Molecular epidemiology of respiratory syncytial virus in hospitalised children in Heidelberg, Southern Germany, 2014-2017.

BACKGROUND: Respiratory syncytial virus (RSV) is the leading cause of hopitalisation in young children with respiratory tract infections (RTI). The aim of this research project was to analyse RSV genotypes and the diversification of RSV strains among hospitalised children in Heidelberg, Germany. METHODS: We prospectively analysed nasopharyngeal swabs (NPS) from children who were hospitalised with acute RTI at the University Hospital Heidelberg, Germany, during winter seasons 2014 to 2017. RSV RT-PCR and RSV sequence analysis of the G gene coding for the attachment glycoprotein were performed. Clinical data was obtained using a standardised questionnaire. RESULTS: RSV was detected in 405 out of 946 samples from hospitalised children. Most RSV positive children were below the age of two years (84.4%) and had a lower RTI (78.8%). The majority of RSV positive children was male, significantly younger than RSV negative children with a median age of 0.39 years and with more severe respiratory symptoms. Out of 405 positive samples, 317 RSV strains were successfully sub-grouped into RSV subtypes A (57.4%; 182/317) and B (42.6%; 135/317). Both RSV subtypes cocirculated in all analysed winter seasons. Phylogenetic analysis of 317 isolates revealed that the majority of RSV-A strains (180/182) belonged to the ON1 genotype, most RSV-B strains could be attributed to the BAIX genotype (132/135). ON1 and BAIX strains showed a sub-differentiation into different lineages and we were able to identify new (sub)genotypes. CONCLUSION: Analysis of the molecular epidemiology of RSV from different seasons revealed the cocirculation and diversification of RSV genotypes ON1 and BAIX.

[What rheumatologists can learn from ophthalmologists]. / Was der Rheumatologe vom Augenarzt lernen kann.

Intraocular inflammation with the imprecise and broad umbrella term "uveitis" is a diagnostic and therapeutic challenge in ophthalmology. Uveitis is one of the most common causes of blindness worldwide and due to the associated costs is comparable to diabetic retinopathy. Patients can be affected by uveitis at any age. Any part of the eye may be affected. The symptoms range from complete absence of symptoms, through all types of vision deterioration up to a red and even very painful eye. Uveitis can be strictly unilateral (also alternating from the left to the right eye) or bilateral with a relapsing or chronic course. The transitions are smooth and the differential diagnoses are very broad. In addition to infectious forms and ocular syndromes restricted to the eye, it also includes those with extraocular systemic diseases, such as ankylosing spondylitis or sarcoidosis. All commonly administered immunosuppressive treatment strategies in rheumatology can be used for non-infectious forms in addition to local and regional forms of treatment. The diagnostic and therapeutic impulses of this interdisciplinary interface between rheumatology and ophthalmology is discussed in more detail in this article.

Transition to adult diabetes care in Germany-High risk for acute complications and declining metabolic control during the transition phase.

OBJECTIVE: There is evidence that transition from pediatric to adult health care is frequently associated with deterioration of health in youths with type 1 diabetes (T1D). The aim of this study was to compare metabolic control, acute complications and microvascular complications in adolescents and young adults before and after transfer to an adult treatment center with respect to the time between first visit in the adult center and last visit in pediatric treatment. METHODS: All data were collected during routine care and retrieved from the German/Austrian DPV database. We analyzed data as of March 2017. RESULTS: We found 1283 young adults with available data of the last pediatric treatment year and the first year after transition to adult care. HbA1c increased significantly from 8.95% (74 mmol/mol) before to 9.20% (77 mmol/mol) in the first year after transition. Frequency of DKA with hospitalization (0.10-0.191 per annum, P < .0001) and severe hypoglycemia (0.23-0.46 per annum, P = .013) doubled during transition. Microvascular complications increased dramatically depending on the time between first visit in adult treatment and last visit in pediatric care. We could not find a significant correlation of this rise of microvascular complications to the duration of transition (short or long). CONCLUSION: This phase of life bears a high risk for detrimental outcome in young adults with T1D. Structured transition programs with case management are therefore needed to improve the transition process and outcomes.

Treatment of young patients with HNF1A mutations (HNF1A-MODY).

