Depolarization induces insulin-like growth factor binding protein-2 expression in vivo via NMDA receptor stimulation

The effect of depolarization and N-methyl-D-aspartate (NMDA) receptor blockade on insulin-like growth factor-I (IGF-I), IGF binding protein-2 (IGFBP-2) and IGFBP-4 expression was analysed in vivo. Depolarization was induced in adult rat brains by applying 3 M KCl to the exposed cortex for 10 min. A subgroup of animals also received daily injections of MK-801. Four days after KCl exposure, the brains were analysed by in situ hybridization, immunohistochemistry and TUNEL. A significant upregulation of IGFBP-2 mRNA and protein was detected in astrocytes after KCl exposure This upregulation was reduced by MK-801 treatment. No alterations in IGF-I or IGFBP-4 mRNA levels were noted. We did not detect TUNEL positive cells, morphological signs of necrosis or apoptosis, or neuronal loss in the depolarized zone. Taken together, these findings indicate that upregulation of IGFBP-2 by depolarization is mediated by NMDA receptors, and, as no neuronal damage was detected, astrocytic NMDA receptors may be responsible for this upregulation.     

Total and free insulin-like growth factor I, insulin-like growth factor binding protein 3 and acid-labile subunit reflect clinical activity in acromegaly

The aim was to evaluate, markers of disease activity in acromegaly in relation to perceived disease activity. Thirty-seven consecutively treated, acromegalic patients, classified by clinical symptoms as inactive (n=16), slightly active (n=10) and active (n=11), entered the study. When evaluating the inactive and the active groups, we found that positive and negative predictive values (PV(pos), PV(neg)) for clinical disease activity of total and free insulin-like growth factor-I (IGF-I) were 0.59, 0.90 and 1.00, 0.82 respectively. Acid-labile subunit (ALS) showed diagnostic merit similar to insulin-like growth factor binding protein-3 (IGFBP-3) with PV(pos) of 0.69 and 0.71 and PV(neg) of 0.91 and 0.92 respectively. We conclude that free IGF-I is more closely related than total IGF-I to perceived disease activity and is as such useful when evaluating previously treated acromegaly for disease activity. Total IGF-I, IGFBP-3 and ALS possess a higher PV(neg) for the clinical disease activity. None of the parameters can at present be claimed to be superior to the others and thus all the measured parameters are recommended to be part of the evaluation of acromegalic patients.     

Roles of insulin-like growth factor-I (IGF-I) and IGF-I binding protein-2 (IGFBP2) and -5 (IGFBP5) in developing chick limbs

Insulin-like growth factor-I (IGF-I) and the IGF-I binding proteins (IGFBPs) which modulate IGF-I action have been implicated in the development of the vertebrate limbs and skeleton. We have examined the distribution of IGF-I, IGFBP2 and IGFBP5 in developing chick limb buds and have investigated their functional roles and relationships during chick limb development. IGF-I and IGFBP2 are co-expressed throughout the lateral plate from which limbs form, although IGFBP2, unlike IGF-I, does not promote formation of rudimentary limb buds from non-limb-forming flank regions in vitro. During limb outgrowth, IGF-I is present in non-AER limb ectoderm, but little IGF-I is present in the AER itself, suggesting that restriction of endogenous IGF-I activity may be required for proper AER function. Consistent with this possibility, the ectoderm of mutant limbless and wingless wing buds, which fail to form an AER, continues to express IGF-I. We also found that the AER contains abundant IGFBP2 but that IGFBP2 is not present in limb subridge mesoderm. In contrast, IGFBP2 is present in the distal mesoderm of mutant limbless or wingless limb buds, which fail to grow out. This suggests that attenuation of IGFBP2 expression is controlled by the AER and that cessation of IGFBP2 expression may be necessary for the proliferation and suppression of differentiation of subridge mesoderm that is required for limb outgrowth to occur. Consistent with this possibility, we found that exogenous IGFBP2 inhibits the anti-differentiative activity of the AER in vitro. We also found that regions of cell death in the limb contain abundant IGF-I-immunoreactive cells, consistent with a role for IGF-I in apoptosis. During skeletogenesis, IGF-I and IGFBP2 are co-localized to the condensing central core of the limb, implicating these factors as potential regulators of the onset of chondrogenic differentiation. Intriguingly, we found that IGF-I and IGFBP2 have opposing effects on chondrogenesis, as IGF-I stimulates but IGFBP2 inhibits accumulation of cartilage matrix by micromass cultures in vitro. Long [R(3)] IGF-I, an analog of IGF-I that cannot bind IGFBPs, is more effective than IGF-I in stimulating matrix accumulation, consistent with a negative role for IGFBP2 in chondrogenesis. As the chondrocytes of the limb mature, IGF-I is present only in terminal hypertrophic chondrocytes, which undergo programmed cell death, while IGFBP2 becomes localized to prehypertrophic and hypertrophic chondrocytes, suggesting involvement in chondrocyte maturation. Consistent with this possibility, we found that exogenous IGFBP2 induces precocious expression of Indian hedgehog, a marker of prehypertrophy, in maturing chondrocytes in vitro. IGF-I and IGFBP2 are also present in the osteoblasts, clasts and nascent matrix of the long bones, consistent with roles in endochondral bone formation. Unlike in rodent limbs, IGFBP5 is not expressed by chick limb ectoderm or AER. IGFBP5 expression is highly localized to developing limb musculature and, later, to the developing skeletal elements where it is expressed by osteoblast precursers and osteoblasts. The results of this study suggest potential novel roles for IGF-I and IGFBP2 in several aspects of limb development including limb outgrowth and AER activity, programmed cell death, chondrogenesis and chondrocyte maturation.     

