Progression-free and overall survival in ovarian cancer patients treated with CVac, a mucin 1 dendritic cell therapy in a randomized phase 2 trial.

BACKGROUND: CAN-003 was a randomized, open-label, Phase 2 trial evaluating the safety, efficacy and immune outcomes of CVac, a mucin 1 targeted-dendritic cell (DC) treatment as a maintenance therapy to patients with epithelial ovarian cancer (EOC). METHODS: Patients (n = 56) in first (CR1) or second clinical remission (CR2) were randomized (1:1) to standard of care (SOC) observation or CVac maintenance treatment. Ten doses were administered over 56 weeks. Both groups were followed for progression-free survival (PFS) and overall survival (OS). RESULTS: Fifty-six patients were randomized: 27 to SOC and 29 to CVac. Therapy was safe with only seven patients with Grade 3-4 treatment-emergent adverse events. A variable but measurable mucin 1 T cell-specific response was induced in all CVac-treated and some standard of care (SOC) patients. Progression free survival (PFS) was not significantly longer in the treated group compared to SOC group (13 vs. 9 months, p = 0.36, hazard ratio [HR] = 0.73). Analysis by remission status showed in the CR1 subgroup a median PFS of 18 months (SOC) vs. 13 months (CVac); p = 0.69 (HR = 1.18; CI 0.52-2.71). However CR2 patients showed a longer median PFS in the CVac-treated group (median PFS not yet reached, >13 vs. 5 months; p = 0.04, HR = 0.32 CI). OS for CR2 patients at 42 months of follow-up showed a difference of 26 months for SOC vs. > 42 months for CVac-treated (as median OS had not been reached; HR = 0.17 (CI 0.02-1.4) with a p = 0.07). CONCLUSIONS: CVac, a mucin 1-dendritic cell maintenance treatment was safe and well tolerated in ovarian cancer patients. A variable but observed CVac-derived, mucin 1-specific T cell response was measured. Notably, CR2 patients showed an improved PFS and lengthened OS. Further studies in CR2 ovarian cancer patients are warranted (NCT01068509). TRIAL REGISTRATION: NCT01068509. Study Initiation Date (first patient screened): 20 July 2010. Study Completion Date (last patient observation): 20 August 2013, the last patient observation for progression-free survival; 29 April 2015, the last patient was documented regarding overall survival.

A longitudinal analysis of maternal serum insulin-like growth factor I (IGF-I) and total and nonphosphorylated IGF-binding protein-1 in human pregnancies complicated by intrauterine growth restriction.

In cord blood and late gestation maternal serum, IGF-I is positively correlated with birth weight, whereas IGF-binding protein-1 (IGFBP-1) is inversely correlated with birth weight. Our goal was to determine whether maternal serum or amniotic fluid concentrations of IGF-I, IGFBP-1, or nonphosphorylated IGFBP-1 (npIGFBP-1) in early gestation predict later fetal growth abnormalities. Maternal serum was collected prospectively across gestation (5-40 wk) from 749 pregnant subjects. Amniotic fluid was collected after amniocentesis during wk 15-26 from 207 subjects. We compared median serum concentrations of IGF-I, IGFBP-1, and npIGFBP-1 in 38 subjects who delivered growth-restricted infants with the control group of 236 subjects with normal weight infants for each gestational age grouping, wk 5-12, 13-23, and 24-34. In the control group median IGF-I concentrations were 14.8, 11, and 15.6 nmol/liter for wk 5-12, 13-23, and 24-34, respectively, compared with 13.7, 14.3, and 10.6 nmol/liter in the intrauterine growth restriction (IUGR) group. Median IGFBP-1 concentrations were 8.5, 30.4, and 24.4 nmol/liter, respectively, in controls, compared with 11.4, 28.6, and 25.5 nmol/liter in the IUGR group. Median npIGFBP-1 concentrations were 6.9, 22, and 17.4 nmol/liter, respectively, in controls, compared with 5.0, 32.1, and 24.2 nmol/liter in the IUGR group. In the control group the median amniotic fluid IGFBP-1 level was 13,160 nmol/liter, and the median npIGFBP-1 level was 15,970 nmol/liter; in the IUGR group these levels were 13,440 and 18,440 nmol/liter, respectively. No clinically useful differences were found between the IUGR and control groups. Our results do not support the use of maternal serum IGF-I or IGFBP-1 or amniotic fluid IGFBP-1 or npIGFBP-1 early in gestation to predict later fetal growth restriction.

Transcriptional and posttranscriptional regulation of intraovarian insulin-like growth factor-binding proteins by interleukin-1beta (IL-1beta): evidence for IL-1beta as an antiatretic principal.