AIM: Children and adolescents with a molecular diagnosis of HNF1A-MODY should be treated with oral sulfonylurea according to current International Society for Pediatric and Adolescent Diabetes (ISPAD) guidelines. METHODS: We surveyed the German-Austrian DPV database of 50 043 people and included 114 patients with a confirmed molecular-genetic diagnosis of HNF1A mutation and diabetes onset at below age 18 years. We analysed hypoglycaemic episodes, metabolic control (HbA1c ) and other clinical variables according to treatment groups. RESULTS: People with HNF1A-MODY were included and analysed according to treatment with insulin alone (n = 34), sulfonylurea (n = 30), meglitinides (n = 22) or lifestyle (n = 28). In those receiving any drug treatment (n = 86), severe hypoglycaemia did not occur with meglitinide and was highest (at 3.6 events per 100 patient-years) with insulin. HbA1c was highest with insulin treatment (insulin = 58 mmol/mol, 7.5%; sulfonylurea = 55 mmol/mol, 7.2%; meglitinides = 52 mmol/mol, 6.9%; P = 0.008), whereas weight (BMI SD score), serum lipids and blood pressure were not different. CONCLUSIONS: Of note, 40% of people with HNF1A-MODY and medical treatment were receiving insulin alone and thus were not being treated in line with up-to-date International Society for Pediatric and Adolescent Diabetes/International Diabetes Federation guidelines, despite insulin treatment being associated with worse metabolic control and the risk of hypoglycaemia. The unlicensed use of oral drugs in patients below age 18 years and adherence by both doctors and patients to the initial insulin treatment might contribute to this finding.

Moderate weight reduction in an outpatient obesity intervention program significantly reduces insulin resistance and risk factors for cardiovascular disease in severely obese adolescents.

Background. Metabolic risk factors like insulin resistance and dyslipidemia are frequently observed in severly obese children. We investigated the hypothesis that moderate weight reduction by a low-threshold intervention is already able to reduce insulin resistance and cardiovascular risk factors in severely obese children. Methods. A group of 58 severely obese children and adolescents between 8 and 17 years participating in a six-month-long outpatient program was studied before and after treatment. The program included behavioral treatment, dietary education and specific physical training. Metabolic parameters were measured in the fasting state, insulin resistance was evaluated in an oral glucose tolerance test. Results. Mean standard deviation score of the body mass index (SDS-BMI) in the study group dropped significantly from +2.5 ± 0.5 to 2.3 ± 0.6 (P < 0.0001) after participation in the program. A significant decrease was observed in HOMA (6.3 ± 4.2 versus 4.9 ± 2.4, P < 0.03, and in peak insulin levels (232.7 ± 132.4 versus 179.2 ± 73.3 µU/mL, P < 0.006). Significant reductions were also observed in mean levels of hemoglobin A(1c), total cholesterol and LDL cholesterol. Conclusions. These data demonstrate that already moderate weight reduction is able to decrease insulin resistance and dyslipidemia in severely obese children and adolescents.

Genetic and clinical characteristics of patients with HNF1A gene variations from the German-Austrian DPV database.

OBJECTIVE: To determine prevalence, genetic and phenotype characteristics of patients with hepatocyte nuclear factor-1α (HNF1A) variants in the Diabetes Patienten Verlaufsdokumenation (DPV) multicentre database and to examine the influence of HNF1A mutation type, or location on clinical phenotypes. PATIENTS AND METHODS: Seventy-one DPV patients were labelled as HNF1A-MODY (MODY3). Forty-four patients carried HNF1A mutations, while 27 patients were found to have HNF1A polymorphisms only. Associations between mutation type/position and age at disease onset, HbAlc, body mass index (BMI), diagnosis, family history and treatment modality were analysed using non-parametric statistics (Wilcoxon test). RESULTS: Patients with HNF1A mutations were 36% male, aged 14.1±5.8 years at diagnosis, and slightly overweight (BMI-SDS: +0.8±1.1). Treatment was lifestyle intervention (20.5%), insulin (35.3%), oral anti-diabetic (OAD, 43%) and both insulin+OAD (15.9%). More patients with missense mutations (60%) than patients with nonsense mutations/frameshift (23.8%) did not use insulin (P=0.03). No differences were found with regard to mutation types, isoform or domain. We identified several previously undescribed mutations in the cohort including c.-158insGGGTTGG in the promoter region, G31X, E41X, Q130X, L162P, R245I, A269P, S355X, Q398X, Q473X, Q495X, E508X, P588fs-insGCCA and P588fs-delAC. Patients carrying HNF1A polymorphisms were significantly younger at diagnosis than patients with HNF1A mutations (10.9±4.2 vs 14.19±5.8 years; P=0.027), and all carried I27L, S487N and A98V (n=3). CONCLUSION: HNF1A-MODY is the second most frequent MODY diagnosis registered in the DPV database, and previously undescribed HNF1A mutations account for about one-third of HNF1A-MODY cases. Patients with HNF1A polymorphisms documented as HNF1A-MODY were misclassified. They may have autoantibody-negative type 1B or type 2 diabetes or may have other MODY types.

