Variation in the clinical management of self-harm by area-level socio-economic deprivation: findings from the multicenter study of self-harm in England.

BACKGROUND: We investigated disparities in the clinical management of self-harm following hospital presentation with self-harm according to level of socio-economic deprivation (SED) in England. METHODS: 108 092 presentations to hospitals (by 57 306 individuals) after self-harm in the Multicenter Study of Self-harm spanning 17 years. Area-level SED was based on the English Index of Multiple Deprivation. Information about indicators of clinical care was obtained from each hospital's self-harm monitoring systems. We assessed the associations of SED with indicators of care using mixed effect models. RESULTS: Controlling for confounders, psychosocial assessment and admission to a general medical ward were less likely for presentations by patients living in more deprived areas relative to presentations by patients from the least deprived areas. Referral for outpatient mental health care was less likely for presentations by patients from the two most deprived localities (most deprived: adjusted odd ratio [aOR] 0.77, 95% CI 0.71-0.83, p < 0.0001; 2nd most deprived: aOR 0.80, 95% CI 0.74-0.87, p < 0.0001). Referral to substance use services and 'other' services increased with increased SED. Overall, referral for aftercare was less likely following presentations by patients living in the two most deprived areas (most deprived: aOR 0.85, 95% CI 0.78-0.92, p < 0.0001; 2nd most deprived: aOR 0.86, 95% CI 0.79-0.94, p = 0.001). CONCLUSIONS: SED is associated with differential care for patients who self-harm in England. Inequalities in care may exacerbate the risk of adverse outcomes in this disadvantaged population. Further work is needed to understand the reasons for these differences and ways of providing more equitable care.

A case of significant loss of mesenteric fat: Educational and clinical relevance.

Mesenteric fat, a type of intraperitoneal adipose tissue, plays a critical role in protection and the immune response. Loss of mesenteric fat is a known consequence of a variety of clinical conditions; however, visual documentation of this rare occurrence is not available in the literature searched. Here we report a case of significant loss of mesenteric fat identified during educational dissection of a 79-year-old male fresh frozen donor cadaver, causing the mesenteric folds to appear transparent. The gross anatomical characteristics, clinical importance, and educational significance of this abnormality are described in this report. Knowledge of this condition may be of interest to clinicians, and documentation could benefit anatomists and educators dissecting and teaching in the laboratory setting.

Tensor fasciae suralis - Prevalence study and literature review.

Tensor fasciae suralis (TFS) is an accessory muscle of the posterior lower extremity. Although TFS has been documented in cadaveric and radiological reports, its prevalence remains unknown. The TFS variant is noteworthy to anatomists, as it may be encountered in the dissection laboratory, and clinicians, as the muscle could potentially cause confusion during physical examination or diagnostic imaging. Multiple variations of TFS have been reported in the literature, suggesting the need for a classification system. We dissected 236 formalin-fixed cadaveric lower limbs to determine the prevalence of TFS. The PubMed and MEDLINE databases were searched to compare the anatomical features of independent TFS case reports. In our prevalence study, the TFS muscle was identified in three lower limbs (1.3%). In total, 38 cases of TFS (32 cadaveric and six radiological) were identified in the literature. Our literature review revealed that the accessory muscle most often arises as a single head from the long head of the biceps femoris, yet many other presentations have been documented. The need for a classification system to distinguish between the subtypes of TFS became apparent during the literature review. Tensor fasciae suralis is a rare muscle, present in only 3 of 236 (1.3%) cadaveric lower limbs dissected in this study. We propose the use of a classification system, based on muscle origin and number of heads, to better organize the subtypes of TFS.

The Association of Early Dietary Supplementation with Vitamin E with the Incidence of Ulcerative Dermatitis in Mice on a C57BL/6 Background: Diet and Ulcerative Dermatitis in Mice.

