Favorable outcome in children and adolescents with a high proportion of advanced phase disease using single/multiple autologous or matched/mismatched allogeneic stem cell transplantations.

We determined the indication, outcome, and risk factors of single and multiple hematopoietic stem cell transplantation(s) (HSCT) in children and adolescents mostly with advanced disease. Forty-one out of 483 patients (8.5 %; median age 9 years) diagnosed at the University of Leipzig with hematological and oncological diseases required HSCT from 1999 to 2011. Patients had overall survival (OS) of 63 ± 10 and 63 ± 16 %, event-free survival (EFS) of 57 ± 10 and 42 ± 16 %, relapse incidence (RI) of 39 ± 10 and 44 ± 18 % and nonrelapse mortality (NRM) of 4 ± 4 and 13 ± 9 % at 10 years after one or more allogeneic and autologous HSCT, respectively. One patient in CR1 and five with advanced disease received two HSCT. Four of the six patients maintained/achieved CR for a median of 13 months. Three died of progression and one of NRM. Two patients had a third HSCT and one survived in CR +231 days after HSCT. Risk factors for OS and EFS were disease stage at HSCT and EBMT risk score. Center (pediatric or JACIE accredited pediatric/adult) was not a determinant for survival. Pediatric single and multiple HSCT are important curative approaches for high-risk malignant diseases with low NRM. Efforts to reduce high RI remain the major aim.

Early related or unrelated haematopoietic cell transplantation results in higher overall survival and leukaemia-free survival compared with conventional chemotherapy in high-risk acute myeloid leukaemia patients in first complete remission.

Between 1996 and 2004, a total of 708 patients were enrolled in the acute myeloid leukaemia (AML) '96 and '02 studies of the East German Study Group (OSHO). Of these, 138 patients (19.5%) had unfavourable cytogenetics defined as complex karyotype, del (5q)/-5, del (7q)/-7, abn (3q26) and abn (11q23). In all, 77 (56%) achieved complete remission 1 (CR1) after induction chemotherapy and were eligible for haematopoietic cell transplantation (HCT). HCT was performed after a median of two cycles of consolidation chemotherapy (CT) in the AML '96 and one cycle in the AML '02 study (P=0.03). After a median follow-up of 19 months, overall survival (OS) at two years was significantly better in the donor group (52+/-9%) versus the no-donor group (24+/-8%; P=0.005). Differences in outcomes were mainly because of a lower relapse incidence in patients after HCT (39+/-11%) compared with a higher relapse incidence in patients undergoing CT (77+/-10%; P=0.0005). Treatment-related mortality was low and not statistically significantly different between the two treatment groups (15+/-7 and 5+/-5% for HCT and chemotherapy, respectively; P=0.49).We conclude that early HCT from related or unrelated donors led to significantly better OS and leukaemia-free survival compared with chemotherapy in patients with unfavourable karyotype.

Presenilin 1 mutations increase amyloid precursor protein production and proteolysis in Xenopus laevis oocytes.

Recent findings suggest that Presenilin 1 (PS1) mutations play a major role in the development of Alzheimer's disease (AD) by increasing the production of the beta amyloid peptide (A beta). The exact mechanism whereby mutations in PS1 lead to this effect is not clear. To examine the question of how PS1 might be involved in amyloid precursor protein (APP) processing, we constructed a chimera of human APP695 fused at the C-terminal to enhanced green fluorescent protein (EGFP). This construct was injected into Xenopus laevis oocytes in the presence of wild type PS1 or one of three PS1 mutations associated with AD. The cellular location of the APP695-EGFP construct was examined by fluorescent confocal microscopy. In addition, membrane fractions of oocytes expressing APP695-EGFP in the presence or absence of wild type or mutant forms of PS1 were evaluated by Western blot analysis. The results show that APP695-EGFP is primarily expressed on the cell surface and undergoes limited cleavage. Specifically, APP695 was cleaved in the A beta domain to generate three distinct C-terminal fragments that correspond in length to stubs expected after cleavage with alpha-, beta- and gamma-secretase, respectively. The presence of wild type PS1 not only increased the production of proteolytic C-terminal fragments of APP, but the production of APP itself. These findings were even more pronounced in the presence of all three PS1 mutations, suggesting that PS1 mutations may lead to over-expression of APP not just increased gamma-secretase activity.

Does long-term continuous administration of pentoxifylline affect platelet function in the critically ill patient?