Entities and frequency of neonatal diabetes: data from the diabetes documentation and quality management system (DPV).

AIMS: The aim of this study was to elucidate the entities and the frequency of neonatal diabetes mellitus (NDM) in a large representative database for paediatric diabetes patients in Germany and Austria. METHODS: Based on the continuous diabetes data acquisition system for prospective surveillance (DPV), which includes 51,587 patients with onset of diabetes before the age of 18 years from 299 centres in Germany and Austria, we searched for patients with onset of diabetes mellitus in the first 6 months of life. RESULTS: Ninety patients were identified, comprising 0.17% of all paediatric cases in the DPV registry. This represented an incidence of approximately one case in 89,000 live births in Germany. A monogenic basis for NDM was established in 30 subjects (seven UPD6, 10 KCNJ11, seven ABCC8, two FOXP3, two PDX1, one INS, one EIF2AK3). Pancreatic hypoplasia or agenesis was reported in 10 patients and seven subjects were classified as having Type 1 diabetes by their centres. Transient neonatal diabetes (TNDM) accounted for approximately 10% of all cases with NDM. No aetiology was defined in 41 subjects, which may reflect incomplete genetic testing or novel genetic aetiologies. CONCLUSION: Based on a large database, we identified a higher rate of NDM in Germany than has been reported previously. Full molecular genetic testing should be performed in all patients diagnosed before 6 months of age.

Transport of insulin-like growth factor-I across endothelial cell monolayers and its binding to the subendothelial matrix.

Cultured human umbilical vein endothelial cells (HUVEC) were used as a model to study transendothelial IGF-I transport, and its deposition into the extracellular matrix (ECM). Specific binding of (125)I-IGF-I to HUVEC monolayers was demonstrated, which was inhibited by aIR-3, a specific antibody directed against the IGF-I receptor. ECM-associated (125)I-IGF-I was approximately 10% of cell-bound IGF-I at 22 degrees C, and increased 4.5-fold at 37 degrees C, indicating that endothelial metabolism is required for the transport. However, neither monensin and cytochalasin B, both of which block endocytosis, nor aIR-3 did inhibit transport of (125)I-IGF-I into the ECM. In order to characterize IGF-I binding to the subendothelial ECM, HUVEC were removed nonenzymatically by treatment with Triton X-100 and ammonia. Specific, saturable binding of (125)I-IGF-I to the isolated ECM was observed, which was protease-sensitive. Antibodies directed against vitronectin inhibited IGF-I binding to the matrix by 35%, while antibodies directed against other ECM proteins had no significant influence on IGF-I binding. Using radioimmunoassays the IGF binding protein-2 was detected in the ECM, while IGFBP-1 and IGFBP-3 were below the detection limits. In order to evaluate functional aspects of IGF-I binding to the matrix, HUVEC were incubated under serum-free conditions in the absence and presence of IGF-I. Under serum-free conditions 48% of cells rounded up and started to detach after 2 hours incubation, while only 23% of the cells started to detach in the presence of IGF-I. These data indicate that IGF-I is transported via a paracellular route across endothelial cells, and becomes bound to the subendothelial ECM. Vitronection seems to be involved in binding of IGF-I to the ECM. ECM-associated IGF-I might play a role in endothelial cell survival and stability.

Tri-iodothyronine treatment in children after cardiac surgery: a double-blind, randomised, placebo-controlled study.