The purpose of this study was to ascertain if prophylactic ingestion of a diet rich in vitamin E would prevent or impede the development of ulcerative dermatitis in mice on a C57BL/6 background. Mice were fed either a standard mouse diet, vitamin E (99 IU/kg), or a mouse diet fortified with vitamin E (3000 IU/kg) after weaning. Cases of ulcerative dermatitis were recorded by individuals unmasked to the diet assignment. The incidence of ulcerative dermatitis in a retrospective cohort of mice on standard diet was compared with the group on the diet fortified with vitamin E. Age was associated with ulcerative dermatitis in standard diet and vitamin E fortified diet groups, r = 0.43, p-value < 0.0001 and r = 0.18, p-value < 0.02, respectively. The average age of incidence for ulcerative dermatitis in the mice fed the standard diet was 89 weeks and for the mice fed the vitamin E diet it was 41 weeks. The unadjusted odds ratio comparing the incidence of ulcerative dermatitis between the two diet groups was 4.6 with a 95% confidence interval of (2.44, 8.58), χ(2) p-value < 0.0001. Therefore, there was an association between the diets and ulcerative dermatitis, with the mice on the vitamin E fortified diet having almost five times the odds of having ulcerative dermatitis compared with mice on the standard diet. Incidence of ulcerative dermatitis was not influenced by sex or genotype. Our study results show that a diet fortified in vitamin E initiated at weaning does not prevent or impede the development of ulcerative dermatitis in mice on a C57BL/6 background and may accelerate development when administered to young mice.

Effect of neonatal modulation of cholesterol homeostasis on subsequent response to cholesterol challenge in adult guinea pig.

Experiments were designed to study whether or not the mechanism of handling dietary cholesterol in adulthood can be modulated by the manipulation of cholesterol homeostasis during neonatal period. The effects of enhancing cholesterol degradation (cholestyramine feeding), high dietary cholesterol intake, and early weaning during neonatal period of guinea pigs on their subsequent plasma cholesterol levels and the response to dietary cholesterol challenged in adulthood were investigated. Pretreatment of neonatal guinea pigs with cholestyramine resulted in (a) a lower plasma cholesterol level, (b) an increased excretion rate of fecal bile acids and total steroids, (c) an expanded bile acid pool, (d) an increased activity of cholesterol 7 alpha-hydroxylase, and (e) no change in the hepatic 3-hydroxy-3-methylglutaryl coenzyme A (CoA) reductase activity when challenged with cholesterol in adulthood. Cholesterol pretreatment during neonatal period resulted in (a) no alteration in the plasma cholesterol level, (b) no alteration in the fecal excretion of steroids, or (c) no alteration in the cholesterol 7 alpha-hydroxylase activity when they were challenged with a high cholesterol diet. Early weaning did not influence the fecal excretion of steroids or cholesterol 7 alpha-hydroxylase activity but resulted in a slight decrease in the hepatic 3-hydroxy-3-methylglutaryl-CoA reductase activity when they were challenged with a high cholesterol diet. These results suggest that stimulation of cholesterol catabolism rather than cholesterol feeding or early weaning during neonatal period can influence the response to dietary cholesterol challenge in adulthood.

Cholestyramine treatment in early life of low-density lipoprotein receptor deficient Watanabe rabbits: decreased aortic cholesteryl ester accumulation and atherosclerosis in adult life.

Effect of cholestyramine treatment in early life of Watanabe heritable hyperlipidemic rabbits (an animal model lacking low-density lipoprotein receptor activity) on subsequent (6 months recovery) occurrence of natural atherosclerotic lesion and arterial cholesterol metabolism was investigated. Initial cholestyramine treatment decreased both plasma total cholesterol and HDL-cholesterol levels which normalized within 4 weeks after treatment was discontinued. At 9 months of age (age of occurrence of spontaneous atherosclerotic lesions), the extent of aortic atherosclerosis in cholestyramine pre-treated animals was modestly lower (P less than 0.05), as compared to controls, with a significant (P less than 0.05) decrease in aortic cholesteryl ester content. Furthermore, at the end of the recovery period aortic activity of acyl-CoA: cholesterol acyltransferase and neutral cholesterol esterase activity was significantly (P less than 0.05) lower in cholestyramine-pretreated animals. These studies show that early cholestyramine pre-treatment in a low-density lipoprotein receptor-deficient animal model causes persistent changes which might influence cholesteryl ester accumulation and atherogenesis in adult life, even after cholestyramine treatment is discontinued.