Intraovarian interleukin-1 (IL-1), a putative intermediary in the ovulatory cascade, has recently been implicated as an antiatretic agent. Given the reported antigonadotropic and thus atretogenic potential of granulosa cell-derived insulin-like growth factor-binding proteins (IGFBPs), we evaluated the ability of IL-1beta to regulate ovarian IGFBP-4 and -5, the IGFBP species elaborated by the rat granulosa cell. Treatment of whole ovarian dispersates of immature rat origin with increasing concentrations of IL-1beta for 96 h resulted in substantial and significant time-dependent inhibition of IGFBP-4 and IGFBP-5 transcripts compared with that in untreated controls. The IL-1 effect proved relatively specific in that no significant alterations in IGFBP transcripts were observed in the presence of select ovarian agonists, including transforming growth factor-alpha, tumor necrosis factor-alpha, endothelin-1, hepatocyte growth factor, keratinocyte growth factor, or basic fibroblast growth factor. The inhibitory effect of IL-1beta on ovarian IGFBP-4 and -5 expression was almost completely reversed in the presence of IL-1 receptor antagonist, suggesting mediation via a specific IL-1 receptor. The addition of actinomycin D to IL-1beta-pretreated whole ovarian dispersates produced a pattern of (IGFBP-4 and -5) messenger RNA decay indistinguishable from that noted for the untreated control group. Medium conditioned by IL-1beta-treated (but not untreated) whole ovarian dispersates displayed a marked diminution in the relative content of the IGFBP-4 and IGFBP-5 proteins (24- and 28- to 29-kDa proteins, respectively). Medium conditioned by IL-1beta-treated (but not untreated) whole ovarian dispersates proteolyzed [125I]IGFBP-5 (but not IGFBP-4) into fragments with apparent molecular masses of 18 and 14 kDa, respectively. In conclusion, our present observations demonstrate the ability of IL-1 to 1) inhibit the steady state levels of transcripts corresponding to IGFBP-4 and -5 in a time-dependent, relatively specific, and receptor-mediated fashion; 2) suppress the accumulation of the corresponding IGFBP proteins; and 3) stimulate the activity of the IGFBP-5-directed (but not IGFBP-4) endopeptidase, a posttranscriptional phenomenon. Our findings also suggest, by inference, that the IL-1beta-mediated inhibition of IGFBP-4 and -5 transcripts is due in part to a decrease in the rate of transcription of the corresponding genes and not to a change in the stability of the relevant messenger RNAs. Consequently, the ability of IL-1 to influence ovarian IGFBP economy appears multifaceted, comprising both transcriptional and posttranscriptional effects. To the extent that IGFBP-4 and -5 constitute atretogenic agents, our present findings support the view that IL-1beta may play an antiatretic role in the context of ovarian physiology.

Insulin-like growth factor binding protein-I levels are strongly associated with insulin sensitivity and obesity in early pubertal children.

In conditions associated with insulin resistance, insulin-like growth factor binding protein-I (IGFBP-I) levels have been shown to correlate inversely with insulin levels. Puberty is associated with insulin resistance and thus provides a model for comparing the relationship of IGFBP-I to both insulin levels and measures of insulin sensitivity. Our study population consisted of 104 healthy pubertal children, age 9.8-14.6 yr. Each subject had his/her insulin sensitivity (Si) assessed by the modified minimal model of Bergman, which employs a frequently sampled i.v. glucose tolerance test. Results showed that IGFBP-I levels were significantly higher in boys than in pubertally matched girls (P < 0.01). There was a strong positive correlation between IGFBP-I levels and Si (r = 0.65, P < 0.0001) and a weaker negative correlation with fasting insulin levels (r = -0.38, P < 0.0001). An inverse relationship was also found between IGFBP-I levels and body mass index (r = -0.46, P < 0.0001) and with IGF-I levels (girls only, r = -0.41, P < 0.003). Consequently, insulin sensitivity, obesity, and IGF-I are important predictors of IGFBP-I levels in pubertal children. It is possible that insulin-mediated suppression of IGFBP-I in obese children may increase free IGF-I levels and thus contribute to somatic growth. The same mechanism may operate in pubertal children, where insulin resistance and growth acceleration occur simultaneously.

The mouse intraovarian insulin-like growth factor I system: departures from the rat paradigm.

Although the rat intraovarian insulin-like growth factor I (IGF-I) system is well documented, the increasing availability of null mouse mutants for components of the IGF system necessitates characterization of the mouse model as well. Therefore, we undertook to define the components of the mouse intraovarian IGF-I system and to examine its operational characteristics. The cellular pattern of ovarian gene expression was comparable in the immature rat and mouse for IGF-I and the type I IGF receptor. In both species, IGF-I messenger RNA (mRNA) is selectively expressed by granulosa cells in growing, healthy appearing follicles. Type I IGF receptor mRNA was also concentrated in granulosa cells, but was uniformly expressed in all follicles large and small, healthy and atretic appearing alike. Cellular patterns of IGF-binding protein (IGFBP) gene expression were similar in mouse and rat, except in the case of IGFBP-2. IGFBP-2 mRNA was localized to the mouse granulosa cell, in contrast to its concentration in the rat thecal-interstitial compartment. This difference in IGFBP expression pattern was also noted in cultured mouse and rat granulosa cells. Although immunoreactive IGFBP-4 (24 and 28 kDa) and IGFBP-5 (29 kDa) were shared by both species, the cultured mouse granulosa cell also featured immunoreactive IGFBP-2 (30 kDa). The mouse paradigm further differed from its rat counterpart in that a maximal dose of FSH, previously shown to suppress the elaboration of rat granulosa cell-derived IGFBPs, was without effect. The addition of IGF-I proved stimulatory to the accumulation of the 28- to 29-kDa IGFBPs, as previously reported for the rat. However, IGF-I proved inhibitory to the accumulation of the 24-kDa IGFBP (presumptive nonglycosylated IGFBP-4); no consistent effect was reported for the rat model. Functional comparisons of mouse and rat ovarian cell cultures revealed qualitatively comparable FSH-stimulated steroidogenesis, disposition of radiolabeled pregnenolone, IGF-I-amplified FSH action, and IGFBP-mediated antigonadotropic activity. These findings indicate that the mouse intrafollicular IGF-I system differs from the rat paradigm in both the makeup and regulation of granulosa cell-derived IGFBPs as well as in the intensity and character of the steroidogenic process. Studies employing the mouse model must take into account these important distinctions relative to the more established rat paradigm.

