Reversed-phase HPLC based assay for selective and sensitive endotoxin quantification - part II.

The impact of naturally occurring 3-deoxy-d-manno­oct-2-ulsonic acid (Kdo) derivatives on endotoxin (ET) quantification was investigated for six ET standards. In our recently published chemical Kdo-DMB-LC ET assay (Bucsella et al., Anal. Methods, 2020, 12,4621) [1], the rare, ET specific sugar acid Kdo is used for ET quantification of S-type ETs. The ET content is calculated based on an external Kdo standard or a representative ET standard. In absence of a specific ET standard, the calculation is based on the reference standard ET (RSE) structure or on a worst-case scenario. This scenario overestimates the total ET content of typical S-type ET preparations by a factor of four. Mainly R-type ETs contain in addition to Kdo also Kdo-s non-stoichiometrically modified with phosphoethanolamine (PEtN), galactose (Gal) or L­glycero-d-manno-heptose (Hep) in substantial quantities. These Kdo species are separated from the unmodified Kdo. All Kdo and Kdo species follow an exponential hydrolytic release from the ET core in dependence on the hydrolysis time. Hydrolysis kinetics for identical Kdo species are the same for all ET standards. Kdo-Gal was released fastest followed by unsubstituted Kdo, Kdo-PEtN, and Kdo-Hep. Between 90 and 150 min a plateau of maximum content is obtained for all Kdo-s. That allows in case of a representative ET standard, ET quantification based on the most present Kdo derivative, here mainly unsubstituted Kdo. If no representative ET standard is available Kdo and all Kdo species must be considered for ET quantification. With that the Kdo-DMB-LC assay is applicable for R- and S-type ETs.

Application of a novel chemical assay for the quantification of endotoxins in bacterial bioreactor samples.

A novel chemical assay, the so-called Kdo-DMB-liquid chromatography (LC) assay, was used for the accurate and cost-effective determination of the endotoxin content in supernatants of Gram-negative bacteria bioreactor samples. During mild acid hydrolysis, the endotoxin-specific sugar acid 3-deoxy-D-manno-oct-2-ulsonic acid (Kdo) is quantitatively released. Kdo is reacted with 1,2-diamino-4,5-methylenedioxybenzene (DMB) to obtain the highly fluorescent derivate Kdo-DMB. It is separated from the reaction mixture by reversed phase-(U)HPLC and detected by fluorescence. From the Kdo content the endotoxin content of the sample is calculated. For three batch cultivations of Escherichia coli K12 and a fed-batch cultivation of Pseudomonas putida KT2440, the evolution of the endotoxin content in dependence on the cultivation time was monitored. Under optimal, constant cultivation conditions a linear correlation between the endotoxin content and the easy-to-access bioreactor parameters optical density at 600 nm and dry cell weight was found for both endotoxin kinds. Under stress cultivation conditions the E. coli K12 cultivation showed a stronger increase of the endotoxin content at harvest in comparison to optimal conditions. Optical density and dry cell weight may be used for production reactors as an economic real-time estimation tool to determine the endotoxin content at different cultivation time points and conditions. The optical density can further be used to establish straightforward sample dilution schemes for endotoxin quantification in samples of unknown endotoxin content. The endotoxin content [ng mL-1] measured by the Kdo-DMB-LC assay and the endotoxin activity [EU mL-1] obtained by the compendial Limulus Amoebocyte Lysate assay show a high correlation for the bacterial bioreactor samples tested.

Novel RP-HPLC based assay for selective and sensitive endotoxin quantification.

The paper presents a novel instrumental analytical endotoxin quantification assay. It uses common analytical laboratory equipment (HPLC-FLD) and allows quantifying endotoxins (ETs) in different matrices from about 109 EU per mL down to about 40 EU per mL (RSE based). Test results are obtained in concentration units (e.g. ng ET per mL), which can then be converted to commonly used endotoxin units (EU per mL) in case of known pyrogenic activity. During endotoxin hydrolysis, the endotoxin specific rare sugar acid KDO is obtained quantitatively. After that, KDO is stoichiometrically reacted with DMB, which results in a highly fluorescent derivative. The mixture is separated using RP-HPLC followed by KDO-DMB quantification with a fluorescence detector. Based on the KDO content, the endotoxin content in the sample is calculated. The developed assay is economic and has a small error. Its applicability was demonstrated in applied research. ETs were quantified in purified bacterial biopolymers, which were produced by Gram-negative bacteria. Results were compared to LAL results obtained for the same samples. A high correlation was found between the results of both methods. Further, the new assay was utilized with high success during the development of novel endotoxin specific depth filters, which allow efficient, economic and sustainable ET removal during DSP. Those examples demonstrate that the new assay has the potential to complement the animal-based biological LAL pyrogenic quantification tests, which are accepted today by the major health authorities worldwide for the release of commercial pharmaceutical products.