Physiological concentrations of insulin induce cellular desensitization to the mitogenic effects of insulin-like growth factor I.

Insulin and insulin-like growth factor I (IGF-I) are structurally related peptides capable of stimulating a variety of metabolic and mitogenic processes. In this study, we investigated the interaction between these peptides and their receptor-mediated pathways in an untransformed cell line. Cultured bovine fibroblasts specifically bound IGF-I and insulin, and each peptide could stimulate DNA synthesis and cell replication through its own receptor. Preincubation of bovine fibroblasts with concentrations of insulin that did not bind to the IGF-I receptor resulted in complete but reversible cellular desensitization to IGF-I-stimulated mitogenesis. Preincubation with as little as 0.1 nM insulin was sufficient to inhibit subsequent IGF-I action. Various insulin analogs produced desensitization in direct relation to the affinity of the insulin for the insulin receptor. Desensitization required > 4 h of cell exposure to insulin and was blocked in the presence of cycloheximide. Neither serum-stimulated mitogenesis nor IGF-I-stimulated glucose uptake were affected by insulin pretreatment. 125I-labeled IGF-I affinity cross-linking experiments indicated that preincubation with insulin did not affect labeling of the 130,000-M(r) alpha-subunit of the IGF-I receptor, but was associated with the loss of IGF-I- and insulin-inhibitive bands at M(r) = 100,000, 85,000, 58,000, and 34,000. These studies suggest that insulin, via interaction with insulin receptors on bovine fibroblasts, regulates IGF-I action at a step distal to IGF-I receptor binding and are consistent with desensitization occurring at an intracellular step in the mitogenic pathway shared by insulin and IGF-I.

Regulation of insulin-like growth factor binding protein-3 messenger ribonucleic acid expression by insulin-like growth factor I.

Insulin-like growth factor binding protein-3 (IGFBP-3) is an important modulator of the anabolic and mitogenic actions of the insulin-like growth factor (IGF) peptides. Previous studies have shown that the IGFs themselves can elevate levels of IGFBP-3 in vivo and in vitro. However, the regulatory mechanisms responsible for IGF-induced increases in IGFBP-3 are unclear. In this study we examined the expression of messenger RNA (mRNA) encoding IGFBP-3 in cultured bovine and human fibroblasts, two cell lines that secrete IGFBP-3 under IGF-I control. Northern analysis of bovine fibroblast RNA hybridized with a specific bovine IGFBP-3 complementary DNA probe indicated a single 2.8-kilobase (kb) transcript readily detectable within 2 h in IGF-I- or insulin-treated, but not in untreated, cells. IGFBP-3 mRNA abundance was maximal around 6 h, and remained elevated after 24 h of treatment. Secreted IGFBP-3 protein appeared more slowly. By Western ligand blotting, IGFBP-3 was not detected in medium from bovine fibroblasts incubated with IGF-I for 2, 4, or 6h, but was apparent after 24 h IGF-I treatment. Induction of IGFBP-3 mRNA was blocked when RNA synthesis was inhibited by actinomycin D. Furthermore, IGFBP-3 mRNA and protein was induced by different IGF-I analogs in direct relation to the ability of the peptides to bind to the type I IGF receptor, indicating a receptor-mediated process. GH had no effect on IGFBP-3 mRNA or protein levels in these cells. In contrast to its effect in bovine fibroblasts, IGF-I had no significant effect on steady state levels of IGFBP-3 mRNA in cultured human fibroblasts. A human IGFBP-3 complementary DNA probe hybridized to a single 2.8-kilobase mRNA species abundant in normal and SV40-transformed human fibroblasts under all culture conditions, and IGFBP-3 protein was secreted by these cells in the absence of exogenous stimuli. In human fibroblast cultures, IGF-I rapidly increased levels of IGFBP-3 in the medium without influencing transcript levels. Steady state levels of induced or constitutively expressed IGFBP-3 mRNA did not change significantly after 6h in the presence of actinomycin D, even though general RNA synthesis was inhibited more than 98%. These data demonstrate that expression of mRNA encoding IGFBP-3 is differentially controlled by IGF-I in bovine and human fibroblasts. Whereas cultured human fibroblasts may be suitable to study posttranscriptional regulation of IGFBP-3 availability, cultured bovine fibroblasts may provide a useful model system to probe the molecular mechanisms of IGFBP-3 gene expression and regulation by IGF-I.