Direct administration of insulin-like growth factor to fetal rhesus monkeys (Macaca mulatta).

A potential treatment for the amelioration of fetal growth failure is insulin-like growth factor-I (IGF-I). To address concerns of safety and efficacy, IGF-I (80 microg/kg; GroPep Pty.) was administered i.p. to healthy rhesus monkey fetuses via ultrasound guidance every other day between gestational days (GD) 110-120 and 130-140 (third trimester; term = approximately GD 165 +/- 10; n = 6). Pregnancies were monitored sonographically, and fetal/maternal blood samples were collected for complete blood counts, immunophenotyping, and biochemical analyses. Blood samples, external measures of the fetus and newborn, and tissue and organ weights were collected at fetal necropsy (GD 150; n = 2) or at term delivery of neonates (GD 160; n = 4). The results of these investigations have shown no evidence of hypoglycemia in the fetus or dam during the course of treatment. Circulating concentrations of fetal, but not maternal, IGF-I increased with treatment (approximately 80 to approximately 1015 ng/ml), and there was no evidence of a change in serum IGF-II or an increase in IGF binding protein-3 compared with historical control values. Fetal lymphocytes and select red cell parameters increased, and a significant elevation in circulating B cells and CD4/CD8 ratios in fetal lymph nodes was shown. Although no changes were detected in body weights, increases in thymic, splenic, and kidney weights and small intestine lengths occurred. Thus, administration of IGF-I to the fetal monkey is safe and results in 1) transient increases in circulating IGF-I, 2) a significant effect on fetal hematopoietic and lymphoid tissues, and 3) an increase in select fetal organ weights and measures. These data suggest that IGF-I may represent a potential candidate for therapeutic treatment of growth-compromised human fetuses in utero.

Adjunctive growth hormone during ovarian hyperstimulation increases levels of insulin-like growth factor binding proteins in follicular fluid: a randomized, placebo-controlled, cross-over study.

GH increases circulating insulin-like growth factor I (IGF-I), which can promote the growth and differentiated function of ovarian granulosa and theca cells. Reported studies of GH as an adjunct to menotropin stimulation in women, largely those with ovarian dysfunction, have not consistently shown a benefit of GH, despite increases in serum and follicular fluid IGF-I. We hypothesized that changes in intrafollicular IGF-binding proteins (IGFBPs), which can antagonize IGF actions on granulosa cells, may underlie the inconsistent effects of GH. In the present study of GH, administered in double-blind, placebo-controlled, cross-over fashion to regularly cycling women undergoing in vitro fertilization, we found that follicular fluid levels of IGFBP-1, -3, and -4 and serum levels of IGFBP-3, as well as follicular fluid and serum IGF-I, were significantly increased in the GH-treated cycles, when compared with the placebo cycle of the same patient. We suggest that the net increase in intrafollicular IGFBPs in GH cycles may mitigate the potential beneficial effect of increased IGF-I.

Two-year treatment of growth hormone (GH) receptor deficiency with recombinant insulin-like growth factor I in 22 children: comparison of two dosage levels and to GH-treated GH deficiency.