Analytical Platforms at Swiss Universities of Applied Sciences.

Numerous projects and industrial and academic collaborations benefit from state-of-the-art facilities and expertise in analytical chemistry available at the Swiss Universities of Applied Sciences. This review summarizes areas of expertise in analytical sciences at the University of Applied Sciences and Arts Northwestern Switzerland (FHNW), the University of Applied Sciences and Arts Western Switzerland (HES-SO), and the Zurich University of Applied Sciences (ZHAW). We briefly discuss selected projects in different fields of analytical sciences.

Pediatric prolactinoma: initial presentation, treatment, and long-term prognosis.

Prolactinoma is a rare pituitary adenoma secreting prolactin. Studies on diagnostics, treatment, and prognosis in pediatric prolactinoma patients are rare. We analyzed clinical presentation, response to treatment, and prognosis of 27 pediatric prolactinoma patients (10 m/17 f. based on patients' records. Tumors included 6 microadenomas (tumor volume: median 0.2 cm3, range 0.01-0.4 cm3; serum prolactin at diagnosis: median 101 ng/ml, range 33-177 ng/ml), 15 macroadenomas (volume: median 3.3 cm3, range 0.4-25.8 cm3; prolactin: median 890 ng/ml, range 87-8624), and 3 giant adenomas (volume: median 44.5 cm3, range 38.6-93.5 cm3; prolactin: median 4720 ng/ml, range 317-10,400); data for 3 patients were not available. Dopamine agonist treatment (n = 22) was safe and effective, leading to reductions in tumor size (p < 0.01) and prolactin levels (p < 0.01). Threat to vision was the indication for decompressing surgery in three of seven operated patients. No patient was irradiated. Long-term functional capacity was not impaired when compared with other sellar masses (n = 235). CONCLUSION: In pediatric prolactinoma, diagnosis is based on hyperprolactinemia and imaging. Dopamine agonist treatment is effective and safe. Overall survival and functional capacity as a measure of quality of survival were not impaired, indicating an optimistic prognosis. Surgery should be considered only in emergency situations of threatened visual function, not presenting a fast response to dopamine agonist treatment. Severe side effects of medication and lack of efficacy should be considered as contraindications. What is Known: • In pediatric prolactinoma-a very rare pediatric neuroendocrinological disease-gender-related differences in terms of clinical presentation at initial diagnosis are known. • Due to the rareness of the disease, reports on long-term outcome and prognosis after childhood-onset prolactinoma based on prospective follow-up are not published. What is New: • Dopamine agonist treatment is efficient and safe for tumor volume reduction in pediatric prolactinoma and surgical interventions are recommended only for decompression of the optic chiasm in case of threat to vision. In case of inefficient response to medication, side effects or parental refuse, alternative therapeutic options should be considered. • Quality of life in terms of survival and functional capacity was not impaired in pediatric prolactinoma patients when compared with 235 long-term survivors of different sellar masses.

Quality of life and growth after childhood craniopharyngioma: results of the multinational trial KRANIOPHARYNGEOM 2007.