Phorbol ester tumor promoters regulate insulin-like growth factor-binding protein-4 proteolysis.

Cultured human fibroblasts secrete a specific protease that alters extracellular insulin-like growth factor-binding protein-4 (IGFBP-4) structure and function. This enzyme appears to be secreted in a latent form and requires IGFs for activation. To study regulation of the IGFBP-4 protease, we treated normal adult human fibroblasts with various hormones and growth regulatory factors, and collected the human fibroblast-conditioned medium (HFCM) for analysis of IGFBP-4 protease activity. The IGFBP-4 protease assay involved incubation of 50 microliters HFCM with or without 5 nM IGF-II for 6 h at 37 C under cell-free conditions; IGF-activated IGFBP-4 hydrolysis was assessed by Western ligand blotting. In HFCM from cells treated with vehicle, GH, insulin, epidermal growth factor, steroid hormones, or forskolin, IGF-II induced the select loss of detectable IGFBP-4 during the assay. In contrast, IGFBP-4 levels were maintained when HFCM from cells treated with phorbol ester tumor promoters was incubated with IGF-II under cell-free conditions. Hydrolysis of [125I]IGFBP-4 to 18,000 and 14,000 mol wt fragments also was prevented in HFCM from cells treated with phorbol esters. Phorbol esters had no effect on endogenous or exogenous IGFBP-4 proteolysis when added directly to HFCM during the assay, however. Treatment of cells with actinomycin-D or cycloheximide could prevent a phorbol ester-induced block of IGF-dependent IGFBP-4 proteolysis. These data suggest that phorbol ester tumor promoters stimulate human fibroblasts to produce and secrete an inhibitor of the IGFBP-4 proteolytic reaction. Alterations in IGFBP-4 protease activity could affect local IGF action through regulation of IGFBP-4 availability.

Regulation of insulin-like growth factor binding protein-5 messenger ribonucleic acid expression and protein availability in rat osteoblast-like cells.

PTH treatment of UMR 106-01 rat osteosarcoma cells increased 20- to 100-fold medium levels of a discrete insulin-like growth factor binding protein (IGFBP) with M(r) of 29K. Northern analysis of UMR cellular RNA hybridized with a specific IGFBP-5 complementary DNA probe indicated a 6.0-kilobase transcript induced within 2 h in PTH-treated cells. IGFBP-5 messenger RNA (mRNA) abundance was maximal around 6 h and remained elevated after 24 h of treatment. Another rat osteosarcoma cell line (ROS 17/2.8) did not express IGFBP-5 mRNA and did not secrete 29K IGFBP. Induction of IGFBP-5 mRNA by PTH was blocked when RNA synthesis in UMR cells was inhibited by actinomycin D (Bu)2cAMP mimicked the effect of PTH on IGFBP-5 mRNA expression and protein secretion. In addition, a monoclonal antibody against IGF-I (Sm 1.2) inhibited the PTH-induced increase in medium IGFBP-5 without influencing IGFBP-5 transcript levels. Direct addition of IGF-I to UMR cell cultures increased medium IGFBP-5 levels approximately 14-fold, with a modest effect on IGFBP-5 mRNA levels. Studies comparing IGF-I, IGF-II, different IGF-I analogs, and insulin indicated that the predominant IGF effect on IGFBP-5 accumulation was type I IGF receptor independent. Thus, in UMR 106-01 cells, PTH and IGF-I increase extracellular concentrations of IGFBP-5 via distinct but coordinate mechanisms; PTH acts primarily to induce IGFBP-5 mRNA expression through a cAMP-mediated mechanism, and IGF-I appears to interact directly with IGFBP-5 protein to promote its accumulation.

Insulin-like growth factor-II enhancement of human fibroblast growth via a nonreceptor-mediated mechanism.