We have reported 1-yr results of a double blind, placebo-controlled trial of recombinant human insulin-like growth factor I (rhIGF-I) replacement in 16 children from the Ecuadorian GH receptor-deficient (GHRD) population. This report extends observations of rhIGF-I efficacy at two dosage levels [120 micrograms/kg BW twice daily (n = 15) and 80 micrograms/kg twice daily (n = 7)] over 2 yr, compares biochemical responses [serum IGF-I and IGF-binding protein-3 (IGFBP-3)] and their relationship to growth effects, and compares treatment effects of rhIGF-I in GHRD to rhGH in idiopathic GH deficiency (GHD). There were no baseline differences between the low and high dose groups for growth velocity (GV), bone age (BA), SD score for height, or percent mean body weight for height (MBWH). Over 2 yr of rhIGF-I treatment, there were no differences in GV or in changes in height SD score, height age (HA), or BA between the two groups; a subgroup of six subjects at the higher dose followed for a third year continued at the second year GV. The higher dose resulted in a greater change in percent MBWH. GV in yr 1 and 2 for the entire group and in yr 3 for a subgroup were greater for GH-treated GHD (n = 11). The GHD group showed a greater change in SD score for height and HA, but did not differ from the rhIGF-I-treated GHRD group in the change in BA (delta BA) or delta HA/delta BA over 2 yr. There was a greater change in percent MBWH in GHRD. There were no differences between dosage groups for serum IGF-I levels at baseline or the near-normal trough levels 12 h after rhIGF-I injection; these individual levels correlated with HA gain in yr 1 and 2. IGFBP-3 levels were markedly low, with no changes of significance with treatment. Comparable growth responses to the two dosage levels and the biochemical changes indicate a plateau effect at or below 80 micrograms/kg BW twice daily. The growth response and favorable trough levels of IGF-I despite the overall lack of increase in circulating IGFBP-3 levels suggest an alternative mechanism for sustaining IGF-I levels and avoiding rapid clearance of rhIGF-I. The greater increase in MBWH with treatment of GHRD than with treatment of GHD may reflect comparable effects on lean body mass without the lipolytic effects of GH in the GHRD subjects. The difference in growth response between rhIGF-I-treated GHRD and rhGH-treated GHD groups is consistent with the hypothesis that 20% or more of GH-influenced growth is due to the direct effects of GH on bone. Nonetheless, the comparable delta HA/delta BA suggests similar long term effects of replacement therapy in the two conditions.

Activin-attenuated expression of transcripts encoding granulosa cell-derived insulin-like growth factor binding proteins 4 and 5 in the rat: a putative antiatretic effect.

Given the suggestion that intraovarian insulin-like growth factor (IGF)-binding proteins (IGFBPs) may constitute markers of follicular atresia, we investigated the possibility that activin, a putative antiatretic principle, may modulate granulosa cell-derived IGFBPs. Untreated granulosa cells cultured for 72 h exhibited a progressive increase in the steady-state levels of transcripts corresponding to IGFBP-4 and IGFBP-5 (1.5-fold and 12-fold, respectively). Transcript levels corresponding to IGFBP-5 were consistently higher than their IGFBP-4 counterparts. Treatment with activin-A (50 ng/ml) for 72 h produced significant (p < 0.05) decrements in the steady-state levels of IGFBP-4 and IGFBP-5 transcripts (46% and 79%, respectively) as compared to controls. Thus, treatment with activin-A appears to be capable of blocking the spontaneous increase in IGFBP-4 and IGFBP-5 transcripts exhibited by untreated cultured granulosa cells. Consistent activin-A-induced decrements were also observed in the accumulation of the IGFBP-5 (but not the IGFBP-4) protein. Dose-response analysis revealed monophasic dose dependence (half maximal inhibitory doses of 16.2 and 7.8 ng/ml for IGFBP-5 and IGFBP-4 transcripts, respectively). The addition of increasing concentrations of the putative activin-binding protein, follistatin, produced dose-dependent reversal of the activin-A effect on IGFBP transcripts (IGFBP-5 > IGFBP-4). Activin-B was as effective as activin-A in reducing IGFBP-4 transcripts (31% decrement, p < 0.05) whereas it had little or no effect on IGFBP-5 transcripts (21% decrement, p > 0.1). No apparent effect was observed for the corresponding proteins. Activin-A action was specific in that treatment with transforming growth factor (TGF)-beta1, inhibin-A, or Müllerian-inhibiting substance (MIS)--all related peptides--failed to produce statistically significant alterations in the steady-state levels of IGFBP-4 and IGFBP-5 transcripts. Taken together, these observations reveal that activin-A exerts a substantial, relatively rapid, follistatin-neutralizable, dose- and time-dependent inhibitory effect on granulosa cell-derived IGFBP transcripts (IGFBP-5 > IGFBP-4). Other members of the TGFbeta superfamily (e.g., inhibin-A, TGFbeta1, and MIS) were without significant effect on the expression of IGFBP-4 and IGFBP-5. To the extent that the inhibition of IGFBP-4 and IGFBP-5 expression is associated with, and possibly causally related to, the promotion of follicular health, the present observations are in keeping with the proposition that activin may play an antiatretic role in the dynamic process of follicular selection.

The effect of GH therapy on the immunoreactive forms and distribution of IGFBP-3, IGF-I, the acid-labile subunit, and growth rate in GH-deficient children.