CONTEXT: Quality of life (QoL) after childhood-onset craniopharyngioma (CP) is frequently impaired due to tumor and/or treatment-related factors such as endocrine deficits and hypothalamic involvement/lesions. PATIENTS AND METHODS: In a multinational trial, we prospectively analyzed parental and self-assessment of CP patient QoL at 3 months, 1 and 3 years after CP diagnosis related to growth hormone (GH) substitution. 47 of 194 CP recruited between 2007 and 2015 in KRANIOPHARYNGEOM 2007 were analyzed for QoL 1 and 3 years after CP diagnosis. QoL was assessed by Pediatric Quality of Life (PEDQOL) questionnaire and PEDQOL scores of parental and self-assessed QoL during 3 years follow-up after CP diagnosis were analyzed. RESULTS: Parents estimated QoL of their children worse than patients did themselves. GH substitution had no relevant effect on short-term weight and height development. CP patients GH-treated at 3 years follow-up presented at baseline (1 year after diagnosis, before GH substitution) with reduced self-assessed QoL when compared with GH non-treated CP. QoL stabilized during 1-3 years of follow-up in GH-treated patients, whereas non GH-treated patients experienced decreases in autonomy (p = 0.03), cognition (p = 0.01), and physical function (p = 0.04). CONCLUSIONS: Parents assess QoL in CP survivors worse than their children. GH substitution should be considered as a therapeutic option to ameliorate imminent impairments of QoL after CP.

Intracystic interferon-alpha in pediatric craniopharyngioma patients: an international multicenter assessment on behalf of SIOPE and ISPN.

BACKGROUND: Craniopharyngiomas are frequent hypothalamo-pituitary tumors in children, presenting predominantly as cystic lesions. Morbidity from conventional treatment has focused attention on intracystic drug delivery, hypothesized to cause fewer clinical consequences. However, the efficacy of intracystic therapy remains unclear. We report the retrospective experiences of several global centers using intracystic interferon-alpha. METHODS: European Société Internationale d'Oncologie Pédiatrique and International Society for Pediatric Neurosurgery centers were contacted to submit a datasheet capturing pediatric patients with cystic craniopharyngiomas who had received intracystic interferon-alpha. Patient demographics, administration schedules, adverse events, and outcomes were obtained. Progression was clinical or radiological (cyst reaccumulation, novel cysts, or solid growth). RESULTS: Fifty-six children (median age, 6.3 y) from 21 international centers were identified. Median follow-up from diagnosis was 5.1 years (0.3-17.7 y). Lesions were cystic (n = 22; 39%) or cystic/solid (n = 34; 61%). Previous progression was treated in 43 (77%) patients before interferon use. In such cases, further progression was delayed by intracystic interferon compared with the preceding therapy for cystic lesions (P = 0.0005). Few significant attributable side effects were reported. Progression post interferon occurred in 42 patients (median 14 mo; 0-8 y), while the estimated median time to definitive therapy post interferon was 5.8 (1.8-9.7) years. CONCLUSIONS: Intracystic interferon-alpha can delay disease progression and potentially offer a protracted time to definitive surgery or radiotherapy in pediatric cystic craniopharyngioma, yet demonstrates a favorable toxicity profile compared with other therapeutic modalities-important factors for this developing age group. A prospective, randomized international clinical trial assessment is warranted.

First experiences with neuropsychological effects of oxytocin administration in childhood-onset craniopharyngioma.

PURPOSE: The hypothalamic hormone oxytocin plays a major role in regulation of behavior and body composition. Quality of survival is frequently impaired in childhood craniopharyngioma patients due to sequelae such as behavioral deficits and severe obesity caused by tumor or treatment-related hypothalamic lesions. METHODS: In our pilot cross-sectional study, we analyzed emotion recognition abilities and oxytocin concentrations in saliva and urine before and after single nasal administration of 24 IU oxytocin in 10 craniopharyngioma patients. Four craniopharyngioma presented with grade I lesions (limited to anterior hypothalamic areas) and 6 craniopharyngioma with grade II lesions (involving mammillary bodies and posterior hypothalamic areas). Emotional tasks were assessed before and after administration of oxytocin using the Geneva multimodal emotion portrayals corpus and the Multidimensional Mood Questionnaire. RESULTS: All patients presented with detectable levels of oxytocin before administration. Nasal administration of oxytocin was well-tolerated and resulted in increased oxytocin concentrations in saliva and urine. After oxytocin administration, craniopharyngioma patients with postsurgical lesions limited to the anterior hypothalamus area showed improvements in emotional identifications compared to craniopharyngioma patients with lesions of anterior and posterior hypothalamic areas. Focusing on correct assignments to positive and negative emotion categories, craniopharyngioma patients improved assignment to negative emotions. CONCLUSIONS: Oxytocin might have positive effects on emotion perception in craniopharyngioma patients with specific lesions of the anterior hypothalamic area. Further studies on larger cohorts are warranted.