The changes of IGF binding proteins after rhGH administration to patients totally dependent on parenteral nutrition

The objective was to study the effect of recombinant human growth hormone (rhGH) administration to patients with chronic malnutrition maintained on total parenteral nutrition (TPN) on the levels of insulin-like growth factor-I (IGF-I) and IGF binding proteins (IGFBPs) during a double-blind trial. After 1 week of TPN the patients were randomized into group I (placebo) or group II (rhGH). Samples were collected on the first day (start of the TPN) to measure basal values, the seventh day to study the effect of TPN and the 10th, 14th and 21st days to evaluate the rhGH effect. Basal laboratory evaluation, nutritional status and glucose tolerance were assessed using standard laboratory techniques. Radioimmunoassays were used to analyse IGF-I, free IGF-I (fIGF-I) and IGFBP1-3. Electrophoresis with Western ligand blotting and Western immunoblotting was applied to find the pattern of IGFBPs. TPN had no effect on the circulating IGF-I concentration and the pattern of IGFBPs present in the studied groups of patients. The rhGH administration led to significant increases of IGF-I, total IGFBP-3, glycosylated IGFBP-3 (39, 42 kDa) and the 29 kDa fragment of IGFBP-3 and the decrease of IGFBP-2 during the trial (P<0.05). The mean levels of IGFBP-1, fIGF-I and the parameters of nutritional status in group II during the trial were not significantly influenced by rhGH. However, it has been found that IGFBP-1 and fIGF-I levels were correlated with the levels of the weekly nitrogen balance of each patient in group II at the end of the trial. In spite of the significant changes of IGF-I, IGFBP-2, total IGFBP-3 and IGFBP-3 (29 kDa proteolytic fragment) after rhGH administration to patients with malnutrition, maintained on parenteral nutrition, the increase of nitrogen balance was seen only in patients who decreased their IGFBP-1 and increased bioavailable IGF-I as reflected by measurement of fIGF-I. The levels of IGFBP-1 may provide a useful marker of IGF-I bioavailability in monitoring the efficiency of the rhGH therapy in malnourished patients.     

Maternal nutrition affects the ability of treatment with IGF-I and IGF-II to increase growth of the placenta and fetus, in guinea pigs

The aim of this study was to investigate how administration of IGF-I and IGF-II, during early to mid pregnancy, affects maternal growth and body composition as well as fetal and placental growth, in ad libitum fed, and in moderately, chronically food restricted guinea pigs. From day 20 of gestation, mothers (3-4 months old) were infused with IGF-I, IGF-II (565 microg/day) or vehicle for 17 days and then killed on day 40 of gestation. Maternal organ weights, fetal and placental weights were assessed. Treatment with IGFs did not alter body weight gain and had small effects on body composition in the mothers. Both IGF-I and IGF-II increased fetal and placental weights in ad libitum fed dams and IGF-I increased placental weight in food restricted dams. In conclusion, treatment with IGF-I during the first half of pregnancy stimulates placental growth in both ad libitum fed and food restricted guinea pigs without affecting maternal growth while fetal growth is stimulated by IGF treatment only in ad libitum fed animals.     

