal antibody (mAb)-activated peripheral blood mononuclear cells (PBMCs). Secretion of IGF-I, IGF-II, and IGFBP-2 by PBMCs from healthy adult donors was determined by radioimmunoassays (RIAs). The PBMC proliferative response after stimulation with anti-CD3 mAb and exposure to increasing concentrations of IGF-I, IGF-II, IGFBP-2, and anti-IGFBP-2 were determined by bromodeoxyuridine enzyme-linked immunosorbent assay. Observations were tested for significance by paired t-tests. We demonstrate an increase in IGFBP-2 secretion associated with both activation of PBMC by anti-CD3 mAb and increasing cell density. Incubation with exogenous IGFBP-2 increased the proliferation of PBMCs, whereas anti-IGFBP-2 had an antiproliferative effect on PBMCs that was reversed by simultaneous exposure to IGFBP-2. The stimulatory activity of IGFBP-2 (1-10 ng/ml) on anti-CD3 mAb-activated PBMCs was similar to that of IGF-I and IGF-II (1-100 ng/ml), with the mean increase in PBMC proliferative response ranging between 150% and 160% for IGFBP-2 (p = 0.03), 150% and 170% for IGF-I (p < 0.01), 133%-161% for IGF-II (p < 0.01), and 157% and 175% for IGF-I + IGF-II (p < 0.01). Thus, our data strongly suggest a role for IGFBP-2 as a local growth factor contributing to the proliferation and activation of mononuclear cells.

The insulin-like growth factors and insulin-signalling systems: an appealing target for breast cancer therapy?

There is compelling evidence from epidemiological studies in humans, as well as in vitro and in vivo experimental observations including transgenic animal models, for a role of the IGF/insulin signalling system in cancer tumourigenesis. In this review focused on breast cancer, we review the experimental evidence, discuss the cellular and molecular mechanisms of tumourigenicity by the IGFs and insulin and various possible therapeutic strategies based on the mechanisms discussed.

Bone formation following intramedullary femoral reaming is decreased by indomethacin and antibodies to insulin-like growth factors.

OBJECTIVE: We aimed to: 1). compare rates of in vitro bone formation following reamed and nonreamed intramedullary fixation in a murine model of femoral fracture healing; and 2). examine whether antibodies to insulin-like growth factor (IGF) I, IGF II, or indomethacin (an inhibitor of the inflammatory process) affect bone formation following intramedullary reaming. DESIGN: Experimental study. PARTICIPANTS: Twenty-four C57 black mice were randomized to two groups: reamed ( = 12), and nonreamed intramedullary nail insertion ( = 12). INTERVENTION: In the reamed group, the femoral canals were successively reamed with 30-, 27-, 25-, and 23-gauge stainless steel pins and stabilized with a 27-gauge pin. In mice randomized to the nonreamed group, a 27-gauge pin was inserted. An external three-point bending force created a midshaft transverse femoral fracture. Seven days postsurgery, each mouse was killed, and the right femur was removed. Following pin removal, the callus was minced, the bone marrow was removed, and both were ultracentrifuged at 1200 rpm for 5 minutes. The supernatent was cocultured with 3-day-old murine calvarial cells in culture media. At day 5 of culture, reamed plasma and calvarial cell cocultures were exposed to either 1.0 micro g/mL of anti-IGF I, 1.0 micro g/mL of anti-IGF II, 2 micro M indomethacin, or served as controls (calvarial cells only). The cells were cultured for a total of 21 days. MAIN OUTCOME MEASUREMENTS: The number of bone nodules was quantified by light microscopy. RESULTS: Reamed pin insertion resulted in 4.1-fold and 8.9-fold increases in the mean number of bone nodules compared to pins inserted without reaming and controls, respectively (399 +/- 40.0 vs. 97.0 +/- 21.0, < 0.001). The positive effect of intramedullary reaming on bone nodule formation was reversed with the administration of antibodies to IGF I and IGF II. The addition of anti-IGF I or anti-IGF II to calvarial, or osteoblastlike, cells treated with supernatent from the callus and bone marrow of mice with prior intramedullary reaming resulted in significant declines in the mean number of bone nodules ( < 0.001). Specifically, treatment of osteoblastlike cells with anti-IGF I or anti-IGF II resulted in 7.0-fold and 5.4-fold declines in mean bone nodule formation compared to cells without such treatment. CONCLUSIONS: Intramedullary reaming prior to pin insertion resulted in a significantly greater number of bone nodules than pin insertion only. Antibodies to IGF I, IGF II, and indomethacin reversed the stimulatory effect of reaming on bone nodule formation, suggesting their role in modulating the course of fracture healing following intramedullary reaming.

