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Background: Children born SGA are predisposed to metabolic abnormalities. While the therapeutic benefit of rhGH therapy in improving height is widely recognised, it can affect carbohydrate metabolism, potentially inducing diabetes. This ongoing, prospective study aims to evaluate the long-term safety and efficacy of Omnitrope® (somatropin) in children born SGA. Here we present data from an interim analysis conducted in April 2017. Methods: Prepubertal children born SGA were recruited according to standard criteria; those included are treated with Omnitrope® and followed at predetermined time intervals until final height is reached. Results: Overall, 278 children were enrolled in the study, and 13 have discontinued the study due to non-response to treatment (height velocity standard deviation score [HVSDS] < +1 after 1 year). Mean (SD) age at baseline and latest visit was 7.86 (2.74) and 13.08 (3.36) years, respectively. To date, there have been no confirmed cases of new-onset diabetes mellitus. Mean oral glucose tolerance (2h), HbA1c, and fasting glucose levels have remained relatively stable from baseline to latest visit; however, mean (SD) fasting insulin levels increased from 35.65 (34.69) pmol/L at baseline to 53.68 (35.93) pmol/L after 1 year and to 70.46 (45.60) pmol/L at latest visit. Auxological measurements indicate improvements in height parameters from baseline to latest visit, including mean height SDS (net gain Δ+1.65) and peak-centred HVSDS (baseline: -2.11; Year 1: +4.16; Year 2: +2.23; latest visit, +0.19). Similarly, mean (SD) IGF-I SDS increased from -1.08 (1.02) at baseline to 0.64 (1.21) after 1 year and to 0.53 (1.27) at latest visit. A total of 2075 adverse events (AEs) have been reported in 242 patients; 99% of these AEs were of mild-to-moderate intensity. Serious AEs (n=125) have been reported in 68 patients; 4 of these (in 3 patients) were suspected as possibly related to study treatment, and only one resulted in study drug being permanently discontinued. Conclusions: In this long-term study of Omnitrope® in children born SGA, no patient has developed new-onset diabetes during rhGH treatment and there have been no concerning or clinically relevant safety findings. In addition, Omnitrope® treatment has been effective, as documented by all height parameters. Patients receiving rhGH treatment should be closely monitored for detrimental changes in carbohydrate metabolism.

It has been hypothesized that a stimulation of regulatory cytokines by microbial compounds reduces autoimmune as well as atopic diseases. Farm-related contact to microbial compounds protects from allergies, but no data on the association between farm contact and type 1 diabetes is available. The aim of this study was to test this association. A case-control study was conducted in five children’s hospitals. Regular contact to farm animals and potential confounders were assessed using a postal questionnaire. Eligible subjects were all prevalent cases with type 1 diabetes registered in the hospitals and hospital-based control subjects (response rate 91%). Included were children aged 6–16 years living in rural areas with German nationality (242 case and 224 control subjects). Regular contact to stables (adjusted odds ratio 1.2 [95% CI 0.5–2.7]) was not associated with type 1 diabetes. In addition, regular contact to specific farm animals was not associated with case status. There was a tendency for an inverse relationship between allergic rhinitis and type 1 diabetes (0.6 [0.3–1.1]; P = 0.11). There was no evidence that early exposure to farm animals largely decreases the risk in children for developing type 1 diabetes.

BACKGROUND AND OBJECTIVE To investigate the burden and the financial and professional consequences for mothers and fathers after the onset of diabetes in their child in relationship to age at onset and family structure. PATIENTS AND METHODS All families of children with an age at onset < 14 years and a diabetes duration < 10 years treated at four large pediatric diabetes centers received a structured questionnaire (burden of diabetes, professional position and career development, financial consequences for both parents, demographic data). RESULTS 580 families with 583 children with type 1 diabetes (46 % girls, diabetes duration 5.0 +/- 3.2 years, age at onset 6.9 +/- 3.9 years) participated. 42 % of the children had an age at onset below 6 years. 11 % had single parents. Before the onset of diabetes in their children 93 % of the fathers worked full-time, thereafter 4 % changed their employment. Mothers worked at onset full-time in 22 % and part-time in 38 %; thereafter 31 % reduced their working time or stopped working. 33 % of the mothers reported handicaps in their professional career development, especially those with a child with age at onset below 6 years (44 %). Negative financial consequences were present in 44 % of the families. The day to day burden on a scale from 1 to 5 decreased both in mothers and in fathers with increasing age at onset. The individual burden was higher in mothers (3.6 +/-1.1) than in fathers (2.8 +/- 1.1) (p = 0.000). CONCLUSIONS Initiatives to reduce the burden on families with a child with diabetes are urgently needed. Particularly the social and professional integration of mothers with younger children at diabetes onset need to be improved through support measures outside the family.