Deciphering the complex interactions of the various components of the insulin-like growth factor (IGF) system [IGF-I and -II peptides, type I and II IGF receptors, and IGF-binding proteins (IGFBPs)] is important for our understanding of cell growth regulation. We report here that IGF-II can enhance IGF-I-stimulated cell proliferation independent of direct IGF-II interaction with type I or II IGF receptors. Human fibroblasts cultured in serum-free medium for 40 h were relatively resistant to the mitogenic effects of added IGF-I. However, preexposure of the cultures to low concentrations of IGF-II enhanced IGF-I action several-fold. IGF-II by itself had no stimulatory effect and did not influence [Gln3,Ala4,Tyr15,Leu16]IGF-I or insulin-stimulated DNA synthesis. IGF-II did not directly interact with type I IGF receptors, as [Leu27]IGF-II, an IGF-II analog that does not bind type I IGF receptors, could mimic IGF-II's potentiating effect. Type II IGF receptors also were not involved because 1) [Gln6,Ala7,Tyr18,Leu19,Leu27]IGF-II, an analog with normal receptor binding, had no effect; and 2) beta-galactosidase, a competitive inhibitor of IGF-II receptor binding, did not influence IGF-II potentiation of IGF-I action. Enhanced cell responsiveness to IGF-I appears to be due to IGF-II-induced changes in pericellular IGFBP-3 and IGFBP-4. These data support the hypothesis that IGF-II can potentiate the action of IGF-I by disrupting the IGFBP barrier at the cell surface, thereby increasing IGF-I availability for type I IGF receptor interaction.

Effect of glucocorticoid on insulin-like growth factor (IGF) regulation of IGF-binding protein expression in fibroblasts.

Insulin-like growth factor (IGF)-binding proteins (IGFBPs) can determine IGF biological competence at the cellular level. IGF-I itself has been shown to be an important peptide regulator of local IGFBP availability. Glucocorticoid also has major effects on IGFBP expression. In the present study, we assessed integrated IGF-I and glucocorticoid regulation of IGFBP messenger RNA (mRNA) and protein expression in two fibroblast model systems. In bovine fibroblasts, IGF-I treatment induced IGFBP-3 and IGFBP-5 mRNA and protein secretion, and had a moderate effect on IGFBP-4 expression. Dexamethasone had little effect on the IGF-induced increase in IGFBP-3, but completely blocked the increase in IGFBP-5 expression. Basal IGFBP-4 expression was inhibited by dexamethasone, and this effect was counteracted by IGF-I. IGFBP-2 expression did not vary with IGF-I or dexamethasone treatment in these cells; IGFBP-1 mRNA was not detectable, and IGFBP-6 mRNA was low and inconsistent. In human fibroblasts, IGF-I treatment increased levels of IGFBP-3 and decreased levels of IGFBP-4 without influencing mRNA expression. IGF-I also increased steady state levels of IGFBP-5 mRNA. Dexamethasone alone decreased IGFBP-3, IGFBP-4, and IGFBP-5 mRNA, but it had no significant effect on IGFBP-3, -4, or -5 expression in the presence of IGF-I. Human fibroblast IGFBP-6 expression was stable under the different culture conditions; IGFBP-1 and -2 mRNA were not detected. These data demonstrate that IGF peptide and glucocorticoid individually modulate IGFBP expression and indicate that glucocorticoid has distinct effects on IGF regulation of IGFBP depending upon the particular IGFBP and the underlying mechanism of IGF regulation. Bovine fibroblasts may provide a useful model system to probe the molecular mechanisms of IGFBP-3, -4, and -5 gene expression and regulation by IGF-I and glucocorticoid, whereas human fibroblasts may be suitable for studying posttranscriptional interactions.

Factors regulating insulin-like growth factor-binding protein-3 binding, processing, and potentiation of insulin-like growth factor action.