We have previously shown that the major correlates of growth following growth hormone (GH) therapy in growth hormone-deficient (GHD) children are changes in circulating insulin-like growth factor-I (IGF-I) and IGF binding protein-3 (IGFBP-3), suggesting a synergistic interaction between IGF-I and IGFBP-3 (1). The first aim of this project was to examine the molecular forms of IGFBP-3 and the acid-labile subunit (ALS), and to assess the changes in these molecular forms during GH administration to GHD children. Plasma samples from prepubertal GHD patients, prior to therapy and during the first year of GH treatment, were subjected to Western ligand and immunoblot analysis. Densitometric analysis of Western ligand blotting (WLB) showed a 76% increase in IGFBP-3 (p = 0.02), but a 56% decrease in 36-kDa IGFBP-2 (p = 0.03) during GH therapy. Western immunoblot (WIB) analysis of IGFBP-3 revealed the presence of intact (40- to 45-kDa doublet) as well as a proteolyzed (28-kDa) form of IGFBP-3 in the serum of GHD and healthy children. Both immunoreactive forms of IGFBP-3 increased by 64% during GH therapy (intact p = 0.003; proteolyzed p = 0.0001). WIB analysis of the ALS showed an 84-to 86-kDa doublet, which increased by 41% with GH therapy (p = 0.01). The response to GH therapy, as measured by the height velocity standard deviation score (SDS) adjusted for bone age, correlated with the percent change in total IGFBP-3 (r = 0.772, p = 0.002 by WIB), intact IGFBP-3 (r = 0.845, p = 0.0005 by WLB; r = 0.541, p = 0.05 by WIB), and proteolyzed IGFBP-3 (r = 0.703, p = 0.007), as well as with the percent change in ALS (r = 0.813, p = 0.014). The second aim of this project was to assess the changes in distribution of the immunoreactive forms of IGFBP-3 and IGF-I among the ternary (ALS/IGFBP-3/IGF) complex, the binary (IGFBP-3/IGF) complex, and uncomplexed IGF during the first year of GH therapy, and to explore further the correlation with growth response to GH. Plasma samples, prior to therapy and after the first year of GH treatment, were separated by neutral size-exclusion chromatography and then subjected to IGFBP-3 immunoradiometric assay (IRMA), IGFBP-3 WIB, and IGF-I IRMA analysis. IGFBP-3 increased in both the ternary (p < 0.0001) and binary (p = 0.01) complexes, but there was a shift in the percentage of IGFBP-3 from the binary to the ternary complex during GH therapy. Both intact and proteolyzed forms of IGFBP-3 were found in both the ternary and binary complexes, but the shift occurred primarily for the proteolyzed (28-kDa) form (p = 0.001). There was a significant increase in IGF-I in the ternary (p = 0.001) and binary (p = 0.005) complexes, but not in uncomplexed IGF-I. The percentage of IGF-I in the ternary complex increased (p = 0.006), whereas the percentage of uncomplexed IGF-I decreased (p = 0.02), during GH therapy. Growth rate, assessed by the height velocity SDS for bone age, correlated best with the changes in ternary complex IGFBP-3 (r = 0.72, p = 0.01) and ternary complex IGF-I (r = 0.56, p = 0.10). In conclusion, GH treatment of GHD children results in significant increases of intact, proteolyzed, and total IGFBP-3, as well as an increase in ALS, which all correlate with the growth response to GH therapy. In addition, GH treatment results in increases in ternary complex IGFBP-3 and IGF-I, which also correlate with the response to therapy. We suggest that the formation of the ternary complex may be a determining factor in the somatic growth response.

Immunoblot studies of the acid-labile subunit (ALS) in biological fluids, normal human serum and in children with GH deficiency and GH receptor deficiency before and after long-term therapy with GH or IGF-I respectively.

OBJECTIVE: The aims of this investigation were (a) to study the presence of immunoreactive forms of the acid-labile subunit (ALS) in different human biological fluids, (b) to define the age dependence of serum ALS in normal children and adults and (c) to compare the regulation of ALS by GH or IGF-I in children with GH deficiency (GHD) and GH receptor deficiency (GHRD) before and after 1 year of therapy with GH or IGF-I, respectively. DESIGN AND PATIENTS: Selected human biological fluids from different consenting volunteers and serum from 68 normal children and 5 adults were analysed. Four children diagnosed as GHD and 7 children diagnosed as GHRD were treated with recombinant human (rh) GH at a dosage of 0.05 mg/kg/day s.c. or rhIGF-I at a dosage of 120 micrograms/kg twice daily s.c., respectively, for 12 months. MEASUREMENTS: Immunoreactive forms of ALS were studied by Western immunoblot using a specific rabbit antiserum derived against synthetic human ALS and quantified by laser densitometry analysis. Serum from children with GHD or GHRD were sampled before and at 6 and 12 months of therapy; serum from these patients had been also assayed at baseline for determination of IGF-I and IGF binding protein (IGFBP)-3 by radioimmunoassay and immunoradiometric assay, respectively. RESULTS: An immunoreactive 85 kDa doublet of ALS was detected in serum, plasma, follicular, peritoneal and synovial fluid, but not in urine, seminal plasma, amniotic or extra-embryonic coelomic fluids. Assessment of serum from newborns to adults revealed an age dependence; the ALS doublet was low, but detectable, in newborns, increased during adolescence and remained constant in adulthood. ALS levels were significantly lower in GHD (P = 0.02) and in GHRD children (P = 0.001) than in age-matched controls. Treatment with rhGH in GHD children produced a 2.7-fold increase in serum ALS concentrations at 6 months of therapy (P = 0.01), which was maintained after 1 year of treatment (P = 0.006), leading to normalization of ALS concentrations. In contrast, administration of rhIGF-I to GHRD children failed to increase and normalize serum ALS levels either at 6 or 12 months of therapy. CONCLUSIONS: Immunoreactive forms of acid-labile subunit are present in serum and plasma, as well as in follicular, peritoneal and synovial fluids, suggesting that acid-labile subunit can either cross the capillary barrier or be secreted locally. Acid-labile subunit concentrations are age-dependent with a sharp increase during adolescence, and are reduced in GH deficient and GH receptor deficient children. While treatment with rhGH is able to increase and normalize acid-labile subunit concentrations in GH deficient children, therapy with rhIGF-I fails to increase serum acid-labile subunit levels in GH receptor deficient patients. These data suggest that acid-labile subunit is directly GH-regulated, and that IGF-I cannot increase acid-labile subunit levels, as assessed by Western immunoblot.