RETRACTION Novel perspectives on hypothalamic-pituitary dysfunction as a risk factor for non-alcoholic fatty liver disease.

Minerva Endocrinologica is retracting the paper by Anika HOFFMANN, Hermann L. MÜLLER, "Novel perspectives on hypothalamic-pituitary dysfunction as a risk factor for nonalcoholic fatty liver disease" (Minerva Endocrinologica 2017 June;42(2):132-44).

Nuchal Skinfold Thickness: A Novel Parameter for Assessment of Body Composition in Childhood Craniopharyngioma.

CONTEXT: Hypothalamic obesity, cardiovascular disease (CVD), and relapse/progression have a major impact on prognosis in childhood-onset craniopharyngioma (CP). We analyzed nuchal skinfold thickness (NST) on magnetic resonance imaging performed for follow-up monitoring as a novel parameter for body composition (BC) and CVD in CP. OBJECTIVE: The objective of the study was to identify the association of NST with body mass index (BMI), waist to height ratio (WHtR), functional capacity, and blood pressure (BP) in CP and controls. DESIGN: This was a cross-sectional and longitudinal prospective study in CP patients. SETTING: The study was conducted at HIT-Endo, KRANIOPHARYNGEOM 2000/2007. PATIENTS: Participants included 94 CP patients and 75 controls. INTERVENTIONS: There were no interventions. MAIN OUTCOME MEASURES: Association of NST with BC and BP in 43 CP and 43 controls was measured. RESULTS: NST correlated with BMI SD score (SDS; r = 0.78; P < .001; n = 169) and WHtR (r = 0.85; P < .001; n = 86) in the total cohort and CP patients (NST-BMI SDS: r = 0.77, P < .001, n = 94); NST-WHtR: r = 0.835, P < .001, n=43) and controls (NST-BMI SDS: r = 0.792, P < .001, n = 75; NST-WHtR: r = 0.671, P < .001, n = 43). In CP, systolic BP correlated with NST (r = 0.575, P < .001), BMI SDS (r = 0.434, P = .004), and WHtR (r = 0.386, P = .011). Similar results were observed for diastolic BP in CP. In multivariate analyses, NST had a predictive value for hypertension in postpubertal CP and controls (odds ratio 6.98, 95% confidence interval [1.65, 29.5], P = .008). During a longitudinal follow-up, changes in NST correlated with changes in BMI SDS (P < .001) and WHtR (P = .01) but not with changes in BP and functional capacity. CONCLUSIONS: Because monitoring of magnetic resonance imaging and BC is essential for follow-up in CP, NST could serve as a novel and clinically relevant parameter for longitudinal assessment of BC and CVD risk in CP.

Oxytocin in survivors of childhood-onset craniopharyngioma.

Quality of survival of childhood-onset craniopharyngioma patients is frequently impaired by hypothalamic involvement or surgical lesions sequelae such as obesity and neuropsychological deficits. Oxytocin, a peptide hormone produced in the hypothalamus and secreted by posterior pituitary gland, plays a major role in regulation of behavior and body composition. In a cross-sectional study, oxytocin saliva concentrations were analyzed in 34 long-term craniopharyngioma survivors with and without hypothalamic involvement or treatment-related damage, recruited in the German Childhood Craniopharyngioma Registry, and in 73 healthy controls, attending the Craniopharyngioma Support Group Meeting 2014. Oxytocin was measured in saliva of craniopharyngioma patients and controls before and after standardized breakfast and associations with gender, body mass index, hypothalamic involvement, diabetes insipidus, and irradiation were analyzed. Patients with preoperative hypothalamic involvement showed similar oxytocin levels compared to patients without hypothalamic involvement and controls. However, patients with surgical hypothalamic lesions grade 1 (anterior hypothalamic area) presented with lower levels (p = 0.017) of oxytocin under fasting condition compared to patients with surgical lesion of posterior hypothalamic areas (grade 2) and patients without hypothalamic lesions (grade 0). Craniopharyngioma patients' changes in oxytocin levels before and after breakfast correlated (p = 0.02) with their body mass index. Craniopharyngioma patients continue to secrete oxytocin, especially when anterior hypothalamic areas are not involved or damaged, but oxytocin shows less variation due to nutrition. Oxytocin supplementation should be explored as a therapeutic option in craniopharyngioma patients with hypothalamic obesity and/or behavioral pathologies due to lesions of specific anterior hypothalamic areas. Clinical trial number: KRANIOPHARYNGEOM 2000/2007(NCT00258453; NCT01272622).