In this study, we investigated the effects of various biochemical and pharmacological agents on insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) cell binding and action in cultured bovine fibroblasts. When cells were preincubated for 48 h with 50 nM recombinant human (rh) IGFBP-3, IGF-I-stimulated [3H]aminoisobutyric acid ([125H]AIB) uptake was enhanced 2- to 3-fold. The addition of cytoskeletal disrupting agents during the preincubation with rhIGFBP-3 did not affect IGFBP-3 potentiation of IGF-I action, nor did a variety of serine, aspartate, and metalloproteinase inhibitors. On the other hand, ammonium chloride and chloroquine, weak bases that neutralize the pH of acidic cell compartments, blocked IGFBP-3 potentiation of IGF-I-stimulated [3H]AIB uptake. Chloroquine and ammonium chloride had no effect alone and did not inhibit IGF-I receptor binding or action in the absence of rhIGFBP-3. Bafilomycin A, a specific inhibitor of ATP-dependent hydrogen ion pumps, also inhibited IGFBP-3 potentiation of IGF-I-stimulated [3H]AIB uptake. Competitive [125I]IGF-I binding and affinity cross-linking experiments suggested structure/function changes in cell-bound IGFBP-3 that were altered in the presence of chloroquine and bafilomycin. Heparin markedly decreased initial IGFBP-3 cell adherence, but could not promote dissociation of IGFBP-3 from cells after the 48-h preincubation. Moreover, heparin did not inhibit IGFBP-3 potentiation of IGF-I action. In summary, these data indicate that IGFBP-3 undergoes specific pH-dependent structural and/or environmental modifications that mediate the enhancing effect of IGFBP-3 on IGF-I action in bovine fibroblasts. They also suggest that IGFBP-3 binding to heparin-like molecules on the cell surface is not directly involved in this process.

Insulin-like growth factor (IGF) binding protein-3 potentiation of IGF action is mediated through the phosphatidylinositol-3-kinase pathway and is associated with alteration in protein kinase B/AKT sensitivity.

Cell-association and processing of insulin-like growth factor binding protein-3 (IGFBP-3) by cultured bovine fibroblasts results in markedly enhanced type I IGF receptor signaling at a step distal to ligand binding. The purpose of the present study was to determine the intracellular mediators of IGFBP-3's potentiating effect. Preincubation of cultured bovine fibroblasts with 50 nM IGFBP-3 had no effect alone, but enhanced by 3- to 4-fold IGF-I-stimulated 3H-aminoisobutryric acid (AIB) uptake. IGFBP-3-induced potentiation was specifically prevented if an inhibitor of phosphatidylinositol 3 (PI3)-kinase activation (LY294002), but not an inhibitor of mitogen-activated protein kinase activation (PD98059), was present during the preincubation period. IGFBP-3 did not directly activate the downstream effector of PI3-kinase, protein kinase B (PKB)/Akt. However, the sensitivity of PKB/Akt to activation by IGF-I was increased by 2- to 4-fold with IGFBP-3 pretreatment. This increased sensitivity was accompanied by altered mobility of PKB/Akt on SDS-polyacrylamide gels, suggestive of a diminished phosphorylation state. Consistent with this, okadaic acid, a potent serine/threonine phosphatase inhibitor, was able to block the potentiation effect of IGFBP-3 and prevent the altered mobility of the PKB/Akt molecule in response to IGFBP-3 treatment. PKB/Akt immunoprecipitated from IGFBP-3-pretreated cells was no longer recognized by an antibody specific for phosphorylated threonine followed by proline. These data indicate that IGFBP-3 modulates type I IGF receptor signaling through an effect on PI-3-kinase pathway substrates and suggest a novel mechanism of dephosphorylation whereby PKB/Akt is transformed into a more sensitive substrate of type I IGF receptor signaling.

Identification and regulation of the IGFBP-4 protease and its physiological inhibitor in human trophoblasts and endometrial stroma: evidence for paracrine regulation of IGF-II bioavailability in the placental bed during human implantation.