Objective: The adrenogenital syndrome (AGS) is familiar to pediatricians but less so to gynecologists. The aim of this study was to evaluate the gynecologic history and status of adult females with AGS. Methods: We attempted to contact 100 patients with AGS treated at our institution between 1958 and 1996. Forty-five patients completed a questionnaire; 21 additionally underwent a gynecologic examination. Results: Fifteen patients had the salt-wasting form, 26 the simple virilizing form, and 4 the late-onset form of AGS. The mean age of menarche was 14.8,13.9, and 16.5 years, respectively. Before reconstructive surgery, genitalia had been Prader intersexual stage I in 8, stage II in 10, stage IV in 14, and stage V in 2 patients. Menstrual irregularities were reported by 75% and urologic problems by 33 % of the women. Thirty-three patients (73%) were sexually active. Sixteen patients became pregnant and those with the simple virilizing or late-onset form of the condition delivered a total of 26 children (13 vaginal, 2 forceps, and 11 cesarean deliveries). Conclusions: These data confirm the long-term concerns associated with AGS. However, pregnancy and vaginal delivery are feasible in many of these women despite previous genital surgery.

Hashimoto encephalopathy (HE) is a rare steroid-responsive encephalopathy associated with elevated antithyroid antibodies and is a well recognised complication of autoimmune thyroid disease. The clinical picture is pleomorphic, presenting with variable symptoms like coma, seizures, neuropsychiatric changes (impairment of cognitive functions, behavioural and mood disturbances, hallucinations) or focal neurological deficits. HE is mainly diagnosed in adults, but also a rare differential diagnosis of encephalopathy or epilepsy in children. The diagnosis is often overlooked at presentation but is crucial as it is a treatable disease. We report on the youngest patient described up to now presenting with progressive epilepsy resistant to anticonvulsive treatment and unclear encephalopathy related to Hashimoto thyroiditis.

A longer acting recombinant FVIII is expected to serve patients' demand for a more convenient prophylactic therapy. We have developed BAX 855, a PEGylated form of Baxter's rFVIII product ADVATE™ based on the ADVATE™ manufacturing process. The conjugation process for preparing BAX 855 uses a novel PEG reagent. The production process was adjusted to yield a rFVIII conjugate with a low PEGylation degree of about 2 moles PEG per FVIII molecule. This optimised modification degree resulted in an improved PK profile for rFVIII without compromising its specific activity. PEGylation sites were identified by employing various HPLC- and MS-based methods. These studies not only indicated that about 60% of the PEG chains are localised to the B-domain, which is cleaved off upon physiological activation during the coagulation process, but also demonstrated an excellent lot to lot consistency with regard to PEGylation site distribution. Detailed biochemical characterization further showed that PEGylated FVIII retained all the physiological functions of the FVIII molecule with the exception of binding to the LRP clearance receptor which was reduced for BAX 855 compared to ADVATE™. This might provide an explanation for the prolonged circulation time of BAX 855 as reduced receptor binding might slow-down clearance. Preclinical studies showed improved pharmacokinetic behaviour and clinically relevant prolonged efficacy compared to ADVATE™ without any signs of toxicity or elevated immunogenicity. The comprehensive preclinical data package formed the basis for approval of the phase 1 clinical study by European authorities which started in 2011.

We report on 46,XX true hermaphroditism and 46,XX maleness coexisting in the same pedigree, with maternal as well as paternal transmission of the disorder. Molecular genetic analysis showed that both hermaphrodites as well as the 46,XX male were negative for Y-chromosomal sequences. Thus, this pedigree is highly informative and allows the following conclusions: first, the maternal as well as paternal transmission of the disorder allows the possibility of an autosomal dominant as well as an X-chromosomal dominant mode of inheritance; second, testicular determination in the absence of Y-specific sequences in familial 46,XX true hermaphrodites as well as in 46,XX males seems to be due to the varying expression of the same genetic defect; and third, there is incomplete penetrance of the defect.

Homozygous or double heterozygous protein-C deficiency can present at birth with purpura fulminans or later in life with venous thrombosis. Two homozygous patients who had previously sustained thrombotic episodes were investigated at a time when they were asymptomatic and not receiving antithrombotic therapy. The plasma levels of protein-C antigen and activity in both individuals were approximately 20% of normal. We administered a highly purified plasma-derived protein C concentrate to these individuals and monitored levels of several markers of in vivo coagulation activation. Assays for protein-C activation (activated protein C and protein C activation peptide) showed a sustained increase from reduced baseline levels, whereas thrombin generation (as measured by prothrombin fragment F1 + 2) gradually decreased over about 24 hours into the normal range. These investigations provide direct evidence that protein C is converted to activated protein C in vivo, and that the protein-C anticoagulant pathway is a tonically active mechanism in the regulation of hemostatic system activation in humans.