Characterization of an insulin-like growth factor binding protein-5 protease produced by rat articular chondrocytes and a neuroblastoma cell line.

Both articular cartilage and the central nervous system are target organs for insulin-like growth factors (IGFs). We have previously described the hormonal regulation of IGF binding proteins (IGFBPs) in the conditioned media (CM) of rat articular chondrocytes and in a rat neuroblastoma cell line (B104). In the studies presented here, we have investigated the role of IGFBP-5 proteases in these complex systems. Proteolysis of [125I] IGFBP-5 was maximal after 2-3 h incubation with CM of either cell type and did not further increase, even with an incubation of 12 h. Assessment of the effect of pH on protease activity showed that proteolysis was active between pH 6 and pH 9, but not at more acidic pH. Among the various protease inhibitors, serine protease inhibitors [benzamidine (100 mM), aprotinin (1 mg/ml), PMSF (10 mM)] and metalloprotease inhibitors [EDTA (1 mM), 1,10-phenanthroline (10 mM)] were the most effective in inhibiting the proteolysis of IGFBP-5, whereas aspartic and cysteine protease inhibitors were ineffective. These results indicate that the IGFBP-5 protease in the conditioned medium of rat articular chondrocytes and B104 cells belongs to a family of serine-metallo proteases. Interestingly, divalent cations, such as Zn+2 (1 mM) and Ca+2 (10 mM) also inhibited the IGFBP-5 proteolysis. This effect was not observed with monovalent ions, such as Na+ and K+. We also examined the effect of IGFs on IGFBP-5 protease activity, and found that IGF-I and -II inhibited the proteolysis in cell-free conditioned medium, while des(1-3) IGF-I was less effective. The IGFs may act to protect [125I] IGFBP-5 from the proteases in the CM, although the precise mechanism remains unknown. Thus, IGFBP-5 protease activity produced by both rat articular cartilage and B104 cells is a serine-metallo protease, that is inhibited by divalent cations and in the presence of IGF.

Regulation of postnatal intestinal maturation by growth hormone: studies in rats with isolated growth hormone deficiency.

During the 3rd wk of postnatal life in the rat, dramatic maturational changes occur in the structure and function of the small intestine, enabling the animal to make the transition from milk to solid food. To investigate the role of GH in the regulation of this complex process, we studied postnatal intestinal maturation in the spontaneous dwarf rat, a strain of Sprague-Dawley rats with an autosomal recessive mutation in the GH gene resulting in complete but isolated GH deficiency. GH-deficient and GH-normal littermates were studied at d 7 and 14 (suckling) and d 23 (postweaned). The body weight of GH-deficient animals was inhibited by 60% at each age. Longitudinal growth of the small intestine was not inhibited, suggesting that longitudinal small bowel growth is independent of GH regulation. Mucosal cell mass was significantly lower in GH deficiency at all ages studied, and digestive hydrolase capacity per cm of intestine was significantly lower in GH-deficient postweaned animals. However, epithelial cell mass increased markedly in association with weaning and the maturation of lactase, sucrase, and aminooligopeptidase proceeded normally in GH deficiency. These data suggest that, although GH is not required for normal postnatal intestinal maturation, the mucosal epithelial hypoplasia found in GH-deficient animals suggests that GH or GH-dependent factors act as an intestinal mucosal growth factor whose function is to promote the homeostatic or steady-state regulation of mucosal epithelial growth.

Insulin-like growth factor-binding proteins (IGFBPs) and their regulatory dynamics.

The IGFBPs are a family of homologous proteins that have co-evolved with the IGFs and that confer upon the IGF regulatory system both functional and tissue specificity. IGFBPs are not merely carrier proteins for IGFs, but hold a central position in IGF ligand-receptor interactions through influences on both the bioavailability and distribution of IGFs in the extracellular environment. In addition, IGFBPs appear to have intrinsic biological activity independent of IGFs. The current status of research on IGFBPs is reviewed herein. Following a brief introduction to the entire IGF/IGFBP system, separate sections for each of the six cloned mammalian IGFBPs, the most extensive for IGFBP3, cover selected topics that emphasize the dynamics of IGFBPs--that is, their regulation in cells, their functionally important post-translational modifications, and their interactions in the cellular microenvironment--and how these dynamics influence physiological function.

Assays for insulin-like growth factors and their binding proteins: practicalities and pitfalls.

Insulin-like growth factors (IGFs) are growth hormone-dependent anabolic peptides that circulate in biologic fluids complexed to a family of IGF binding proteins. Measurement of the serum concentrations of IGF peptides and IGF binding proteins has proved to be an effective means of evaluating functional growth hormone status and makes it possible to establish a diagnosis of IGF deficiency.