The IGF family plays an important role in implantation and placental physiology. IGF-II is abundantly expressed by placental trophoblasts, and IGF binding protein (IGFBP)-4, a potent inhibitor of IGF actions, is the second most abundant IGFBP in the placental bed, expressed exclusively by the maternal decidua. Proteolysis of IGFBP-4 results in decreased affinity for IGF peptides, thereby enhancing IGF actions. In the current study, we have identified the IGFBP-4 protease and its inhibitor in human trophoblast and decidualized endometrial stromal cell cultures, and we have investigated their regulation in an effort to understand control of IGF-II bioavailability at the placental-decidual interface in human implantation. IGFBP-4 protease activity was detected in conditioned media (CM) from human trophoblasts and decidualized endometrial stromal cells using (125)I-IGFBP-4 substrate. Identification of the IGFBP-4 protease as pregnancy-associated plasma protein-A (PAPP-A) was confirmed by specific immunoinhibition and immunodepletion of the IGFBP-4 protease activity with specific PAPP-A antibodies. The IGFBP-4 protease activity was IGF-II-dependent in trophoblast CM. In decidualized stromal CM, PAPP-A/IGFBP-4 protease activity was also IGF-II-dependent, but was evident only when IGF-II was added in molar excess of the predominant IGFBP in decidualized stromal cell CM, IGFBP-1, supporting bioavailable IGF-II as a key cofactor of IGFBP-4 proteolysis by PAPP-A. Cultured first and second trimester human trophoblasts (n = 5) secreted PAPP-A into CM with mean +/- SEM levels of 172.4 +/- 32.8 mIU/liter.10(5) cells, determined by specific ELISA. PAPP-A in trophoblast CM (n = 3) and did not change in the presence of IGF-II (1-100 ng/ml). Cultured human endometrial stromal cells (n = 4) secreted low levels of PAPP-A (6.25 +/- 3.6 mIU/liter.10(5) cells). A physiological inhibitor of PAPP-A, the proform of eosinophil major basic protein (proMBP), was detected in trophoblast CM at levels of 1853 +/- 308 mIU/liter.10(5) cells, determined by specific ELISA, and was nearly undetectable in CM of human endometrial stromal cells. Upon in vitro decidualization of endometrial stromal cells with progesterone, PAPP-A levels in CM increased nearly 9-fold without a concomitant change in proMBP. In contrast to the experiments with trophoblasts, IGF-II and the IGF analogues, Leu(27) IGF-II, and Des (1-6) IGF-II, resulted in a dose-dependent decrease of PAPP-A levels in decidualized endometrial stromal CM by 70-90%, and a dose-dependent increase in proMBP of 14- to 41-fold. The data demonstrate conclusively that the IGF-II-dependent IGFBP-4 protease of human trophoblast and decidual origin is PAPP-A. Furthermore, the differential regulation of decidual PAPP-A and proMBP by insulin-like peptides supports a role for trophoblast-derived IGF-II as a paracrine regulator of these maternal decidual products that have the potential to regulate IGF-II bioavailability at the trophoblast-decidual interface. Overall, the data underscore potential roles for a complex family of enzyme (PAPP-A), substrate (IGFBP-4), inhibitor (proMBP), and cofactor (IGF-II) in the placental bed during human implantation.

Cholesterol excretion studies in familial hypercholesterolemic children and their normolipidemic siblings.

Twenty children ages 3 to 17 yr, eight with normal lipids and 12 with familial hypercholesterolemia were studied on a metabolic unit for 14 days to evaluate fecal bile acid and fecal neutral sterol excretion. The diet contained a moderately low cholesterol content, 180 to 200 mg/day. Stools were collected in three separate, 3-day pools. Fecal bile acids and fecal neutral sterols were measured using two stool markers and thin-layer, and gas-liquid chromatography techniques. Fecal neutral sterol and fecal bile acid excretion were the same for normal and familial hypercholesterolemic children on a mg/kg basis. Fecal neutral sterols in familial hypercholesterolemic children decreased with age, p less than 0.001; fecal bile acid excretion also appeared to decrease with age, but less significantly, p less than 0.07. Although the familial hypercholesterolemic children have significantly increased plasma and potentially elevated tissue or total body cholesterol, the excretion of fecal bile acids and fecal neutral sterols did not differ between familial hypercholesterolemic and normal children.

Effect of enhancement of cholesterol degradation during neonatal life of guinea pig on its subsequent response to dietary cholesterol.