A human plasma-derived butyrylcholinesterase preparation manufactured on the industrial scale is described.The human butyrylcholinesterase (hBChE) product was extensively investigated for its purity using immunological and electrophoretic methods and characterized by thorough glycoproteomic approaches. A comprehensive preclinical testing programme addressing safety and pharmacokinetic parameters supplemented the biochemical characterization.The high-purity hBChE preparation is tetrameric and has high specific activity and molecular integrity of the protein backbone. Acute toxicity studies and in vivo thrombogenicity studies provided evidence of a sufficient safety margin for use in humans.Extensive preclinical safety and pharmacokinetic testing confirmed that this hBChE preparation can be used for further efficacy testing as a bioscavenger for toxic organophosphate compounds in appropriate animal models and ultimately in humans.

BACKGROUND Impaired bone health has been advocated after solid organ transplantation in adult and pediatric patients. Osteoporosis accompanied by fractures have been found also in heart transplantation recipients. METHODS Parameters of bone mineral density (BMD) and bone geometry were measured in 34 patients (15 females, mean age: 14.6 ± 5.5 years) 5.28 ± 5.16 years after heart transplantation (n=30) and heart-lung transplantation (n=4) using peripheral quantitative computed tomography. Transplantation had been performed because of dilated cardiomyopathy in 25, congenital heart disease in five, idiopathic pulmonary hypertension in three patients, and arrhythmogenic right ventricular dysplasia in one patient at a mean age of 9.4 ± 6.1 years. RESULTS Trabecular BMD (z scores -0.82 ± 0.50, P

Baxter has developed a recombinant therapy for treating von Willebrand's disease. Recombinant VWF is co-expressed with the rFVIII in CHO cells used to produce the rFVIII product Advate. This rVWF is used as a drug component for a rVWF-rFVIII complex drug product. CHO cells produce partially processed and partially un-processed rVWF still containing the pro-peptide. In order to make a consistent preparation containing mature and processed rVWF only rVWF is exposed to recombinant furin to remove the pro-peptide. Recombinant VWF and furin are produced under serum- and protein-free conditions. It is highly purified by a series of chromatographic steps and formulated in a protein-free buffer and has a homogeneous multimer distribution. The specific activity is higher in rVWF than in commercial plasma-derived VWF-FVIII complex products. SDS agarose electrophoretic analysis shows the presence of ultra-high molecular weight multimers. The FVIII-binding capacity and affinity of rVWF to FVIII is comparable to VWF in plasma. Carbohydrate analysis shows an intact glycosylation pattern. Recombinant VWF binds to collagen and promotes platelet adhesion under shear stress. It stabilizes endogenous FVIII in VWF-deficient knock-out mice as seen by a secondary rise in murine FVIII.

The present study investigates whether fetal adrenal transplants into the omentum of adrenalectomized rats will be integrated into the recipient's endocrine system to provide competent adrenocortical function. The results demonstrate that fetal adrenals graft with a rich vascular supply, mature histologically, and produce increasing levels of corticosterone. When bilateral adrenalectomy is performed in the recipient, survival is prolonged and addisonian crisis can be prevented. Moreover, adrenocorticotrophic hormone levels decrease with increasing levels of corticosterone, indicating that the fetal grafts are integrated into the physiological pituitary-adrenocortical feedback system.

In summary our results show that FVIII-VWF concentrates promote platelet adhesion under flow at high shear stress in a dose-dependent manner.I n addition our results indicate that platelet adhesion is effectively promoted by FVIII-VWF concentrates even if they lack the highest molecular weight multimers. There was no correlation between the VWF-mediated platelet adhesion to collagen and the number of multimers.

Osteopenia and growth retardation have been described in children with chronic arthritis. GH has an impact on both. In the present controlled study, we used peripheral quantitative computed tomography to evaluate forearm bone mass, density, and geometry as well as forearm muscle and fat area in 17 patients with juvenile idiopathic arthritis (JIA) receiving treatment with GH for 3.8 +/- 1.1 yr compared with an untreated age- and sex-matched control group (n = 17). All patients had a mean age of 15.3 +/- 2.5 yr and a mean duration of illness of 8.2 +/- 4.4 yr. Height, weight, body mass index, bone parameters, and muscle area were significantly decreased in both groups compared with those in healthy age-matched children. Compared with untreated JIA patients, GH-treated JIA patients had significant higher bone mineral content as well as total cross-sectional area (CSA), cortical CSA, and muscle CSA. Fat CSA was lower in the GH-treated group. A significant difference between groups for height-corrected cortical and muscle areas was only seen in male patients. Cortical CSA relative to muscle CSA was not different between groups. These findings are compatible with an anabolic effect of GH on muscle and bone development.