OBJECTIVE: The methylxanthine derivative pentoxifylline (PTX) is one of those promising substances which are under current investigation to modify or limit inflammatory response. Anti-aggregation activity has also been described that may contribute to the beneficial effects of this substance. Long-term effects on platelet function have not been elucidated yet. DESIGN: Prospective, randomized study. SETTING: Clinical investigation on a surgical intensive care unit of a university hospital. PATIENTS: 26 trauma patients and 26 patients suffering from sepsis secondary to major operations were consecutively studied. INTERVENTIONS: The patients prospectively received either 1.5 mg/kg per h pentoxifylline continuously for 5 days (after a loading dose of 600 mg) (trauma-PTX, n = 13; sepsis-PTX, n = 13) or saline solution as placebo (trauma-control; n = 13; sepsis-control, n = 13). MEASUREMENTS: On the day of admission (trauma patients) or day of the diagnosis of sepsis and at 12:00 p.m. during the next 5 days, platelet aggregation induced by adenosine diphosphate (ADP 2.0 mumol/l), collagen (4 microliters/ml), and epinephrine (25 mumol/l) was determined by a turbidimetric method from arterial blood samples. Standard coagulation screen was also monitored. MAIN RESULTS: In untreated trauma and sepsis patients, maximum platelet aggregation induced by all three agonists decreased during the first few days after inclusion in the study [trauma: ADP - 17.1 +/- 8.0 rel% (% change from baseline); sepsis: ADP -26.1 +/- 5.6 rel%]. In due course, maximum platelet aggregation recovered, reaching the baseline value or even exceeding it (trauma patients). In the PTX-treated patients, platelet aggregation was significantly less impaired (sepsis group: ADP -4.4 +/- 3.3 rel%) or even increased beyond baseline values in the first few days of the study (trauma group: ADP 16.1 +/- 8.0 rel%). Fibrinogen plasma levels were lower in the non-treated control groups (p < 0.05) than in the PTX groups. CONCLUSIONS: Continuous infusion of PTX for 5 days did not impair platelet function in critically ill patients. In both trauma and sepsis patients, the usual deterioration in platelet function was even attenuated, which may be due to the effects of PTX on cytokine release (e.g., reduction in tumor necrosis factor and interleukin-1), improvement in microcirculation, or additional fibrinolytic effects.

Influence of long-term continuous intravenous administration of pentoxifylline on endothelial-related coagulation in critically ill patients.

OBJECTIVE: To determine the influence of pentoxifylline on endothelial-associated coagulation. DESIGN: Prospective, randomized, placebo-controlled study. SETTING: A surgical intensive care unit of a university hospital. PATIENTS: Consecutive patients (n = 60) with trauma or sepsis secondary to major (nontrauma) surgery. All patients received controlled mechanical ventilation. INTERVENTIONS: According to a randomized sequence, the patients either received pentoxifylline continuously over 5 days (1.5 mg/kg/hr iv) (trauma-pentoxifylline group [n = 15], sepsis-pentoxifylline group [n=15] or saline solution as placebo (trauma-control group [n = 15], sepsis-control group [n = 15]. MEASUREMENTS AND MAIN RESULTS: In addition to the standard coagulation screen, thrombomodulin, protein C, (free) protein S, and thrombin-antithrombin plasma concentrations were measured by enzyme-linked immunosorbent assays. Intensive care therapy, hemodynamics, and changes of Acute Physiology and Chronic Health Evaluation II score were comparable for pentoxifylline-treated and nontreated patients. An average dose of 2.5 g/day of pentoxifylline (range 2.2 to 2.9) was infused into the pentoxifylline-treated patients. At baseline, plasma thrombomodulin concentrations were higher in the septic patients than in the trauma patients. Thrombomodulin plasma concentrations increased significantly more in the control patients (trauma: from 38.9 +/- 10.5 to 59.9 +/- 10.1 ng/mL; sepsis: from 49.7 +/- 12.1 to 72.3 +/- 11.2 ng/mL) than in the pentoxifylline-treated patients (trauma: from 37.9 +/- 11.9 to 50.2 +/- 9.2 ng/mL; sepsis from 51.9 +/- 10.1 to 63.3 +/- 10.2). Starting from similar baseline values, protein C concentration increased significantly more in the sepsis-pentoxifylline patients (from 52.0 +/- 11.1% to 69.1 +/- 11.1%) than in the untreated septic patients (from 50.1 +/- 10.0% to 52.5 +/- 9.5%). There were no significant differences between the pentoxifylline-treated and nontreated groups for protein S and thrombin-antithrombin concentrations. Standard coagulation parameters (fibrinogen, activated partial thromboplastin time, antithrombin III) did not differ between these groups either. CONCLUSIONS: Continuous intravenous administration of pentoxifylline for 5 days beneficially influenced the thrombomodulin/protein C/protein S system in both the trauma and septic patients.

Decreased protein synthetic activity of the hypothalamic tuberomamillary nucleus in Alzheimer's disease as suggested by smaller Golgi apparatus.