Inhibition of human osteoblast marker gene expression by retinoids is mediated in part by insulin-like growth factor binding protein-6

All-trans -retinoic acid (atRA) inhibits osteoblast marker gene expression and markedly increases expression of insulin-like growth factor binding protein-6 (IGFBP-6) in human osteoblasts. The possibility that IGFBP-6 inhibits the osteoblast phenotype and also mediates the inhibitory effect of atRA on osteoblast marker gene expression was explored using an antisense approach. Stable human osteoblast-like osteosarcoma SaOS-2 cells were prepared that expressed antisense IGFBP-6 RNA under basal and atRA-stimulated conditions. The functional expression of IGFBP-6 antisense RNA was confirmed by measuring IGFBP-6 mRNA by Northern analysis or by measuring IGFBP-6 protein in the conditioned media (CM) by radioimmunoassay. Antisense clones produced less mRNA and had less IGFBP-6 protein in the CM than controls. IGFBP-6 protein levels in the CM were inversely correlated with alkaline phosphatase (ALP) activity, whereas IGFBP-3 and IGFBP-4 protein levels were not. We reasoned that atRA would have little or no effect on ALP activity in IGFBP-6 antisense clones if atRA mediated its inhibitory effects by recruiting IGFBP-6. In the majority of IGFBP-6 antisense clones with the lowest IGFBP-6 mRNA and CM protein levels and only modest changes in other IGF system components, atRA did not significantly decrease ALP activity. These findings provide evidence that atRA recruits IGFBP-6 to inhibit the human osteoblast phenotype.     

Thrombocytosis and thrombocythemia

Thrombocytosis is caused by three major pathophysiological mechanisms: (1) reactive or secondary thrombocytosis; (2) familial thrombocytosis; and (3) clonal thrombocytosis, including essential thrombocythemia and related myeloproliferative disorders. Recent work has begun to elucidate the abnormal megakaryocytopoiesis of essential thrombocythemia, which is associated with paradoxically elevated plasma levels of thrombopoietin. The clonal nature of all cases of essential thrombocythemia has been challenged. Thrombotic complications are the major causes of morbidity and mortality in this disease. Indications for platelet cytoreduction and antiplatelet therapy, as well as complications of treatment, are being clarified.     

Heritable thrombophilia and childhood thrombosis

Thrombotic problems are rare during childhood but are increasingly recognized, particularly in tertiary care paediatric populations, and represent a different spectrum of disorders to those seen in adults. An understanding of the aetiological factors involved in the pathogenesis of these events is important both for prevention and management. A number of inherited prothrombotic defects have been shown to be independent risk factors for thromboembolism in adult studies, and may also contribute to thrombotic events in childhood. Homozygous deficiencies of naturally occurring inhibitors of coagulation are clearly associated with major prothrombotic disorders, often presenting in the perinatal period. The association of other inherited prothrombotic disorders with thrombosis in childhood is less well defined. The prevalence of heritable thrombophilia varies in different clinical settings and the risks associated with individual defects has only been addressed in a small number of studies to date. Additional acquired risk factors are also present in a high percentage of cases and again differ from those seen in adult thrombosis. Further studies are required to assess the risks associated with heritable thrombophilia during infancy and childhood, and to define the place of thrombophilia screening in paediatric practice.     

Stem cell factor: laboratory and clinical aspects

Stem cell factor is an essential haemopoietic progenitor cell growth factor with proliferative and anti-apoptotic functions. Molecular biologists have now dissected some of the various pathways through which this cytokine signals to the nucleus. At the same time, new molecules have become available which can inhibit SCF signalling. This provides an exciting prospect for the treatment of Kit+ malignancies such as acute myeloblastic leukaemia. The capacity of SCF to synergize with other cytokines has been exploited in the ex vivo expansion of haemopoietic progenitors and dendritic cells, which may also hold therapeutic promise. In this review the last 5 years' literature on these issues is reviewed and collated.     

Reduced serum insulin-like growth factor-1 (IGF-1) and IGF-binding protein-3 levels in adults with inflammatory bowel disease

In the present study, the changes in circulating IGF-1 and its binding protein IGFBP-3 were determined in adult patients with active inflammatory bowel disease (IBD) in order to assess the effect of this inflammatory condition on the IGF system. IGF-1 and IGFBP-3, as well as interleukin-6 (IL-6) were measured in serum obtained from 22 consecutive newly diagnosed patients (mean age 41.3 years) with active IBD, including 10 patients with Crohn's disease (CD), and 12 with ulcerative colitis (UC). For comparison the same parameters were determined in 30 healthy volunteers matched for age, sex and Body Mass Index (BMI). Serum IGF-1 and IGFBP-3 levels were similar in the two subgroups of patients and the values from all patients were combined for comparison with those from the control group. The mean (+/- SD) serum IGF-1 concentration (178 +/- 91 ng/ml) in the patients with IBD was lower compared with that in the controls (227 +/- 79 ng/ml, P<0.035). Similarly, the mean IGFBP-3 concentration in the patients was lower than in the controls (1.6 +/- 0.6 ng/ml vs 3.2 +/- 0.7 ng/ml respectively, P<0.001), Serum IL-6 levels were higher in the patients compared with the controls (5.5 +/- 4.2 vs 0.65 +/- 0.11 pg/ml, P<0.0001). The reduced IGF-1 and IGFBP-3 levels in patients with active IBD suggest that this systemic inflammatory condition is associated with a degree of acquired GH resistance, possibly induced by inflammatory cytokines.     

