Major differences in clinical presentation, diagnosis and management of men and women with autosomal inherited bleeding disorders.

BACKGROUND: In recent years, more awareness is raised about sex-specific dilemmas in inherited bleeding disorders. However, no large studies have been performed to assess differences in diagnosis, bleeding phenotype and management of men and women with bleeding disorders. Therefore, we investigated sex differences in a large cohort of well-defined patients with autosomal inherited bleeding disorders (von Willebrand disease (VWD), rare bleeding disorders (RBDs) and congenital platelet defects (CPDs)). METHODS: We included patients from three nationwide cross-sectional studies on VWD, RBDs and CPDs in the Netherlands, respectively the WiN, RBiN and TiN study. In all studies a bleeding score (BS) was obtained, and patients filled in an extensive questionnaire on the management and burden of their disorder. FINDINGS: We included 1092 patients (834 VWD; 196 RBD; 62 CPD), of whom 665 (60.9%) were women. Women were more often referred because of a bleeding diathesis than men (47.9% vs 36.6%, p = 0.002). Age of first bleeding was similar between men and women, respectively 8.9 ± 13.6 (mean ±sd) years and 10.6 ± 11.3 years (p = 0.075). However, the diagnostic delay, which was defined as time from first bleeding to diagnosis, was longer in women (11.6 ± 16.4 years) than men (7.7 ± 16.6 years, p = 0.002). Similar results were found when patients referred for bleeding were analyzed separately. Of women aging 12 years or older, 469 (77.1%) had received treatment because of sex-specific bleeding. INTERPRETATION: Women with autosomal inherited bleeding disorders are more often referred for bleeding, have a longer diagnostic delay, and often require treatment because of sex-specific bleeding. FUNDING: The WiN study was supported (in part) by research funding from the Dutch Hemophilia Foundation (Stichting Haemophilia), Shire (Takeda), and CSL Behring (unrestricted grant).

No accumulation of a prophylactic dose of nadroparin in moderate renal insufficiency.

BACKGROUND: Low-molecular-weight heparins (LMWHs) have been shown to accumulate in patients with renal insufficiency, especially in therapeutic dosages. Although no appropriate studies have been conducted for prophylactic dosages of nadroparin, dose reduction is sometimes recommended, especially for high prophylactic dosages. We assessed accumulation of a prophylactic dose of 5700 IU subcutaneous nadroparin once daily in patients with renal insufficiency. METHODS: We conducted a prospective cohort study and measured peak anti-Xa activity four hours after subcutaneous nadroparin injection on day 1, 3, 5 and if possible day 10 in adults with and without renal insufficiency defined as a glomerular filtration rate (GFR) below or above 50 ml/min/1.73 m2. Patients with a GFR below 10 ml/min/1.73 m2 were excluded. RESULTS: We included 14 patients in each group. In the group with renal failure 12 patients had a GFR between 30 and 50 ml/min/1.73 m2. Peak anti-Xa activity showed a high interindividual variability, but was fairly constant within each patient. There was no rise in peak anti-Xa activity on day 3 and 5 after consecutive administration. In the group with normal renal function, peak anti-Xa activity declined on day 5 compared with day 1 (p = 0.005). CONCLUSION: Prophylactic dosages of nadroparin showed no accumulation in patients with a GFR between 30-50 ml/min/1.73 m2. Dose reduction in this group could lead to suboptimal thromboprophylaxis. Due to underrepresentation of patients with a GFR < 30 ml/min/1.73 m2 (n = 2), we cannot give recommendations for this group.

Cell polarity of the insulin-like growth factor system in human intestinal epithelial cells. Unique apical sorting of insulin-like growth factor binding protein-6 in differentiated human colon cancer cells.