BACKGROUND: Serum thyroid hormone concentrations decline transiently during critical illness and after surgical procedures. We investigated prospectively the endocrine and haemodynamic effects of tri-iodothyronine treatment after cardiopulmonary bypass operations in children with congenital cardiac malformations. METHODS: We did a randomised, double-blind, placebo-controlled trial, in which 40 children (median age 0.6 years; range 2 days to 10.4 years) were randomly assigned placebo (saline) or one daily infusion of tri-iodothyronine (2 microg/kg bodyweight on day 1 after surgery and 1 microg/kg bodyweight on subsequent postoperative days up to 12 days after surgery. Before and 2 h, 24 h, and 72 h after the first infusion, plasma concentrations of thyroid hormones were measured by RIA, and systolic cardiac function was evaluated by echocardiography. During the postoperative course intensive-care measures were assessed by use of the therapeutic intervention scoring system. FINDINGS: In all patients, postoperative plasma concentrations of thyrotropin, thyroxine, free thyroxine, tri-iodothyronine were abnormally low and plasma concentrations of reverse tri-iodothyronine were raised. After start of treatment, tri-iodothyronine was significantly higher in patients given tri-iodothyronine than in those receiving placebo, whereas thyrotropin, thyroxine, free thyroxine, and reverse tri-iodothyronine remained similar in the two groups. At discharge, thyroid hormones of all patients were within the normal range, but thyrotropin secretion increased to plasma concentrations higher than those seen before treatment. The mean change of cardiac index was significantly higher in children given tri-iodothyronine (20.4% [SD 19.6] vs 10.0% [15.2]; p=0.004). Systolic cardiac function improved most in patients given tri-iodothyronine after longer cardiopulmonary bypass operations. Overall, patients given tri-iodothyronine had significantly lower mean treatment scores. INTERPRETATION: Treatment of children with tri-iodothyronine after cardiopulmonary bypass operations raises tri-iodothyronine plasma concentrations and improves myocardial function especially in patients with low postoperative cardiac output without adverse events, and without delaying postoperative recovery of thyroid function. Furthermore, tri-iodothyronine reduces the need for postoperative intensive care.

Lymphocytic hypophysitis with central diabetes insipidus and consequent panhypopituitarism preceding a multifocal, intracranial germinoma in a prepubertal girl.

UNLABELLED: We report the clinical course of a prepubertal girl with central diabetes insipidus (DI) and consequent panhypopituitarism evolving over a period of 10 years due to lymphocytic hypophysitis and subsequent germinoma. Two years after the diagnosis of central DI was established, MRI revealed a thickened pituitary stalk. Later pituitary enlargement and increasing thickening of the pituitary stalk impinging on the optic chiasm required a trans-sphenoidal biopsy which disclosed active hypophysitis with lymphocytic infiltrates and necrosis. High dose dexamethasone treatment only temporarily halted the disease process. Therefore, stereotactic radiation therapy was performed as a rescue treatment and MRI findings almost reversed. However, the subsequent MRI showed multiple intracranial lesions identified histologically as a germinoma and a standard chemotherapy and radiation was performed. CONCLUSION: The diagnosis of diabetes insipidus in children requires long-term follow up beyond the pubertal age in order to establish the underlying cause. In contrast to lymphocytic hypophysitis in adults, lymphocytic hypophysitis in prepubertal children may represent the first sign of a host reaction to an occult germinoma.

Interaction of IGF-I and 1 alpha, 25(OH)2D3 on receptor expression and growth stimulation in rat growth plate chondrocytes.