The effect of feeding cholestyramine to neonatal guinea pigs on their subsequent plasma cholesterol levels and response to dietary cholesterol were studied. Male neonatal guinea pigs were suckled for 6 days. One group was maintained on a 1.1% cholestyramine diet for 6 weeks and the control group weaned normally. Both groups of guinea pigs were then fed a standard diet of Guinea Pig Chow for 6 weeks. During the standard diet period bile acid and neutral sterol excretion rates were significantly higher in the group previously treated with cholestyramine than the control group despite the similarity in plasma cholesterol levels. When both groups of guinea pigs were subjected to a 0.5% cholesterol diet for 4 weeks, plasma cholesterol levels were significantly lower in the group previously treated with cholestyramine than the control group. The plasma cholesterol levels continued to be significantly lower in the group previously treated with cholestyramine after an additional four weeks on standard diet. These results suggest that stimulation of cholesterol catabolism in the neonatal period can influence the subsequent response to dietary cholesterol.

Human hepatoma cells synthesize and secrete insulin-like growth factor Ia prohormone under growth hormone control.

Nucleotide sequencing of cDNAs encoding human insulin-like growth factor I (IGF-I) predicts the existence of two different prohormone forms of IGF-I. The E peptide regions extend the carboxy-terminus of the 70 amino acid core IGF-I molecule (BCAD domains) by either an additional 35 (IGF-Ia) or 77 (IGF-Ib) amino acids. Employing antiserum directed against a peptide sequence unique to the E peptide region of IGF-Ia prohormone, we have identified EIa immunoreactive material (IR-EIa) in the conditioned medium of a human hepatoma cell line, HepG2. Human growth hormone (GH) had dose-dependent stimulatory effects on IR-EIa secretion; incubation of HepG2 cells with GH at maximal concentrations (1-5 micrograms/ml) increased specific IR-EIa in 24 h conditioned medium 3-fold. The addition of human placental lactogen, insulin, IGF-I, dexamethasone, beta-estradiol, or progesterone had no significant effect. Acid chromatography of HepG2 cell conditioned medium revealed a single elution peak of IR-EIa corresponding to M(r) = 12,000-20,000. There was no immunologically detectable 7500 M(r) IGF-I peptide in acid-chromatographed conditioned medium under either basal or stimulated conditions. Biosynthetic labelling of HepG2 cell products with [35S]Trans label and immunoprecipitation with antisera specific to the E or to the AD regions of the IGF-Ia molecule indicated a single species of approx. 14,000 M(r). These data indicate that the E peptide region of IGF-Ia is translated and released as part of the larger molecule in cultured HepG2 cells, and that the levels of this prohormone are regulated by GH.

Characterization and partial purification of the insulin-like growth factor (IGF)-dependent IGF binding protein-4-specific protease from human fibroblast conditioned media.

Insulin-like growth factors (IGFs) are one of the most potent stimulators of cell growth. IGFs are modulated by six high affinity binding proteins (IGFBPs) which are, in turn, regulated through post-translational modifications such as proteolysis. In the conditioned media of human fibroblasts, IGFBP-4 is cleaved by an apparently novel IGFBP-4-specific protease that requires IGF for functional activity. We have used several biochemical manipulations, including size exclusion chromatography, native gel electrophoresis, chaotropic salt precipitation, hydrophobic interaction chromatography, ion exchange chromatography, isoelectric focusing, lectin affinity chromatography, and metal chelating affinity chromatography to both characterize and partially purify the IGF-dependent IGFBP-4 protease. Our results indicate that this protease is a highly glycosylated, Zn+2 binding metalloprotease with a native molecular weight greater than 200 kDa.

In vivo and in vitro toxicity of phospholipase C from Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces phospholipase C (PLC), a heat-labile hemolysin. Histopathological analysis of PLC-treated mice revealed that the primary target organs involved in PLC-induced toxicity were the liver and kidney. Mice treated i.v. with PLC demonstrated significant tubular epithelial necrosis of the kidney with hematuria, while when given i.p. they exhibited hepatonecrosis with cellular infiltration. Splenomegaly was also a consistent finding. Results from in vitro studies indicate that PLC is toxic for mouse peritoneal cells and human leukocytes.