Fusiform dilatation of the internal carotid artery in childhood-onset craniopharyngioma: multicenter study on incidence and long-term outcome.

PURPOSE: Fusiform dilatations of the internal carotid artery (FDCA) represent a vascular complication following surgery for suprasellar tumors in children. Incidence rate and long-term prognosis of FDCA in terms of survival rates, vascular complications, and quality of survival are unknown for patients with childhood-onset craniopharyngioma. METHODS: Magnetic resonance imaging (MRI) results of 583 patients with childhood-onset craniopharyngioma, recruited from 2001 to 2015 in the German Childhood Craniopharyngioma Registry, were reviewed for FDCA. Risk factors for FDCA and long-term outcome after FDCA were analyzed. RESULTS: Fourteen of 583 patients (2.4 %) developed FDCA based on reference assessment of MRI. FDCA occurred ipsilateral to the surgical approach and was not related to degree of resection, hypothalamic involvement, or irradiation. The median time interval between first detection of FDCA and initial surgery was 0.79 years (range 0.01-5.56 years). During a median follow-up of 6.47 years (range 1.2-21.9 years) after first detection of FDCA, no bleeding or cerebrovascular events were observed in any patient. Irradiation was not related to FDCA. Survival rates and functional capacity were similar in patients with and without FDCA. Clinically the FDCA was unapparent in all cases and not treated. CONCLUSION: FDCA is a rare complication related to surgical treatment of childhood-onset craniopharyngioma without major impact on prognosis and clinical course of the disease. CLINICAL TRIAL NUMBER: KRANIOPHARYNGEOM 2000-NCT00258453; KRANIOPHARYNGEOM 2007-NCT01272622.

History before diagnosis in childhood craniopharyngioma: associations with initial presentation and long-term prognosis.

OBJECTIVE: Childhood craniopharyngiomas (CP) are often diagnosed after a long duration of history (DOH). Tumor size, hypothalamic involvement (HI), and obesity are associated with reduced overall survival (OS) and functional capacity (FC). The effect of DOH and specific symptoms in history on presentation at initial diagnosis and long-term prognosis are unknown. DESIGN: Retrospective analysis of patients' records and prospective longitudinal follow-up. METHODS: Histories of 411 CP patients recruited in HIT Endo, KRANIOPHARYNGEOM 2000 were retrospectively evaluated for DOH, symptoms, and characteristics. The effect of specific manifestations and DOH on clinical presentation and tumor characteristics at time of initial CP diagnosis and long-term outcome were analyzed. Main outcome measures were 10-year OS and progression-free survival (PFS), FC, and BMI during longitudinal follow-up. RESULTS: Median DOH was 6 months (range: 0.1-108 months) and correlated with age at diagnosis. Tumor size, HI, degree of resection, and BMI at diagnosis were not related to DOH. In multivariate analysis adjusted for age at diagnosis, only hydrocephalus was found to have a relevant influence on DOH. Visual and neurological deficits were associated with larger initial tumor size and impaired 10-year OS. Weight gain and growth failure were observed with longest DOH. PFS and FC were not related to any specific symptom. Endocrine deficits at diagnosis were associated with long DOH. CONCLUSIONS: CP is frequently diagnosed after long DOH, especially in older children. However, DOH was not associated with tumor size, HI, survival, or FC. Visual and neurological deficits necessitate rapid diagnostic workup.

Nonalcoholic fatty liver disease and fatigue in long-term survivors of childhood-onset craniopharyngioma.