Growth plate cartilage cell express receptors for, and are affected by both IGF-I and 1 alpha, 25(OH)2D3. The studies were undertaken to investigate interaction between these two hormone systems, that is, (i) to study effects of 1 alpha, 25(OH)2D3 on IGF-type 1 receptors (IGFIR), on IGF-I stimulated cell replication, colony formation, and on alkaline phosphatase activity (AP), and conversely, (ii) to study the effect of IGF-I on vitamin D receptor (VDR) expression on 1 alpha, 25(OH)2D3 stimulated growth parameters and on AP activity. Freshly isolated rat tibial chondrocytes were grown in monolayer cultures, (serum-free) or in agarose stabilized suspension cultures (0.1% FCS). Vitamin D receptor and IGFIR were visualized by immunostaining with the monoclonal antibody (mAb) 9A7 gamma and mAb alpha IR3, respectively, and quantitated by RT-PCR for mRNA and by Scatchard analysis using [3H]-1,25(OH)2D3 and [125I]-alpha IR3. Cell proliferation was measured by [3H]-thymidine incorporation, growth curves in monolayer cultures, and by colony formation in agarose-stabilized suspension cultures. IGF-I dose-dependently increased [3H]-thymidine incorporation. 1 alpha, 25(OH)2D3, but not 1 beta, 25(OH)2D3 was stimulatory at low ((10-12 M) and slightly inhibitory at high (10-8 M) concentrations. The effect of IGF-I was additive to that of 1 alpha, 25 (OH)2D3 [IGF-I 60 ng/ml, 181 +/- 12.7; 1 alpha, 25(OH)2D3 10(-12) M, 181 +/- 9.8%, IGF-I + 1 alpha, 25(OH)2D3, 247 +/- 16.7%, P < 0.05 by ANOVA] and specifically obliterated by polyclonal IGF-I antibody (AB-1). Interaction could also be confirmed in suspension cultures. IGFIR mRNA and [125I]-alphaIR3 binding was increased by low (10(-12) m) but not by high (10(-8) M) concentrations of 1 alpha, 25(OH)2D3. Homologous up-regulation by IGF-I (60 ng/ml) was specifically inhibited by AB-1 and markedly amplified by coincubation with 1 alpha, 25(OH)2D3 (10(-12)m). Immunostaining with alpha IR3 showed specific IGFIR expression in rat growth cartilage, but not liver tissue. Stimulation of chondrocytes with 1 alpha, 25(OH)2D3 or IGF-I suggested some increase of receptor expression in single cells, but the predominant effect was increased recruitment of receptor positive cells, Vitamin D receptor expression was markedly stimulated (fourfold) by IGF-I (60 ng/ml), but not IGF-II and inhibited by actinomycin D. This study shows that IGF-I and 1 alpha, 25(OH)2D3 mutually up-regulate their respective receptors in growth plate chondrocytes. In parallel, they have additive effects on cell proliferation and colony formation suggesting independent effector pathways.

Ligand blot analysis of insulin-like growth factor-binding proteins using biotinylated insulin-like growth factor-I.

A nonradioactive method for the detection of insulin-like growth factor-binding proteins (IGFBPs) was developed utilizing human recombinant insulin-like growth factor-I (IGF-I) biotinylated with N-hydroxysuccinimido-biotin. Human plasma samples were separated by 15% SDS polyacrylamide gel electrophoresis, and proteins were transferred to nitrocellulose by Western blotting. The nitrocellulose sheets were incubated overnight with IGF-I-biotin at 4 degrees C. The next day streptavidin-peroxidase was added for 1 h, and IGFBPs were visualized by an enhanced chemiluminescence system. Five characteristic bands with molecular weights of 41 and 39 (IGFBP-3), 34 (IGFBP-2), 30 (IGFBP-1) and 24 kD (IGFBP-4) were detected. Binding was specific for IGFs, since unlabeled IGF-I inhibited IGF-I-biotin binding. IGFBP ligand blots with biotinylated IGF-I and (125)I-IGF-I yielded comparable results. The suitability of the new assay for clinical purposes was demonstrated by several clinical examples. In summary, a rapid, reliable, nonradioactive assay for qualitative analysis of IGFBPs has been developed.

Transient secondary hypothyroidism in children after cardiac surgery.

Thyroid hormone status was assessed in 132 children with congenital heart defects undergoing cardiac surgery (median age 3.1 y; range 2 d to 16.2 y). Plasma TSH, thyroxine (T4), free thyroxine (fT4), triiodothyronine (T3), reverse triiodothyronine (rT3), thyroglobulin (Tg), and urinary iodine excretion were measured before and every other day after cardiac surgery (d 1-21). After surgery we observed strikingly low plasma concentrations of TSH (0.4 mU/L; 0.2-1.3), T3 (0.6 nmol/L; 0.3-1.2), T4 (48.9 nmol/L; 12.9-82.4), IT4 (12.9 pmol/L; 5.1-19.3), and Tg (9.4 micrograms/L; 1.5-20.6), whereas rT3 plasma concentrations increased (0.13 pmol/L; 0.05-0.3; n = 40). The maximal post-operative changes of TSH and rT3 preceded changes of T3, T4, fT4, and Tg. Postoperative urinary iodine excretion increased significantly (n = 109). Thyroid hormone plasma concentrations were lowest after cardiopulmonary bypass operations and in patients treated with dopamine. In patients with postoperative T3 plasma concentrations less than 0.6 nmol/L (n =52) the period of mechanical ventilation and intensive care treatment was significantly prolonged. Furthermore, the cumulative doses of inotropic and vasoactive catecholamines and furosemide were significantly higher in this patient group. Our results demonstrate transient secondary hypothyroidism in children after cardiac surgery that may contribute to postoperative cardiac and respiratory dysfunction and may delay recovery. Possible benefits of thyroid hormone replacement therapy need to be thoroughly examined.