High-dose growth hormone treatment of short children born small for gestational age.

The effect of GH administration was evaluated over 2 yr in 50 short, prepubertal, non-GH deficient children born small for gestational age, who had been randomly allocated to a group receiving no treatment or daily sc GH treatment at a dose of 0.2 or 0.3 IU/kg. At the start of the study, mean age was 5.2 yr, bone age was 4.0 yr, height SDS was -3.5, height velocity SDS was -0.8, weight SDS was -2.7, and body mass index SDS was -1.9. Catch-up growth was observed in none of the untreated and all of the treated children. The response to GH treatment included a near doubling of growth velocity and of weight gain and a mean height increment of more than 2 SDS. GH treatment was associated with a distinct acceleration of bone maturation. The differences between the growth responses evoked by the two GH doses were minor. The prepubertal GH-induced catch-up growth was associated with elevated serum concentrations of insulin, insulin-like growth factor-I, insulin-like growth factor binding protein-3, and osteocalcin, whereas insulin-like growth factor-II levels remained unaltered. GH treatment was well tolerated. In conclusion, high-dose GH administration over 2 yr is emerging as a potential therapy to increase the short stature that results from insufficient catch-up growth in young children born small for gestational age. The long-term impact of this approach remains to be delineated.

Transcriptional and post-translational regulation of insulin-like growth factor-binding protein-5 in rat articular chondrocytes.

The aim of this study was to assess the regulation of insulin-like growth factor-binding proteins (IGFBPs) by IGFs in primary cultures of rat articular chondrocytes (RAC). Employing Western ligand blotting, immunoprecipitation and Northern blot analysis, RAC were found to secrete IGFBP-5 (29 kDa) and IGFBP-4 (24 kDa) as the predominant IGFBPs, as well as IGFBP-2 (32-30 kDa) and IGFBP-3 (43-39 kDa) as the minor species. Treatment of cells with IGF-I and IGF-II resulted in a dose-dependent increase of IGFBP-5 and a small increase in IGFBP-4 in conditioned media (CM). Des(1-3) IGF-I and [Gln6, Ala7, Tyr18, Leu19] IGF-II ([QAYL] IGF-II), which bind to the type 1 IGF receptor but not to IGFBPs, also induced IGFBP-5 peptide, although the increase was less than with IGF-I or IGF-II treatment of RAC. [Leu27] IGF-II, which does not bind to the type 1 IGF receptor but binds to IGFBPs, resulted in little induction of IGFBP-5, while [QAYL-Leu27] IGF-II, which has reduced affinity for both the type 1 IGF receptor and IGFBPs, did not increase IGFBP-5. These data suggest that the increase in IGFBP-5 in CM is modulated by both the type 1 IGF receptor and the interaction between IGFs and IGFBPs. Northern blotting analysis showed that IGF-I, IGF-II and des(1-3) IGF-I treatment of RAC increased steady state levels of IGFBP-5 mRNA, suggesting that the IGF-mediated increase in IGFBP-5 is transcriptionally modulated. Interestingly, the increase in IGFBP-5 peptide levels and mRNA were not parallel, suggesting the possibility of post-translational modifications of IGFBP-5, such as those seen with IGFBP-5 protease. IGFBP-5 protease activity was detectable in untreated CM, whereas treatment with IGF-I and IGF-II partially protected IGFBP-5 from proteolysis. In summary, treatment of RAC with IGF-I and IGF-II results in dose-dependent increases in both IGFBP-5 peptide in the CM and mRNA levels. These changes are mediated by interactions via the type 1 IGF receptor as well as IGFBPs, both transcriptionally and post-translationally.

Identification and characterization of insulin-like growth factors (IGFs), IGF-binding proteins (IGFBPs), and IGFBP proteases in human synovial fluid.

The insulin-like growth factors (IGFs) and IGF-binding proteins (IGFBPs) in human synovial fluid play an important role in maintaining articular cartilage metabolism. In this study we measured the concentrations of IGF-I, IGF-II, and IGFBP-3 in normal human synovial fluid by RIA and characterized the IGFBPs by Western ligand blot (WLB), Western immunoblot, and immunoprecipitation. We also extended the study and compared normal synovial fluid to synovial fluids from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). The concentrations of IGF-I, IGF-II, and IGFBP-3 in normal synovial fluid were 19 +/- 3 (mean +/- SE), 194 +/- 14, and 349 +/- 65 ng/mL, respectively. In synovial fluid of patients with OA, IGF-I levels were elevated, whereas IGF-II was decreased, and the IGFBP-3 level was similar to the control value. In patients with RA, both IGF-I and IGFBP-3 were elevated, whereas IGF-II remained unchanged. WLB and immunoprecipitation of normal synovial fluid revealed IGFBP-1 (26-29 kDa), IGFBP-2 (32 kDa), IGFBP-3 (42- to 39-kDa doublet), and IGFBP-4 (24 kDa); the IGFBP-3 doublet was very faint. In RA synovial fluid, all IGFBPs were dramatically increased, whereas little change was seen in the synovial fluid of OA. Western immunoblot against IGFBP-3 revealed a prominent 30-kDa immunoreactive fragment of IGFBP-3 in synovial fluids of normal adults as well as in those of RA and OA patients. This was concurrent with detectable IGFBP-3 protease activity, which was characterized to be of the metallo- and serine protease family. Thus, in normal synovial fluid, there is a balance of circulating IGF, IGFBP, and proteases to modulate the bioactivity of IGF. In pathological states, the increased IGF-I concentrations were accompanied by an increase in IGFBP-I concentrations were accompanied by an increase in IGFBP-3 levels in synovial fluid. These findings suggest that alteration of the IGF and IGFBP axis in pathological states may be important for understanding the underlying pathophysiology of disordered articular growth and metabolism.