To investigate the burden and the financial and professional consequences for mothers and fathers after the onset of diabetes in their child in relationship to age at onset and family structure.All families of children with an age at onset14 years and a diabetes duration10 years treated at four large pediatric diabetes centers received a structured questionnaire (burden of diabetes, professional position and career development, financial consequences for both parents, demographic data).580 families with 583 children with type 1 diabetes (46 % girls, diabetes duration 5.0 +/- 3.2 years, age at onset 6.9 +/- 3.9 years) participated. 42 % of the children had an age at onset below 6 years. 11 % had single parents. Before the onset of diabetes in their children 93 % of the fathers worked full-time, thereafter 4 % changed their employment. Mothers worked at onset full-time in 22 % and part-time in 38 %; thereafter 31 % reduced their working time or stopped working. 33 % of the mothers reported handicaps in their professional career development, especially those with a child with age at onset below 6 years (44 %). Negative financial consequences were present in 44 % of the families. The day to day burden on a scale from 1 to 5 decreased both in mothers and in fathers with increasing age at onset. The individual burden was higher in mothers (3.6 +/-1.1) than in fathers (2.8 +/- 1.1) (p = 0.000).Initiatives to reduce the burden on families with a child with diabetes are urgently needed. Particularly the social and professional integration of mothers with younger children at diabetes onset need to be improved through support measures outside the family.

Die Keimdrusen haben endokrine und exokrine Funktionen. Sie bilden mannliche und weibliche Sexualhormone sowie Keimzellen. Bei Anlagestorungen oder Fehlfunktionen in der Embryonal- und Fetalzeit kann ein intersexuelles Genitale entstehen. Die Keimdrusen konnen primar oder sekundar erkrankt sein. Dies ist diagnostisch erfassbar und hat unterschiedliche therapeutische Konsequenzen. Die Keimdrusen werden uber einen Regelkreis mit Ruckkopplung (Hypothalamus-Hypophysen-Gonaden-Achse) gesteuert. Als Signalstoffe wirken hier das Luteinisierende-Hormon-Releasing-Hormon (LHRH) aus dem Hypothalamus, die Gonadotropine luteinisierendes Hormon (LH) und follikelstimulierendes Hormon (FSH) der Hypophyse sowie die in den Keimdrusen gebildeten Sexualhormone und Inhibine. Eine fruhzeitige Aktivierung dieser Achse fuhrt zu Pubertas praecox. Verspatete Aktivierung fuhrt zu Pubertas tarda, ausbleibende Aktivierung zu einem bleibenden Hypogonadismus. Bei Schadigungen der Keimdrusen werden die Bildung und Funktion der Keimzellen stets eher beeintrachtigt als die Bildung der Sexualhormone.

Adrenocortical tumours (ACT) are a rare but important cause of virilisation in infancy and childhood. Four cases of virilising ACT are presented. Two girls (age 0.9 years and 3.9 years) and two boys (age 6.2 years and 6.4 years) had symptoms and signs of virilisation before the age of 6 years. Diagnosis of a virilising adrenal tumour was confirmed by laboratory tests, diagnostic imaging and histology. However, one female patient was misdiagnosed and treated for 3 months as atypical congenital adrenal hyperplasia. Ultrasonography of the adrenal region could not visualise the tumour in three out of four cases. The most sensitive method of diagnostic imaging was MRI. In all cases, treatment consisted of complete surgical resection of the adrenal tumour by open abdominal surgery. Immunohistochemistry was performed in all patients and in two patients there was an overexpression of p53, indicating p53 mutation and in three cases the ki67 proliferation index was greater than 5%. The classification of ACT in childhood is extremely difficult. Histology scores adapted from adrenal tumours in adults and molecular markers are under investigation, but there is still not enough clinical experience since ACT are so rare. Conclusion:long-term follow-up is mandatory not only because of the uncertainty in classification of adrenocortical tumours, but also for observation of growth and pubertal development.

Unter Pubertas praecox versteht man das vorzeitige Auftreten sekundarer Geschlechtsmerkmale — bei Madchen im Alter von unter 8 Jahren und bei Knaben im Alter von unter 9 Jahren. Besonders in den USA ist eine Verschiebung zu einem fruheren Pubertatsbeginn zu verzeichnen, uber eine Neudefinition wird diskutiert.Nach Schatzung liegt die Inzidenz der Pubertas praecox zwischen 1:5000 und 1:10 000. Interessanterweise sind zehnmal mehr Madchen als Knaben betroffen.

Severe congenital protein C deficiency is a very rare disease which is most often caused by a homozygous genetic defect and is inherited as an autosomal recessive trait. The prevalence of homozygous protein C deficiency has been estimated at 1 in every 160,000 to 360,000 births (Miletich, 1987). Double heterozygous defects can also result in severe protein C deficiency. Protein C deficiency is manifested by the occurrence of severe, often life-threatening thromboembolic disease. Affected children typically develop symptoms of purpura fulminans, cerebral thrombosis and/or retinal or vitreous hemorrhage/thrombosis during the first days or weeks of their life. In the absence of an effective replacement therapy, treatment options have been limited and of suboptimal efficacy.