OBJECTIVE: Hypothalamic obesity in childhood craniopharyngioma (CP) patients carries a high risk for development of metabolic syndrome. In metabolic syndrome, the development of nonalcoholic fatty liver disease (NAFLD) is known. The aim of this study is to detect the risk for NAFLD in childhood-onset CP. DESIGN: This cross-sectional study included liver computed tomography (CT); ultrasound analysis of abdomen; measurements of serum parameters, height, weight and body composition; and daily medication of patients with childhood-onset CP. METHODS: A total of 384 patients recruited in trials HIT Endo and KRANIOPHARYNGEOM 2000 were analyzed. Ninety-four survivors were included by fulfilling the criteria of proven hypothalamic involvement (HI), a minimum time interval of 5 years between diagnosis and study, and a minimum age of 18 years at the time of evaluation. A total of 19 patients agreed to participate. To quantify the degree of steatosis hepatis, analyses of liver density were performed once by non-contrasted CT of liver sections. RESULTS: NAFLD occurs in about 50% of CP patients with HI and is associated with elevated liver enzymes and homeostasis model assessment index. BMI is not an effective predictive factor but body fat mass measured by near-infrared spectroscopy (NIRS) is. Over half of CP patients (60%) with NAFLD are treated with stimulating agents, with risk of hepatic side effects. CONCLUSIONS: NAFLD is a major adverse late effect in childhood-onset CP. NIRS rather than BMI should be used to measure body composition and predict NAFLD. Stimulating agents for treatment of fatigue and daytime sleepiness in CP should be prescribed judiciously.

Survival, hypothalamic obesity, and neuropsychological/psychosocial status after childhood-onset craniopharyngioma: newly reported long-term outcomes.

BACKGROUND: Quality of life (QoL) and long-term prognosis are frequently, and often severely, impaired in craniopharyngioma (CP) patients. Knowledge of risk factors for long-term outcome is important for optimization of treatment. METHODS: Overall survival (OS) and progression-free survival (PFS), body mass index (BMI), neuropsychological status (EORTCQLQ-C30, MFI-20), and psychosocial status were analyzed in 261 patients with childhood-onset CP diagnosed before 2000 and longitudinally observed in HIT-Endo. RESULTS: Twenty-year OS was lower (P = .006) in CP with hypothalamic involvement (HI) (n = 132; 0.84 ± 0.04) when compared with CP without HI (n = 82; 0.95 ± 0.04). OS was not related to degree of resection, sex, age at diagnosis, or year of diagnosis (before/after 1990). PFS (n = 168; 0.58 ± 0.05) was lower in younger patients (<5 y at diagnosis) (n = 30; 0.39 ± 0.10) compared with patients aged 5-10 years (n = 66; 0.52 ± 0.08) and older than 10 years (n = 72; 0.77 ± 0.06) at diagnosis. PFS was not associated with HI, degree of resection, or sex. HI led to severe weight gain during the first 8-12 years of follow-up (median BMI increase: +4.59SD) compared with no HI (median increase: +1.20SD) (P = .00). During >12 years of follow-up, patients with HI presented no further increase in BMI. QoL in CP patients with HI was impaired by obesity, physical fatigue, reduced motivation, dyspnea, diarrhea, and nonoptimal psychosocial development. CONCLUSIONS: OS and QoL are impaired by HI in long-term survivors of CP. HI is associated with severe obesity, which plateaus after 12 years. OS/PFS are not related to degree of resection, but gross-total resection should be avoided in cases of HI to prevent further hypothalamic damage, which exacerbates sequelae.

Eating behavior, weight problems and eating disorders in 101 long-term survivors of childhood-onset craniopharyngioma.

BACKGROUND: As a result of hypothalamic involvement and/or treatment-related hypothalamic damage, up to 75% of childhood craniopharyngioma patients develop hypothalamic obesity. METHODS: Eating behavior was analyzed in 101 survivors of childhood craniopharyngioma, recruited from 1980 to 2001 in the HIT-Endo multicenter study, and in 85 body mass index (BMI)-matched healthy controls using the Inventory for Eating Behavior and Weight Problems (IEG) and the Inventory for Eating Disorders (ESI). RESULTS: Severely obese patients (BMI>8 SD; n=9) presented with pathological eating behavior, more weight problems, and eating disorders, as compared to obese (BMI 3-8 SD; n=44) and normal or overweight patients (BMI<3 SD; n=48). Craniopharyngioma patients with different degrees of obesity showed similar or even less pathological findings as compared to BMI-matched normal controls. CONCLUSION: Severe obesity is associated with pathological eating behavior/disorders in craniopharyngioma patients. As these disorders are not disease-specific, risk factors for hypothalamic obesity should be the focus of further craniopharyngioma research.