In this study, we have used enterocyte-like differentiated HT29-D4 human colonic carcinoma cells cultured in a glucose-free medium (HT29-D4-GAL cells) on semi-permeable supports in order to investigate the polarity of the insulin-like growth factor (IGF) system. We report that these cells secrete endogenous IGF-II predominantly (66%) from the basolateral cell surface where type I IGF receptors are almost all (> 96%) localized. HT29-D4-GAL cells also secrete IGF-binding protein (IGFBP) -2, -4, and -6 as evidenced by Western ligand and immunoblot analyses of conditioned medium. IGFBP-2 and IGFBP-4 are secreted primarily into the basolateral side (71 and 87%, respectively), whereas IGFBP-6 is targeted to the apical surface (76%) as a possible consequence of an active sorting. Finally, HT29-D4-GAL cells are found to display responses to IGF-II added to the basolateral but not the apical membrane side in terms of intracellular tyrosine phosphorylation and long-term stimulation of amino acid uptake. This study indicates (a) that IGF-II is potentially capable of autocrine regulation on the basolateral side of HT29-D4-GAL cell, and (b) that IGFBP-6 has a unique pattern of secretory polarity. It supports the concept that a differential sorting of the various forms of IGFBPs might play a modulatory role in the maintenance of a functional polarity in the differentiated HT29-D4-GAL cells.

Alterations in serum levels of insulin-like growth factors and insulin-like growth-factor-binding proteins in patients with colorectal cancer.

It has been reported that insulin-like growth factor (IGF) II is associated with human primary colorectal tumors and colon-carcinoma cell lines. Here, we examine alterations in circulating levels of IGFs and IGF binding proteins (IGFBPs) in patients with colorectal carcinoma, and compare them to age- and nutrition-adjusted references. We report (i) an increase in serum IGF-II concentrations (about 2-fold), whereas IGF-I concentrations are regarded as normal when aging is taken into account; (ii) an apparent increase in serum IGFBP-3 levels when compared to those of healthy elderly subjects, IGFBP-3 only being detected in the 150-kDa IGFBP ternary complex as in normal serum; (iii) abnormally elevated serum IGFBP-2 levels taking into account the apparent concentrations of IGFBP-3. This simultaneous elevation of IGFBP-3 and IGFBP-2 in the serum of patients with colorectal tumors appears to be unique in that it reflects a break in the inverse relationship between the serum IGFBP-3 and IGFBP-2 levels that is observed in normal and in several physiopathological conditions. Moreover, it enables a distinction to be made between 76.5% (13/17) of patients with colorectal carcinoma and normal adults, age-related healthy aged and malnourished patients. We propose that the disturbed serum IGFBP profile observed in the patients with colorectal cancer may be a consequence of oversecretion of IGF-II by the tumor cells. The usefulness of IGFs and IGFBPs as potential colorectal tumor-associated metabolic markers should be further investigated.

des-(1-3)-IGF-I, an insulin-like growth factor analog used to mimic a potential IGF-II autocrine loop, promotes the differentiation of human colon-carcinoma cells.

HT29-D4 human colon-carcinoma cells have been shown to secrete insulin-like growth factor (IGF)-II and to simultaneously express type-I IGF receptors. However, the sequestration of IGF-II by several molecular forms of IGF-binding proteins (IGFBP) in the culture medium prevents the establishment of an operative IGF-II autocrine loop. IGFBPs secreted by HT29-D4 cells (HT29-D4 IGFBP) comprise isoforms of IGFBP-4 (25, 27 and 30 kDa) and 2 unidentified forms (34.5 and 32-34 kDa). This latter does not bind 125I-IGF-I. The net affinity of HT29-D4 IGFBP is about 12 times stronger for IGF-II (KD approx. 10(-10) M) than for IGF-I. All the HT29-D4 IGFBP molecular forms are unable to bind the N-terminally truncated IGF-I analog, des-(1-3)-IGF-I. In contrast, HT29-D4 cell-surface type-I IGF receptors bind IGF-I and des-(1-3)-IGF-I identically (KD approx. 5 x 10(-10) M). We have taken advantage of these particular binding properties to use des-(1-3)-IGF-I to mimic a potential IGF autocrine loop and to observe its biological consequences. Nanomolar concentrations of des-(1-3)-IGF-I induce HT29-D4 cells to develop into a differentiated phenotype, as judged by a substantial carcinoembryonic antigen release and the induction of numerous intercellular cysts with well-organized microvilli. In the same way, des-(1-3)-IGF-I early induces a slight inhibition of HT29-D4 cell proliferation. Based on these findings, we conclude that the type-I IGF receptor primarily controls the differentiation of these colonic cells, and that HT29-D4 cancer cells remain in an undifferentiated state because of their inability to use endogenous IGF-II as an autocrine regulatory factor.