Fertility rates in women with congenital adrenal hyperplasia (CAH) are reported to be poor, but few data are available. We assessed rates and course of pregnancy, mode of delivery and long-term outcome of offspring from women with CAH.A large cohort of women with CAH due to 21-hydroxylase deficiency had initially been diagnosed and followed at one centre. Those women who had given birth were contacted. Information was gathered from hospital records, direct patient contact, structured questionnaire and the Documentation of Pregnancy and Preventive Care Booklets.Between 1978 and 1998, 18 women with CAH (one salt wasting, 12 simple virilizing, five nonclassical) had given birth to 31 children (18 females, 13 males). Delivery was by Caesarean section in 16 out of the 31 children. None of the female newborns was masculinized. Twenty-nine children were born at term, five children were small for gestational age (SGA). Postnatal development was basically normal in all children; 18 are now older than 10 years, seven are between 5 and 10 years old, six are less than 5 years old.Fertility is reduced in females with CAH, especially those with the severe or salt wasting phenotype. In those women with CAH who do conceive, course and outcome of pregnancy is mostly uneventful, although the rate of SGA offspring may be increased. Psychomotor and somatic long-term development of the children was within normal limits.

Because the current demand for alpha-1-protease inhibitor (A1PI) exceeds the available supply, we aimed to develop a process for purification of A1PI from plasma which would achieve the highest possible degree of purity, specific activity and yield.A1PI was purified from Cohn fraction IV-1,4 using ethanol precipitation and Q-Sepharose chromatography. Ceramic hydroxyapatite chromatography was used as a final purification step. Two independent virus-inactivation procedures (chemical and vapour heating) were applied.The resulting A1PI had an unprecedented high specific activity. In addition, the process led to the discovery of a new isoform of A1PI in isoelectric focusing gels.The high specific activity of the A1PI preparation achieved with this process should allow a reduction of the A1PI total protein load necessary to achieve clinically relevant effects.

To investigate the usefulness of factor VIII (FVIII) knockout mice as an animal model of hemophilia A, we characterized the antibody response in FVIII knockout mice to recombinant human FVIII, administered intravenously or subcutaneously with or without adjuvant, and compared results to those in normal mice. Anti-factor VIII antibodies were detected after both intravenous and subcutaneous administration, with the highest titers after subcutaneous administration plus adjuvant. Depending on the administration strategy. knockout mice formed antibodies more rapidly and developed higher titers of inhibitory antibodies (Bethesda) than normal mice, suggesting differences in epitope specificity. Blotting thrombin cleavage products separated by gel electrophoresis showed that both strains developed antibodies against the nonfunctional B domain as well as against functional domains of factor VIII. The antibodies were mainly of the IgG1 subclass and resembled type I antibodies in hemophilia A.

Activated protein C (APC) protects against sepsis in animal models and inhibits the lipopolysacharide (LPS)-induced elaboration of proinflammatory cytokines from monocytes. The molecular mechanism responsible for this property is unknown. We assessed the effect of APC on LPS-induced tumour necrosis factor alpha (TNF-alpha) production and on the activation of the central proinflammatory transcription factor nuclear factor-kappaB (NF-kappaB) in a THP-1 cell line. Cells were preincubated with varying concentrations of APC (200 microg/ml, 100 microg/ml and 20 microg/ml) before addition of LPS (100 ng/ml and 10 microg/ml). APC inhibited LPS-induced production of TNF-alpha both in the presence and absence of fetal calf serum (FCS), although the effect was less marked with 10% FCS. APC also inhibited LPS-induced activation of NF-kappaB, with APC (200 microg/ml) abolishing the effect of LPS (100 ng/ml). The ability of APC to inhibit LPS-induced translocation of NF-kappaB is likely to be a significant event given the critical role of the latter in the host inflammatory response.

The aim of the study was to evaluate the effect of growth hormone (GH) (14 IU/m2/week) on height, body mass index and predicted adult height in children with Prader-Labhart-Willi (PLWS) syndrome and GH deficiency. - By auxological criteria GH deficiency was suspected in 10 patients (age: 7-16 years). In 9 patients two GH provocative testings and MR imaging of the pituitary were performed. IGF-I measurements, bone age determinations, body mass index (BMI), height standard deviation score, height velocity -SDS and predicted adult height (PAH) were determined before and during GH treatment. Nine patients could be followed for up to 2 years, 3 patients for 4 years and 2 patients for 5 years of GH treatment. - Predicted adult height could be calculated in all after 1 year of treatment and was on average 11.6 cm below target height (-17.6 to +2.5 cm). GH treatment in a conventional dose (14 IU/m2/week) in daily subcutaneous injections was performed. Height SDS improved from -3.47 to -2.27 after 2 years of treatment, height velocity SDS from -1.74 to +2.65 after 1 year and remained +1.78 after 2 years. PAH increased on average by 5.1 cm. BMI was unchanged during 2 years. Bone age development was appropriate with +2.13 years after 2 years. In 2 patients who were treated with GH for up to 5 years and in 1 who was treated up to 4 years, the positive effect on height SDS and height velocity SDS persisted. - In conclusion GH treatment in a conventional dose (14 IU/m2/week) in PLWS patients with GH deficiency is effective over a period of 2 years in regard to height development and predicted adult height but ony limited in regard to changes in BMI.