IGF-II and IGF-binding proteins increase dramatically during rabbit pregnancy.

During pregnancy, changes in the IGF axis are associated with changes in maternal metabolism and nutrient repartitioning which are necessary to meet the demands of a growing conceptus. The aim of this study was to assess the IGF axis, maternal weight changes and food intake in female New Zealand White rabbits (n = 7) prior to breeding (day 0) and serially throughout pregnancy until term (day 30-31). The total weight of the pregnant does progressively increased from 4.03 +/- 0.06 kg (mean +/- S.E.M.) on day 0 to 4.47 +/- 0.07 kg on day 30 (P < 0.001). Maternal tissue mass (total weight minus estimated conceptus weight) increased until day 18, plateaued to day 22/23, and then significantly declined. On day 30, the maternal tissue mass was not significantly different from the non-pregnant value, such that the final increase in total weight was due to conceptus growth. Although the does were fed ad libitum, food intake did not change until day 29 when it decreased to approximately 50% of previous intake (P < 0.01). Maternal serum IGF-I was 499 +/- 32 ng/ml on day 0, reached a peak of 832 +/- 160 ng/ml on day 21 (P < 0.02), and then declined to 341 +/- 49 ng/ml on day 30. In contrast, serum IGF-II increased dramatically from a non-pregnant level of 85 +/- 14 ng/ml to 16,295 +/- 2488 ng/ml on day 23 (P < 0.001), and then rapidly declined (3335 +/- 954 ng/ml, day 30). Changes in serum IGF-binding proteins (IGFBPs) followed a pattern similar to IGF-II, as assessed by Western-ligand blotting. All IGFBPs, especially the 45-40 kDa IGFBP-3 doublet, increased dramatically between days 12 and 24 of pregnancy, and then declined towards term. In conclusion, we observed unique and dramatic changes in the maternal serum IGF axis that corresponded to periods of maternal weight gain and loss. The tissue source of IGFs and IGFBPs remains undetermined, although it is of note that the time when major changes in the IGF axis were first observed coincided with the time of functional change from yolk sac to placenta in the rabbit.

Collection of blood in heparinized tubes does not alter the molecular distribution or forms of IGFBP-3 and IGF.

The major serum carrier of the insulin-like growth factors (IGFs) is IGF-binding protein-3 (IGFBP-3) that exists in the circulation associated with IGF and an acid labile subunit to form a ternary (158-kDa) complex. It has been reported that heparin disrupts the IGF carrying capacity of the ternary complex and is a potent inhibitor of ternary complex reformation (Clemmons et al., 1983; Baxter, 1990). Thus, the aim of this study was to determine if, in a clinical setting where blood may be collected in both nonheparinized and heparinized tubes, heparin alters the molecular distribution or immunoreactive measurement of IGFBP-3 and IGF-I. Two different collection modalities were examined: protocol 1, blood was drawn and immediately centrifuged and aliquotted; and protocol 2, blood was drawn, left at room temperature for 2 h and then at 4°C overnight prior to centrifugation. Samples were drawn from a normal adult and from a growth hormone-deficient (GHD) child and subjected to neutral size-exclusion chromatography to separate the ternary 158-kDa complex from the binary IGFBP-3-IGF (approx 50 kDa) complex. Fractions were then subjected to Western ligand blot (WLB), western immunoblot (WIB), and measurement of IGFBP-3 by immunoradiometric assay (IRMA), while the IGF distribution was measured by radioimmunoassay (RIA) following acidic size-exclusion chromatography. In both serum and plasma of a normal adult, WLB detected a 45-40-kDa IGFBP-3 doublet eluting primarily within the 158-kDa IGFBP region (i.e., ternary complex). Similarly, assessment of immunoreactive IGFBP-3 by WIB showed a 45-40-kDa IGFBP-3 doublet, as well as a 29 kDa immunoreactive form primarily eluting in the 158-kDa IGFBP region of the chromatography. Measurement of IGFBP-3 by IRMA confirmed these findings. No difference between serum and plasma was detected in either collection protocol. RIA of IGF-I revealed that the ternary complex carried the majority of the circulating IGF-I and that there was no difference between serum and plasma. Assessment of serum and plasma of a GHD child showed reduced serum concentrations of IGFBP-3 but no difference in the IGFBP profiles between serum and plasma. These data demonstrate that the collection of blood in heparinized tubes does not alter the molecular distribution or forms of IGFBP-3 and IGF-I.