Short-term, high-dose testosterone treatment fails to reduce adult height in boys with constitutional tall stature.

UNLABELLED: Height predictions based on three different methods (Bayley-Pinneau [BP], Tanner-Whitehouse Mark II [TW II], Roche-Wainer-Thissen [RWT]) were compared to adult heights in 19 males with constitutional tall stature previously treated with high-dose testosterone oenanthate for 6 months (group A) and 25 untreated tall males (group B). Their chronological ages (CA) at the initial evaluation of tall stature ranged from 12.1 to 16.6 years in group A and from 10.4 to 15.7 years in group B; at the time of assessment of adult height ages ranged from 18.0 to 26.5 years and from 18.4 to 25.1 years, respectively. Height measurements and predicted adult heights were expressed as height standard deviation scores (height SDS) for chronological age using the tables of Reinken and van Oost [14]. Height SDS in group A were 2.8 (range = 1.8-5.4) before testosterone treatment, 3.0 (range = 2.0-4.8) thereafter and finally 3.0 (range = 2.1-4.2) (P = NS) and in group B 2.7 (range = 0.5-4.3) and 2.4 (range = 1.3-3.5) (P = NS). A significant difference between adult height SDS and predicted height SDS according to BP was detected both in group A (3.0; range = 2.1-4.2 vs 3.6; range = 2.4-5.0. P < or = 0.004) and group B (2.4; range = 1.3-3.5 vs 3.0; range = 2.0-4.9; P < or = 0.0002), whereas no significant difference between adult height SDS and predicted height SDS according to TW II and RWT was found in either group. These data indicate that BP height predictions overestimated adult height in our patient group of treated and untreated males with constitutional tall stature. In contrast, the TW II and RWT methods were more accurate in predicting adult height in these patients, but also failed to demonstrate that testosterone therapy in boys with constitutional tall stature can be limited to a 6-month period in order to reduce adult height. CONCLUSION: The widely used height prediction method of BP is inaccurate in boys with constitutional tall stature. High dose testosterone treatment fails to reduce adult height in these individuals when discontinued before complete closure of the epiphyses.

Bilateral renal venous thrombosis in a neonate associated with resistance to activated protein C.

Renal venous thrombosis (RVT) is a serious complication of neonates. In most cases the underlying cause of RVT remains unclear. Here we report a neonate with bilateral RVT and adrenal haemorrhage associated with a heterozygous mutation of the gene encoding for clotting factor V, resulting in resistance to activated protein C. Vigorous thrombolytic therapy with urokinase followed by recombinant tissue plasminogen activator dissolved the thrombus in the inferior vena cava and restored perfusion of both kidneys. However, a haemorrhagic rupture of the right kidney occurred, requiring emergency nephrectomy. Despite reperfusion of the left kidney and resumption of urine output, the patient remained dialysis dependent. Due to persistent adrenal insufficiency, long-term substitution of hydrocortisone was necessary. The patient was prophylactically treated with coumarin during the first 6 months of life and is now waiting for renal transplant at the age of 1 year.

Influence of hysterectomy on the fibrinolytic activity of plasma of women with intact ovarian function.