Diencephalic syndrome in childhood craniopharyngioma--results of German multicenter studies on 485 long-term survivors of childhood craniopharyngioma.

CONTEXT: Childhood craniopharyngiomas (CPs) are known to be associated with an increased risk of excessive weight gain and hypothalamic obesity. Atypical clinical manifestations include the development of a diencephalic syndrome (DS) with a failure to thrive or maintain weight at appropriate body mass index. CASES AND METHODS: In a retrospective study, we analyzed 21 of 485 childhood CP patients (4.3%) who presented with a low weight (< -2 body mass index SD) at the time of diagnosis. Eleven of the 21 patients were identified with a DS due to proven hypothalamic involvement. We show the clinical manifestations of DS and weight development before and after diagnosis in these 11 patients. The first significant differences between patients with low weight at diagnosis and normal-weight patients at diagnosis are observed at 5 years of age. Within the first 2 years after diagnosis, the weight of DS patients and normal-weight patients converge to a similar level. Tumor size does not play a role with respect to DS development. Finally, tumor characteristics of DS patients were compared with magnetic resonance imaging scans of obese CP patients at the time of diagnosis. CONCLUSIONS: DS is a rare clinical manifestation in childhood CP and should be considered as a discrete diagnosis in failure to thrive. DS at the time of diagnosis does not preclude weight gain after diagnosis of a CP with hypothalamic involvement.

A comparison of GFP-tagged clathrin light chains with fluorochromated light chains in vivo and in vitro.

Clathrin triskelia consist of three heavy chains and three light chains (LCs). Green fluorescent protein (GFP)-tagged LCs are widely utilized to follow the dynamics of clathrin in living cells, but whether they reflect faithfully the behavior of clathrin triskelia in cells has not been investigated yet thoroughly. As an alternative approach, we labeled purified LCs either with Alexa 488 or Cy3 dye and compared them with GFP-tagged LC variants. Cy3-labeled light chains (Cy3-LCs) were microinjected into HeLa cells either directly or in association with heavy chains. Within 1-2 min the Cy3-LC heavy chain complexes entered clathrin-coated structures, whereas uncomplexed Cy3-LC did not within 2 h. These findings show that no significant exchange of LCs occurs over the time-course of an endocytic cycle. To explore whether GFP-tagged LCs behave functionally like endogenous LCs, we characterized them biochemically. Unlike wild-type LCs, recombinant LCs with a GFP attached to either end did not efficiently inhibit clathrin assembly in vitro, whereas Cy3- and Alexa 488-labeled LC behaved similar to wild-type LCs in vitro and in vivo. Thus, fluorochromated LCs are a valuable tool for investigating the complex behavior of clathrin in living cells.

Serum-free differentiation of murine embryonic stem cells into alveolar type II epithelial cells.

Alveolar type II (AT2) epithelial cells have important functions including the production of surfactant and regeneration of lost alveolar type I epithelial cells. The ability of in vitro production of AT2 cells would offer new therapeutic options in treating pulmonary injuries and disorders including genetically based surfactant deficiencies. Aiming at the generation of AT2-like cells, the differentiation of murine embryonic stem cells (mESCs) toward mesendodermal progenitors (MEPs) was optimized using a "Brachyury-eGFP-knock in" mESC line. eGFP expression demonstrated generation of up to 65% MEPs at day 4 after formation of embryoid bodies (EBs) under serum-free conditions. Plated EBs were further differentiated into AT2-like cells for a total of 25 days in serum-free media resulting in the expression of endodermal marker genes (FoxA2, Sox17, TTR, TTF-1) and of markers for distal lung epithelium (surfactant proteins (SP-) A, B, C, and D, CCSP, aquaporin 5). Notably, expression of SP-C as the only known AT2 cell specific marker could be detected after serum-induction as well as under serum-free conditions. Cytoplasmic localization of SP-C was demonstrated by confocal microscopy. The presence of AT2-like cells was confirmed by electron microscopy providing evidence for polarized cells with apical microvilli and lamellar body-like structures. Our results demonstrate the differentiation of AT2-like cells from mESCs after serum-induction and under serum-free conditions. The established serum-free differentiation protocol will facilitate the identification of key differentiation factors leading to a more specific and effective generation of AT2-like cells from ESCs.