In an attempt to find a rapid and reproducible method for routine polymerase chain reaction genotyping of transgenic mice, two novel approaches were developed.One approach allowed reproducible amplification from crude lysates of tail snips, using a thermally activated polymerase. In a second approach, for situations in which non-invasive techniques are necessary, oral swab specimens were amplified after DNA extraction by use of an isolation kit. Samples from 10 transgenic factor VIII knockout mice were genotyped after processing by use of these and other methods.False-negative results were not obtained by use of the two novel approaches. Despite their relative simplicity, both approaches yielded results comparable to those obtained by use of procedures known to be reliable, such as organic extraction and a DNA extraction kit.Both approaches are useful for PCR-amplification of DNA from mammalian sources.

Creutzfeldt-Jakob disease typically affects older patients, yet victims of new-variant Creutzfeldt-Jakob disease (nvCJD) are unusually young. Because the cellular prion protein PrP(C) is required for disease development, we investigated age-dependent variability in cell surface PrP(C) expression on various subclasses of human peripheral blood leukocytes (PBL) as a possible susceptibility factor.Three age groups of healthy individuals (mean ages: 6, 33 and 68) were studied by two-color FACS analysis of PBL with fluorescent monoclonal antibodies to PrP(C) and to the lineage markers CD3, CD19, CD4, and CD8. For each subclass marker, surface PrP(C) levels were expressed as mean fluorescence intensity ratios (MFIR) by dividing the geometric mean of the fluorescence of each test antibody by the geometric mean of its isotype-matched control antibody. PrP(C) expression levels in each age and lineage group were compared using appropriate non-parametric tests.We found significant age-related differences in PrP(C) expression on lymphocytes (p=0. 0004). The elderly expressed significantly higher levels than children (p=0.0006) and adults (p=0.0009). PrP(C) expression was also significantly higher in CD3(+) compared to CD19(+ )(p=0.0004) and in CD8(+) compared to CD4(+ )lymphocytes (p=0.0044).If PrP(C) expression on PBL were a significant susceptibility factor for nvCJD, young persons would display higher levels. Instead, the elderly expressed the highest amounts of PrP(C) on PBL. This argues against the hypothesis that variability in cell surface expression of PrP(C) in PBL contributes to the exquisite susceptibility of the young to nvCJD.

Die hormonelle Signalubertragung beeinflusst Wachstum, sexuelle Differenzierung und Pubertatsentwicklung, die Homoostase des Stoffwechsels und die Anpassung an Stresssituationen sowie auch die psychische und soziale Entwicklung. Endokrine Erkrankungen konnen Wachstum und Entwicklung tiefgreifend verandern.

Hemostasis is initiated by tissue factor (TF) exposed on cellular phospholipid (PL) membranes, leading to thrombin generation. The binding of thrombin to thrombomodulin (TM), activates the protein C pathway, resulting in the inactivation of factors Va and VIIIa by activated protein C (APC) and a negative feedback effect on thrombin generation. A new assay system was developed for simultaneous measurement of thrombin and APC generation in defibrinated plasma induced by large unilamellar PL vesicles complexed with full-length recombinant TF (TF:PL). TF:PL preparations with a low TF concentration induced an initial rate of thrombin generation below 100 nM/min, and resulted in less thrombin formation in the presence of TM than in its absence. In contrast, TF:PL preparations with a high concentration of TF induced a higher rate of thrombin generation, and APC-mediated feedback inhibition did not occur, despite maximal APC generation. We used the same TF:PL surfaces to study factor Va inactivation by APC in a non-plasma reaction system, and found an inverse correlation between TF surface density and the rate of factor Va inactivation. This observation suggests a previously unrecognized hemostatic effect of TF, namely a non-enzymatic surface density-based inhibition of the anticoagulant effect of APC. In this model, high concentrations and surface density of TF exert complementary effects by promoting the regular procoagulant cascade and by inhibiting the protein C pathway, thereby maximizing hemostasis after vascular injury.