Diagnosis and management of refractoriness to platelet transfusion

Improvements in the availability and quality of platelet transfusions have markedly reduced the morbidity and mortality associated with intensive myelosuppressive therapy. Alloimmunization and refractoriness to platelet transfusion remains a significant clinical problem, although the incidence of alloimmunization may be declining due to more widespread use of leucocyte depleted products. Alloimmunization can be distinguished from other causes of poor post transfusion increments by the measurement of lymphocytotoxic or antiplatelet antibodies. In addition to medical approaches to reduce the risk of bleeding in individual patients, identification of histocompatible donors can usually be accomplished by HLA matching of donor and recipient, platelet cross matching or a combination of both techniques. There are a number of selection strategies which can be utilized and optimal patient management requires close cooperation and communication between clinicians and blood centers.     

An intravenous loading dose of azathioprine decreases the time to response in patients with Crohn's disease

Azathioprine, an effective therapy for Crohn's disease, is limited by a prolonged time to response. The aim of this study was to determine the safety and utility of a loading dose of azathioprine to decrease the time to response in patients with Crohn's disease. Twelve patients were studied: 6 with 13 fistulae and 6 with inflammatory disease. All patients received an intravenous infusion of azathioprine (50 mg/h for 36 hours). Response was determined by physical and radiographic examination for fistulae and by the Crohn's Disease Activity Index for inflammatory disease. Erythrocyte concentrations of azathioprine metabolites were measured by chromatography. Seven of 13 fistulae closed by week 4, and three had a temporary decrease in drainage. One fistula improved at week 16. Two fistulae failed to improve. Four of 6 patients with inflammatory disease achieved remission, and 1 improved temporarily. Improvement was rapid (≤4 weeks). Peak concentrations of azathioprine metabolites occurred within 3 days. Clinical response did not correlate with azathioprine metabolite concentrations at the azathioprine dose studied. No adverse events occurred. An 1800-mg intravenous loading dose of azathioprine is safe and may decrease the time to response to ≤4 weeks in patients with Crohn's disease. Correlation between clinical response and azathioprine metabolite concentrations at larger azathioprine doses should be determined.     

Regulation of protein secretion into bile: Studies in mice with a disrupted mdr2 p-glycoprotein gene

Protein is secreted into bile via several independent pathways. The aim of this study was to investigate whether these pathways are influenced by secretion of biliary lipid. Protein secretion and biliary lipid output were studied in wildtype mice (+/+), heterozygotes (+/−), and homozygotes (−/−) for mdr2 gene disruption. Biliary lipid and protein output were varied by infusion with taurocholate (TC) and tauroursodeoxycholate (TUDC). Exocytosis and transcytosis were unaltered in (−/−) mice. Infusion with TC strongly induced secretion of alkaline phosphatase in (−/−) mice but had little effect in (+/−) and (+/+) mice. Infusion with TUDC had little effect on alkaline phosphatase output. In contrast, both TUDC and TC strongly stimulated secretion of aminopeptidase N and lysosomal enzymes in (+/+) mice but had no effect in (−/−) animals. Aminopeptidase N secretion correlated with phospholipid output, but only at high flux. At low flux, aminopeptidase N was secreted independently from both phospholipid and bile salts. The canalicular membrane enzymes alkaline phosphatase and aminopeptidase N are secreted via separate pathways. Part of alkaline phosphatase output is controlled by bile salt hydrophobicity, whereas at high lipid flux, aminopeptidase N secretion seems to be coupled to phospholipid output. Lysosomal enzymes follow the latter pathway.     