Hysterectomies are frequently required operations in gynecology. Several studies have reported an association between premenopausal hysterectomy and the risk of cardiovascular diseases. However, the pathophysiological linkages between these two conditions have not been elucidated. In recent years it has been shown that a decrease in plasma fibrinolytic activity is associated with increased risk of thrombosis. Furthermore, it has been known that the uterus is a very finbrinolytic active organ. In the present study we investigated the hypothesis that hysterectomy may lead to a decrease in plasma fibrinolytic activity, and thereby increase the risk for thromboembolic diseases. Fibrinolytic parameters of plasma were investigated in 26 women before and 6 weeks after premenopausal hysterectomy. Euglobulin lysis time (ELT), a global measure of plasma fibrinolytic activity, and the levels of tissue plasminogen activator (tPA) and plasminogen activator inhibitor type 1 (PAI-1) were not different before and after hysterectomy. The ELT difference before and after venous occlusion, which is a good indicator for the risk of thrombosis, was also not significantly changed after hysterectomy. Estradiol-17 beta, progesterone, LH, FSH and sex hormone binding globulin displayed no significant changes after hysterectomy. Furthermore, the hormone measurements also indicated that the women were premenopausal. There were no correlations between the hormone values and fibrinolytic parameters. These data indicate that premenopausal hysterectomy does not lead to changes in plasma fibrinolytic activity.

The fibrinogen anion-binding exosite of thrombin is necessary for induction of rises in intracellular calcium and prostacyclin production in endothelial cells.

Thrombin stimulation of prostacyclin (PGI2) synthesis by cultured human umbilical vein endothelial cells (HUVEC) requires the active site of thrombin and involves rapid and transient rises in cytoplasmic free calcium [Ca2+]i. In this study, we investigated whether or not the anion-binding exosite for fibrinogen recognition of thrombin (which confers certain substrate specificities) is also necessary for the induction of rises in [Ca2+]i and PGI2 production. Thrombin variants which lack either the catalytic site (DIP-alpha-thrombin) or anion-binding exosite (gamma-thrombin) either alone or in combination failed to induce rises in [Ca2+]i or PGI2 production in HUVEC. To further study the role of the anion-binding exosite of thrombin in the activation of HUVEC, COOH-terminal fragments of hirudin were used. This portion of hirudin interacts with the anion-binding exosite of thrombin and inhibits thrombin-induced fibrinogen coagulation while leaving the catalytic activity of thrombin intact. A 21-amino acid COOH-terminal peptide of hirudin (N alpha-acetyldesulfato-hirudin45-65 or Hir45-65) inhibited thrombin-induced (0.5 U/ml) rises in [Ca2+]i and PGI2 production with IC50 of 0.13 and 0.71 microM, respectively. Similar results were obtained using shorter hirudin-derived peptides. Thus, the fibrinogen anion-binding exosite of thrombin is required for alpha-thrombin-induced rises in [Ca2+]i and PGI2 production in HUVEC.

Identification of endothelial and mesothelial cells in human omental tissue and in omentum-derived cultured cells by specific cell markers.

Human omental tissue has been used as a source for the isolation and cultivation of microvascular endothelial cells, but also for mesothelial cells. Since both cell types have several morphologic and functional features in common, concerns were raised whether endothelial cells can be separated from mesothelial cells by the methods described for the isolation of microvascular endothelial cells. In the present study, endothelial cells were identified in the capillaries of native human omentum by several endothelial-cell specific markers. von Willebrand factor was demonstrated by polyclonal and monoclonal antibodies, a lectin-specific ligand by Ulex europaeus I, and an endothelial-cell specific surface epitope by the monoclonal antibody, PAL-E. These markers were not found positive with mesothelial cells of native omentum. Mesothelial cells were identified by monoclonal antibodies against the intermediate filaments, cytokeratin and vimentin. After having demonstrated the specificity of the methods for the distinction between endothelial and mesothelial cells within native omentum, these methods were applied to omentum-derived cells previously claimed to be microvascular endothelial cells. These cultured cells proved to be negative for von Willebrand factor, Ulex europaeus I ligand and PAL-E epitope. In contrast to this, the cultivated cells stained positive to cytokeratin and vimentin. Furthermore, it was shown by immunoprecipitation studies that omentum-derived cells did not synthesize and secrete vWF, indicating the nonendothelial nature of these cells. Finally, electron microscopy demonstrated microvilli on the surface of cultivated omentum-derived cells indicative for the mesothelial origin of these cells. The data presented demonstrate that the cells obtained using the previously published methods for the isolation and cultivation of "microvascular endothelial cells" from omental tissue are of mesothelial and not of endothelial origin. Thus, a great number of data obtained with this type of omentum-derived cells thought to be microvascular endothelial cells need re-evaluation.