A method for defining binding sites involved in protein-protein interactions: analysis of the binding of plasminogen activator inhibitor 1 to the somatomedin domain of vitronectin.

Plasminogen activator inhibitor type-1 (PAI-1) is bound to vitronectin (VN) in plasma and in the extracellular matrix. We previously employed a domain-swapping approach to show that the high-affinity binding site for PAI-1 in VN is contained within residues 12-30 in the amino-terminal somatomedin B (SMB) domain. In this study, we attempt to further delineate the location of this site by employing a novel approach that is based on the use of monoclonal antibodies (Mabs) together with site-directed mutagenesis. Six separate Mabs were identified that bound to the SMB domain and competed with PAI-1 for binding to VN. The relative affinity of each of the Mabs, and of PAI-1 itself, for binding to individual variants of SMB (prepared by alanine scanning mutagenesis), was then determined and compared in competitive binding experiments. Three separate, partially overlapping Mab epitopes within SMB were defined by these studies, and the PAI-1 binding site was localized to the region between residues 24 and 37. When considered together with the domain swapping data, these studies suggest that the PAI-1 binding site is contained within a common seven-residue region (i.e., residues 24-30) in the SMB domain.

Growth promoting effects of human placental lactogen during early organogenesis: a link to insulin-like growth factors.

Many maternally derived factors may be involved in the regulation of embryonic growth but the control mechanisms involved are poorly understood. Human placental lactogen (hPL) has been implicated in playing a role in the control of embryonic growth. Several investigators suggested that there may be a possible link between the effects of this hormone and insulin-like growth factors (IGFs). In order to determine the growth promoting potential of hPL and involvement of IGFs in the mechanism of action of the hormone, 9.5 d rat embryos were cultured in vitro for 48 h in depleted serum in the presence and absence of hPL with additional IGF antisera. The growth supporting capacity of the serum was reduced by removal of low molecular weight molecules by prolonged filtration of the serum using filters with a molecular weight exclusion of 30 kDa. Addition of hPL (3.2-25.6 ng/ml) to depleted serum significantly improved embryonic growth and development, suggesting that the developing embryo may utilise hPL. The presence of antisera against hPL, IGF-I and -II abolished the hPL-induced increase in the development in all parameters suggesting that there may be a possible link between the IGFs and the effects of hPL on rat embryonic development and this hormone may achieve its growth promoting effects via IGFs.

Dysregulation of insulin-like growth factors in a case of generalized acquired lipoatrophic diabetes mellitus (Lawrence Syndrome) connected with autoantibodies against adipocyte membranes.

We report on a 33-year-old male patient with generalized acquired lipodystrophy, insulin resistant diabetes mellitus and acanthosis nigricans (Lawrence Syndrome). First probable symptoms of lipodystrophy (weight loss, shrinkage of subcutaneous fatty tissue, and loss of muscular strength) became evident three years ago, with the onset of diabetes mellitus occurring about six months later. The patient suffered from the following clinical symptoms: IDDM with increasing insulin-requirement, extreme reduction of fatty tissue, fatty liver hepatitis with elevated liver enzymes, glomerulopathy, muscular and neuropathic pains, as well as hypertriglyceridaemia. A basal C-peptide concentration is rather high. Definitely, the endogenous insulin secretion is increased. In other words, insulin resistance is documented. In an effort to identify the pathogenetic mechanisms of lipoatrophic diabetes mellitus in this patient and to develop a therapeutic strategy, antibodies against different tissues and endocrinologic regulation were investigated. It was possible to demonstrate the presence of serum autoantibodies against lipocytes of the subcutis and other tissues, against hepatic stellate cells, together with autoantibodies against different endocrine organs. By studying the basis of diabetic abnormalities relating to the growth hormone (GH), the insulin-like growth factor (IGF) dynamics in this patient, i.e. reductions of GH, IGF-I, IGF-II, IGF-Binding protein (IGF-BP) 2 and IGF-BP 3, were detected. An immunosuppressive treatment strategy was not beneficial.

Identification of insulin-like peptides in cerebral ganglia neurosecretory cells of the mussel Mytilus edulis.