Interruption of a transforming growth factor α autocrine loop in Caco-2 cells by antisense oligodeoxynucleotides

We have previously shown that Caco-2 cell proliferation is driven by basolateral membrane epidermal growth factor receptors. The aim of this study was to investigate whether autocrine production of transforming growth factor α (TGF-α) activates these receptors and stimulates proliferation using antisense oligodeoxynucleotides. Caco-2 cells grown on microporous membranes or Jurkat cells were exposed to conventional or 5′ cholesterol-modified oligodeoxynucleotides synthesized with random, antisense, or missense base sequences. Indices of proliferation were measured, including [³H]thymidine or [³H]uridine uptake for studies of short-term stimulation and the methylthiotetrazole assay as an index of cell number increase over longer periods. Secretion of TGF-α by cells was detected using a soft agar bioassay. Incubation with antisense oligodeoxynucleotides inhibited TGF-α secretion compared with controls. Random and missense oligodeoxynucleotides had no effect on proliferation. The TGF-α antisense oligodeoxynucleotides markedly inhibited proliferation, an effect that was abolished by adding TGF-α to the medium. Oligonucleotides had no effect on Jurkat cells, a lymphocytic cell line lacking epidermal growth factor receptors. Cholesterol-modified oligodeoxynucleotides were more effective and specific than unmodified oligodeoxynucleotides. Caco-2 cell proliferation is driven by autocrine stimulation of epidermal growth factor receptors by TGF-α. This mechanism may be effectively inhibited by antisense oligodeoxynucleotides, particularly those modified by the 5′ attachment of cholesterol.     

Diagnosis of Wilson's disease in an asymptomatic sibling by DNA linkage analysis

The molecular genetic diagnosis of Wilson's disease in the 5-year-old sister of a patient with Wilson's disease is reported. The girl was clinically free of disease and had no conventional biochemical markers of Wilson's disease (i.e., normal ceruloplasmin, normal copper in the serum, normal 24-hour urinary copper excretion). Diagnosis with restriction fragment length polymorphisms and a nonradioactive polymerase chain reaction-based analysis with microsatellite markers showed her to be homozygous for the disease-associated markers. A liver biopsy was performed, and a 20-fold increased liver copper content confirmed the diagnosis. The child was treated with chelation therapy with d-penicillamine. The report of this study clearly shows the advantage of DNA linkage analysis (especially polymerase chain reaction) over conventional laboratory methods for presymptomatic diagnosis of Wilson's disease before irreparable liver and neurological damage occurs. The only limitation of this DNA-based diagnosis is the fact that it is only applicable in siblings of an index patient whose diagnosis was made by phenotypic criteria.     

Intra-abdominal mass associated with gastrointestinal hemorrhage: A new manifestation of bacillary angiomatosis

Bacillary angiomatosis is a recently described vascular proliferative lesion that occurs most commonly in individuals infected with human immunodeficiency virus. Cutaneous lesions are the most frequently described manifestations of bacillary angiomatosis. However, as culture techniques and disease recognition have improved, additional manifestations have been identified in human immunodeficiency virus-infected individuals, including bacillary peliosis hepatis and isolated bacteremia. Two species of the genus Bartonella (formerly Rochalimaea), Bartonella henselae or Bartonella quintana, have been cultured from the cutaneous lesions of bacillary angiomatosis. A new manifestation of Bartonella infection is reported: an intra-abdominal mass presenting with massive gastrointestinal hemorrhage in a patient with human immunodeficiency virus infection. B. quintana was cultured from a percutaneous needle-biopsy specimen obtained from the highly vascularized intra-abdominal mass. The bacillary angiomatosis lesion resolved after 3 months of tetracycline treatment. Recognition of Bartonella infection is extremely important because it is readily treatable with antibiotic therapy.     

Medical costs in community subjects with irritable bowel syndrome

Costs of management of irritable bowel syndrome (IBS) are unknown. The direct medical charges in community subjects with IBS were estimated. An age- and sex-stratified random sample of residents of Olmsted County, Minnesota, ranging in age from 20 to 95 years, was mailed a valid self-report questionnaire. Subjects were categorized as having IBS, having some symptoms but inadequate criteria for IBS, and controls. All charges (in 1992 U.S. dollars) for health services rendered in the year before completing the survey were obtained (except outpatient medications). A total of 88% of subjects with IBS, 86% of subjects with some symptoms of IBS, and 83% of controls incurred direct medical charges during the study year. The odds of incurring charges were 1.6 times greater in subjects with IBS relative to those without symptoms (P < 0.01) adjusting for age, sex, education, marital status, and employment. Overall median charges incurred by subjects with IBS were $742 compared with $429 for controls and $614 for subjects with some symptoms. Among those subjects with nonzero charges, there were significant positive associations with age, higher education, and symptom groups (all P < 0.01) but not sex. The economic impact of IBS is significant. A better understanding of the determinants of these costs is needed so that cost-saving strategies can be implemented.