Heparin stimulates fibrinolysis in mesothelial cells by selective induction of tissue-plasminogen activator but not plasminogen activator inhibitor-1 synthesis.

The regulation of tissue-plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI-1) synthesis was studied in cultured human mesothelial cells derived from omentum (HOMC). Heparin (100 U/ml) as well as pentosan polysulfate (300 micrograms/ml) stimulated tPA synthesis by HOMC 2.9-4.5-fold. Heparin-induced tPA production was dose- and time-dependent and was inhibited by cycloheximide. tPA production by HOMC was also stimulated by phorbol 12-myristate 13-acetate (2.6-fold), fibrin clots (1.9-fold), or batroxobin (1.9-fold). Heparin and pentosan polysulfate did not stimulate PAI-1 production by HOMC, while phorbol 12-myristate 13-acetate (100 nM) increased the concentration of PAI-1 in the conditioned medium by 2.6-fold over 24 h. The interaction of heparin with HOMC was studied by direct binding experiments. Dose-dependent specific binding of biotinylated heparin to HOMC was saturable at about 10 micrograms/ml, the KD was estimated to about 0.15 microM. Biotinylated heparin bound rapidly to HOMC and reached a plateau within 60 min. Unlabeled heparin as well as pentosan polysulfate inhibited binding of biotinylated heparin in a dose-dependent fashion. These data demonstrate that heparin interacts with HOMC, and increases the fibrinolytic capacity in these cells by selectively increasing the production of tPA.

Structural requirements for the extracellular interaction of plasminogen activator inhibitor 1 with endothelial cell matrix-associated vitronectin.

The interaction of plasminogen activator inhibitor-1 (PAI-1) with its binding protein vitronectin (VN) (Declerck, P. J., De Mol, M., Alessi, M.-C., Baudner, S., Paques, E.-P., Preissner, K. T., Müller-Berghaus, G., and Collen, D. (1988) J. Biol. Chem. 263, 15454-15461) in the extracellular matrix (ECM) of cultured human endothelial cells (HUVEC) was studied. Like PAI-1, VN was found associated with the ECM as evidenced by direct antibody binding, by Western blot analysis as well as by diffuse immunofluorescence staining in permeabilized HUVEC. The specific interaction of VN with confluent monolayers of HUVEC was found to be saturable within 2-4 h at 37 degrees C only with respect to binding to the cells, while no saturable binding to the underlying ECM was observed, indicating that the majority if not all ECM-associated VN was derived from the culture medium. In contrast to PAI-1, ECM-associated VN was resistant toward glycine (pH 2.3), guanidine or urokinase treatment, suggesting that VN was tightly associated with the ECM network. Binding of recombinant PAI-1 (rPAI-1) was largely blocked by anti-VN IgG and only partly by anti-collagen IgG but not by antibodies against other ECM components, indicating that VN constitutes the primary binding protein for ECM-associated PAI-1. This contention was supported by ligand blotting experiments in which rPAI-1 was reacted with nitrocellulose replicas of electrophoretically separated ECM components. Protein band(s) (Mr = 63,000-67,000), comigrating with bovine VN (i.e. medium-derived VN) rather than with human VN were identified as major binding component(s). Moreover, binding studies with purified components revealed that PAI-1 did not show any affinity for collagen (type I/III) alone, whereas VN collagen coating was a much better template for PAI-1 binding than VN alone and that conformationally extended VN provides maximal PAI-1 binding capacity. Binding of rPAI-1 to surface-coated VN was saturable and revealed that (unlike urokinase) heparin or the synthetic peptide Gly-Arg-Gly-Asp-Ser did not inhibit PAI-1 binding. Ligand binding of rPAI-1 to nitrocellulose replicas from sodium dodecyl sulfate-polyacrylamide gels containing electrophoretically separated peptides from VN digests documented the association of PAI-1 with Mr = 10,000-20,000 fragments originating from the heparin-binding domain of VN. These results indicate that the exposure of the glycosaminoglycan-binding domain in VN may allow the concomitant binding of PAI-1 and heparin-like molecules to this region of the VN molecule.(ABSTRACT TRUNCATED AT 400 WORDS)