Although proteolytic processing of pro-von Willebrand factor (pro-vWF) resulting in free propeptide and mature vWF is known to be initiated intracellularly, vWF released from endothelial cells may contain a high proportion of incompletely processed pro-vWF. Because pro-vWF is only rarely detectable in normal human plasma, we investigated whether extracellular processing of pro-vWF is possible. A recombinant preparation (rpvWF) containing both pro-vWF and mature vWF subunits was infused into 2 pigs and 1 dog with severe von Willebrand disease, 2 mice with a targeted disruption of the vWF gene, and 2 healthy baboons. Total vWF antigen (vWF:Ag), free propeptide, and pro-vWF were measured using enzyme-linked immunosorbent assay techniques in blood samples drawn before and after infusion. vWF:Ag increased promptly. No pro-vWF could be detected when the first postinfusion sample was drawn after 30 minutes (pigs) or 60 minutes (mice), but pro-vWF was detectable for short periods when postinfusion samples were drawn after 15 minutes (dog) or 5 minutes (baboons). In contrast, free propeptide was increased at the first timepoint measured, suggesting that it was generated from the pro-vWF in the rpvWF preparation. vWF multimers were analyzed in the rpvWF preparation and in plasma samples drawn before and after infusion of rpvWF using ultra-high resolution 3% agarose gels to allow separation of homo- and hetero-forms of the vWF polymers. Within 30 minutes after infusion in the pigs, 1 hour in the dog and the mice, and within 2 hours in the baboons, the multimer pattern had changed to that typically seen in mature vWF. These data indicate that propeptide cleavage from unprocessed vWF can occur extracellularly in the circulation. The enzyme or enzymes responsible for this cleavage in plasma remain to be identified.

Congenital adrenal hyperplasia (CAH) is a common autosomal recessive disease with a wide range of clinical manifestation. In 90-95% of the cases it is caused by 21-hydroxylase deficiency (OMIM #201910) due to mutations of the CYP21 gene (GDB Accession #M12792). In most cases the CYP21-inactivating point mutations are transferred by apparent gene conversions from CYP21P to CYP21. In only a few cases point mutations have been described, which are not present in the pseudogene. Using Southern blot analysis and DNA sequencing we have identified a novel mutation (141delT) of the CYP21 gene in a patient suffering from the salt wasting form of CAH. This results in a premature termination of a truncated protein at amino acid position 51 (L51X), which is likely to result in an enzyme with no activity. This novel mutation has not been reported to occur in the CYP21P alleles and it was not found in the CYP21P alleles in this CAH family.

Dutch Kooiker dogs with hereditary von Willebrand disease (vWD) have undetectable levels of von Willebrand factor (vWF), resulting in spontaneous hemorrhage of mucosal surfaces similar to the clinical picture of vWD in humans. We used this canine model of vWD to study the in vivo effects of a new recombinant von Willebrand factor (rvWF) preparation that contained all species of vWF multimers compared with an rvWF fraction containing only low molecular weight multimers (LMW-rvWF) and with a plasma-derived factor VIII/vWF concentrate (pdvWF). Administration of rvWF in these vWF-deficient dogs resulted in a vWF:Ag half-life of 21.6 hours in one dog and 22.1 hours in a second dog. Administration of pdvWF resulted in a half-life for vWF:Ag of 7.7 hours, and LMW-rvWF, 9 hours. The in vivo recovery of vWF:Ag after administration of rvWF was 59, 64 and 70% in three dogs, respectively; 33% after pdvWF, and 92% after LMW-rvWF. The in vivo recovery of ristocetin cofactor (RCoF) was 78, 110 and 120% for rvWF, and 25% for pdvWF. Both rvWF and pdvWF caused increases in factor VIII. Although no effect was seen on bleeding time at the dosages used, the rate of blood flow from cuticle wounds was reduced after a single bolus administration of rvWF. The rvWF was able to control a severe nose bleed in one dog.

The molecular properties of alpha 1-acid glycoprotein are briefly discussed. This molecule has been shown in in vitro experiments to have both a stabilizing effect on vascular permeability and antiinflammatory properties. We were able to demonstrate these two effects in vivo in guinea pigs (skin, Evan's Blue extravasation) and in rats (paw, carrageenan induced inflammation). Further experiments were performed in rats relating to possible therapeutic indications for alpha 1-acid glycoprotein: (1) inhibitory effect on brain edema formation after experimental stroke, (2) therapeutic effect in the puromycin aminonucleoside-induced minimal change nephrosis, (3) improvement of vital parameters in hemorrhagic-hypovolemic shock, (4) increase in survival rate in septic peritonitis, and (5) promising effects in burn-induced remote lung injury. The high content of sialic acid and the high negative charge of alpha 1-acid glycoprotein are believed to be major contributors to its stabilizing effect on vascular permeability. The protein is bound to the glycocalyx of the endothelial cells (and presumably to structures of the glomerular basement membrane), thereby hindering the passage of other polyanionic molecules through the vascular wall. The antiinflammatory/immunomodulatory effect of alpha 1-acid glycoprotein appears mainly due to suppression of polymorphonuclear neutrophils. This action is dependent on the glycan part of the molecule, which is highly variable (microheterogeneity). It is obvious that there are differences between the different glycan forms as far as the antiinflammatory property of the protein is concerned. Together with data in the literature, the results presented here suggest a variety of potential indications for therapeutic use of alpha 1-acid glycoprotein in humans.