The immunostaining patterns of cerebral ganglia sections from the mussel Mytilus edulis with monoclonal antibodies raised against cerebral ganglia (CG) extracts were compared to those obtained with various polyclonal anti-insulin-like antibodies. One of the monoclonal antibodies (MAB 46) revealed clusters of positive cells in localization comparable to those revealed by the polyclonal antibodies. The nature of the antigen recognized by MAB 46 and the polyclonal antibodies was compared by gel filtration-HPLC of a cerebral ganglia extract. Similar peaks were revealed by the monoclonal and polyclonal antibodies. MAB 46 significantly inhibited the cerebral ganglia induced stimulation of amino-acid incorporation by mantle edge cell suspensions, suggesting that the antigen recognized by MAB 46 is involved in the control of growth.

Insulin-like growth factors enhance phagocytosis by human neutrophils in vitro.

Polymorphonuclear neutrophils (PMNs) were isolated from human blood, and PMN phagocytosis was assessed by measuring the chemiluminescence (CL) response in the presence of ZAP (opsonized zymosin particles containing luminol). The administration of 6.5 nM of insulin-like growth factor I (IGF-I), des(1-3)-IGF-I, IGF-II or insulin to PMNs for 20 min resulted in significant increases of the CL response for all test preparations. Des(1-3)-IGF-I, a truncated IGF-I with low affinity binding to IGF binding proteins (IGFBPs), was the most potent CL stimulator. The CL production evoked by 6.5 nM of des(1-3)-IGF-I was inhibited significantly by both 0.25 and 1.0 nM of EGTA (Ca2+ chelator), or 10 microM nifedipine (Ca2+ channel inhibitor), pertussis toxin (0.05 and 1.0 micrograms/ml) or cholera toxin (5 micrograms/ml). These results suggest that IGF-I and its homologues are potent stimulators of phagocytosis and that this action is modulated by IGFBP, and may require extracellular Ca2+ and/or IGF-I receptor G-protein coupling.

Quantification of antibodies to human growth hormone by high-performance protein G affinity chromatography with fluorescence detection.

The technique of high-performance affinity chromatography (HPAC) is applied to the quantitative determination of antibodies to human growth hormone (hGH) in serum from patients. An affinity column consisting of covalently immobilized protein G on a rigid support is used to capture the antibodies. Texas Red labeled hGH (hGH-TR) is used as a fluorescence probe for detecting the anti-hGH antibodies. Calibration curves are established by using a well-characterized monoclonal antibody to hGH (GHC101). The minimum detectable concentration (MDC) of anti-hGH antibody in serum is 250 ng/mL (this represents 10 ng of anti-hGH injected onto the protein G column). Analytical recoveries are 92-110% for seven replicates with 250-4000 ng/mL of GHC101. A precision of 15% relative standard deviation (RSD) can be achieved at the MDC. The precision is better above the detection limit. The linear dynamic range of the method is approximately 2 orders of magnitude. The total fluorescence recovery from the affinity column is greater than or equal to 96%. Sample analysis times are on the order of 20 min. The HPAC technique gives results in absolute units of concentration that correlate well with binding capacity values determined by radioimmunoassay.

Activation of osteoblast insulin-like growth factor-II/cation-independent mannose-6-phosphate receptors by specific phosphorylated sugars and antibodies induce insulin-like growth factor-II effects.

The phosphorylated monosaccharide, mannose-6-phosphate (M6P), causes a dose-dependent stimulation of alkaline phosphatase production by osteoblasts. The concentrations tested ranged from 0.1 to 30 mM. A maximal effect was reproducibly seen at 10-30 mM, and represented a 30% stimulation over control cells. Glucose-6-phosphate and fructose-1-phosphate also stimulated osteoblast alkaline phosphatase production, but not to the same extent as M6P. Sugar residues such as mannose, mannose-1-phosphate, and fructose-6-phosphate had no effect. The stimulatory effect of M6P is similar to that seen with insulin-like growth factor II(IGF-II). However, increasing doses of IGF-II did not further stimulate or add to the effect of 10 mM M6P. These data indicate that the mechanism for the transduction of the stimulatory signal may be similar for both IGF-II and M6P. They do not address, however, the possibility of separate or similar binding sites for the two agents. A specific polyclonal antibody to the IGF-II/cation-independent mannose-6-phosphate receptor (IGF-II/CI-MPR) elicits the same effects as M6P and IGF-II in these bone cells. Non-immune serum used as a control does not have any effect. These results suggest that activation of the osteoblast IGF-II/CI-MPR by either M6P or a specific antibody can evoke a biological response similar to that observed with IGF-II.