Potential autocrine role of insulin-like growth factor II during suramin-induced differentiation of HT29-D4 human colonic adenocarcinoma cell line.

Suramin, a drug that binds to several types of growth factors, has been previously shown to induce the enterocyte-like differentiation of HT29-D4 human colonic adenocarcinoma cells, suggesting that growth factors are involved in such a process. Undifferentiated HT29-D4 cells release insulin-like growth factor II (IGF-II) into the culture medium that is totally complexed to heterogeneous IGF binding proteins (IGFBP) expressing high affinities for this growth factor (Kda = 0.02 nM and Kdb = 1.4 nM). These complexes do not allow IGF-II to bind to HT29-D4 cell surface type I IGF receptors, as evidenced by using 125I-IGF-II-IGFBP complexes. However, the addition of 40-100 micrograms/ml suramin, i.e., concentrations identical to the ones that are able to induce HT29-D4 cell differentiation, induces the release of IGF-II from IGF-II-IGFBP complexes, thereby allowing IGF-II to bind to the cell surface receptors. At such concentrations, suramin is indeed unable to alter IGF-II binding to HT29-D4 cells, a capacity that is observed only for concentrations higher than 200 micrograms/ml. Thus, suramin might have the unusual capacity to allow the establishment of an IGF-II autocrine loop involved in HT29-D4 cell differentiation. Consistent with this hypothesis is the fact that exogenously applied IGF-I (2.5 micrograms/ml) or agonist monoclonal antibody alpha IR-3 (2.5 micrograms/ml), which can bypass IGFBP present in the culture medium, induces part of HT29-D4 cell differentiation that is characterized by an important carcinoembryonic antigen release and the induction of numerous intercellular cysts with microvilli.

Changes in the cAMP synthesis by fetal thyroid during the 5 last days of the intrauterine life in the rat.

The cAMP synthesis by fetal rat thyroids is stimulated in vitro by thyrotropin (TSH) or forskolin during the 5 last days of intrauterine life. The effects are TSH- or forskolin-dose-dependent with equal responses to 20 mIU.ml-1 TSH or to 1 microM forskolin. The magnitude of the responses to TSH or to forskolin decreases significantly as the fetus is ageing. Since forskolin effects bypass hormone receptors, the adenylate cyclase activity can be concluded to decrease during the end of gestation probably in relation with thyroid iodine accumulation. The responses to TSH and forskolin becoming synergic between 19 1/2 and 21 1/2 days indicate modifications of adenylate cyclase properties probably related to some maturation of the enzyme.

Fetal hypophysis as the main source of serum TSH in fetal rat.

Decapitation performed at days 17-18 leads to a drastic drop (82%) in blood TSH of 19 and 21-day-old rat fetuses below the mother's level. 125I-TSH injected at 21 days into the mother's bloodstream is not found in fetal blood. The fetal hypophysis is the main source of fetal plasmatic TSH.

Forskolin stimulates cAMP production and the onset of the functional differentiation in the fetal rat thyroid in vitro.

Forskolin, a diterpen stimulating the adenylcyclase induces in vitro an accumulation of cAMP in thyroid glands explanted from 17 day-old rat fetuses. Thyroid glands from 15 day-old fetuses exposed to forskolin exhibit, within 24 hr, an active folliculogenesis and 125I fixation in follicular cavities. The results indicate that cAMP probably activates the onset of both morphological and physiological maturation in the fetal rat thyroid.