A new collagen binding assay has been developed for the determination of the functional activity of human von Willebrand factor based on the following principle: pepsin-digested type III collagen from human placenta was covalently immobilized on a microtitre plate. Binding of collagen to the microtitre plate was carried out in neutral phosphate buffer within 1 h. A collagen concentration of 3 micrograms mL-1 was sufficient to achieve optimal coating. After drying, the coated microtitre plates remained stable for months without losing their vWF-binding properties and could be incorporated into a ready-to-use kit. The suitability of various types of collagen for use in this assay was evaluated by simulating the binding of vWF to collagen immobilized on the microtitre plate by using surface plasmon resonance technology with collagen immobilized on a sensor chip. vWF was most effectively bound to pepsin-digested type III collagen from human placenta. The assay thus comprises the following steps: (1) serial dilutions of a vWF reference preparation and vWF-containing samples are prepared and bound to a microtitre plate which is precoated with collagen; (2) vWF is detected with a polyclonal antibody; (3) the substrate reaction is photometrically measured with an ELISA reader. This test is highly specific and sensitive for vWF (detection limit: 10 ng mL-1). The collagen binding activity measured corresponds to the degree of multimerization of vWF. The high sensitivity and the short time needed to carry it out may make it useful for clinical diagnosis and for the measurement of the functional activity of vWF in factor concentrates. In certain applications it may represent a suitable replacement for the ristocetin cofactor assay.

Dutch Kooiker dogs with hereditary von Willebrand disease have undetectable levels of von Willebrand factor (vWF), resulting in spontaneous haemorrhage of mucosal surfaces similar to the clinical picture of von Willebrand disease in humans. We used this canine model of von Willebrand disease to study the in vivo effects of a new recombinant von Willebrand factor (rvWF) preparation that contained all species of vWF multimers compared with a rvWF fraction containing only low molecular weight multimers (LMW-rvWF) and with a plasma-derived factor VIII/vWF concentrate (pdvWF). Administration of rvWF in these vWF-deficient dogs resulted in a vWF:Ag half-life of 21.6 h in one dog and 22.1 h in a second dog. Administration of pdvWF resulted in a half-life for vWF:Ag of 7.7 h, and LMW-rvWF, 9 h. The in vivo recovery of vWF:Ag after administration of rvWF was 59%, 64% and 70% in three dogs, respectively; 33% after pdvWF, and 92% after LMW-rvWF. The in vivo recovery of ristocetin cofactor (RCoF) was 78%, 110% and 120% for rvWF, and 25% for pdvWF. Both rvWF and pdvWF caused increases in FVIII. Although no effect was seen on bleeding time at the dosages used, the rate of blood flow from cuticle wounds was reduced after a single bolus administration of rvWF. The rvWF was able to control a severe nose bleed in one dog.

Dem von-Willebrand-Faktor (vWF) kommt neben seiner Bedeutung als Carrier und Stabilisator fur Faktor VIII eine bedeutende Funktion in der primaren Hamostase zu. Hier fungiert er als Bindeglied zwischen spezifischen Rezeptoren der Thrombozytenoberflache und Komponenten der extrazellularen Matrix wie beispielsweise Collagen. Seine Domanenstruktur wird in Abbildung 1 wiedergegeben. Die Collagenbindungsepitope befinden sich innerhalb eines Disulfid-Loops der A1- bzw. A3-Domane der reifen Untereinheit des vWF [1].

Der Ausfall des Wachstumshormons (STH) wird im allgemeinen im 2.–4. Lebensjahr erkannt. Bei rund der Halfte der Kinder ist eine geburtstraumatische Genese anzunehmen, wobei haufig die Geburt aus Steislage erfolgte. Auserdem gibt es familiare und idiopathische Falle. Die Diagnosestellung erfolgt uber den Insulinbelastungs-, den Argininbelastungs- oder den Clonidintest. Sehr aussagekraftig ist auch das nachtliche STH-Profil im Schlaf mit Werten alle 20–30 min. Fur die Diagnose eines echten STH-Mangels sind wenigstens 2 eindeutig pathologische Tests (maximaler Anstieg von STH in beiden Tests auf einen Wert von unter 10 ng/ml) zu fordern.

In humans, type III von Willebrand disease is caused by deletions or nonsense mutations. In dogs, the underlying genetic defects have not been determined yet. We searched for the genetic defect in four related type III deficient Dutch Kooiker dogs obtained from one breeder. Mutation analysis was performed with total RNA isolated from platelets or whole blood. The complete coding region of the vWf gene was amplified by RT-PCR and sequenced by the cycle sequencing technique. Two homozygous mutations were found, a G--A transition at the first position of the donor splice site sequence of intron 16 (TGgtaagt--TGataagt) and a missense mutation at nt 208 (G--A) (1). The splice site defect resulted in the generation of a transcript containing 46bp of intron sequence and a stop codon at amino acid position 729 in the propeptide region of the vWf protein. This mutation seems to be causative for the type III phenotype. The effect of the missense mutation in exon 3 which causes a change of Val to Ile on the vWD phenotype is unclear. Probably, this transition represents a polymorphism occurring in Dutch Kooiker dogs. Both mutations were not present in 5 healthy mongrel dogs.