The nucleus tuberalis lateralis (NTL) and tuberomamillary nucleus (TM), which are located close together in the tuberal region of the human hypothalamus, are differentially affected by Alzheimer's disease (AD), In the AD, the NTL shows only early cytoskeletal alterations, i.e. pre-tangle stages, while the TM is characterised by advanced Alzheimer's changes, e.g. neurofibrillary degeneration, senile plaques and amyloid deposition. Earlier we showed that the early cytoskeletal alterations in the NTL are not accompanied by changes in protein synthetic activity. The present study was carried out in order to measure the protein synthetic activity of the neighbouring area, the TM, which is severely affected by advanced Alzheimer changes. A polyclonal antibody against MG-160, a conserved membrane sialoglycoprotein of the Golgi apparatus, was used to stain this organelle and using an image analysis system, the size of the Golgi apparatus was measured as an index for synthetic and secretory activity in 15 Alzheimer patients and 21 controls. A significant decrease in the size of the Golgi apparatus was found in the TM neurons in AD, although the cell profile area remained unchanged. These data suggest that the protein synthetic and secretory activity of TM neurons is indeed decreased in AD.

Synthetic lipid-anchored receptors based on the binding site of a monoclonal antibody.

Highly specific ligand receptor interactions generally characterize molecular recognition at cell surfaces and other biological systems. In this study we simulate a membrane receptor by fusing a monoclonal antibody fragment to a phospholipid. A sulfhydryl group in the hinge region of a monoclonal antibody fragment, was covalently linked to derivatives of phosphatidylethanolamines and phosphatidylserine via three different hydrophilic spacer arms. We investigated and characterized these lipid-anchored Fab-fragments which we have named 'Fab-lipids' in liposomal and monolayer systems. Methods for the monomolecular assembling of such films at the air/water interface and techniques used for their manipulation are outlined. We describe two possibilities for building a monomolecular receptor layer, consisting of two-dimensional pattern of oriented Fab-fragments with their artificial hydrophobic anchor embedded in a lipid matrix. In the first method a monomolecular film at the air/water interface was allowed to form from a vesicular suspension and driven into a phase separation, resulting in protein rich domains embedded in a protein depleted phase. This film was transferred onto a solid support in such a way that the established pattern was preserved. Alternatively, a recognition pattern was formed by directly cross-linking the Fab-fragments to preformed planar membranes composed of the reactive spacer-lipids and an inert matrix lipid. Specificity as well as contrast of the binding activity of the receptor layers were qualified using micro-fluorimetry.

A miniaturized micro-fluorescence film balance for protein-containing lipid monolayers spread from a vesicle suspension.

In order to study protein-lipid monolayers at the air/water interface a miniaturized micro-fluorescence film-balance apparatus has been developed and combined with a modified technique of spreading and separating a monolayer from a vesicle suspension. The spreading method provides non-denaturing conditions for protein-lipids. When applied to protein-lipid vesicles, monolayers with incorporated proteins are obtained, and their thermodynamic parameters may be controlled in a well-defined way by film balance techniques. In the apparatus introduced, a movable microscope allows the observation of micro-fluorescence during the tracking of individual domains at the air/water interface of a fixed Langmuir trough. After the control of parameters such as subphase temperature, surface pressure and lateral molecule distribution, a monolayer may be transferred and immobilized on a planar solid support, making it accessible to optical surface-sensitive measuring methods as well as to electron microscopy and scanning probe techniques.

Immobilized proteins in buffer imaged at molecular resolution by atomic force microscopy.

Samples of supported planar lipid-protein membranes and actin filaments on mica were imaged by atomic force microscopy (AFM). The samples were fully submerged in buffer at room temperature during imaging. Individual proteins bound to the reconstituted membrane were distinguishable; some structural details could be resolved. Also, surface-induced, self-assembling of actin filaments on mica could be observed. Monomeric subunits were imaged on individual actin filaments. The filaments could be manipulated on or removed from the surface by the tip of the AFM. The process of the decoupling of the filamentous network from the surface upon changing the ionic conditions was imaged in real time.

Two-dimensional recognition pattern of lipid-anchored Fab' fragments.

A two-dimensional pattern of oriented antibody fragments was formed at the air-water interface and transferred onto a solid support. The Fab'-fragments of a monoclonal antibody against the hapten dinitrophenyl (DNP) were covalently linked via a hydrophilic spacer to phospholipid vesicles. A monomolecular lipid-protein layer at equilibrium with these vesicles was allowed to form at the air-water interface. The monolayer was separated from the vesicle phase and transferred to a Langmuir-Blodgett trough. By cooling and compressing, the previously homogeneous lipid-protein film was driven into a two-dimensional phase separation resulting in protein-rich domains and a second phase consisting mainly of lipid. This film was transferred onto a solid support in a way that preserved the protein-lipid pattern. The specificity as well as the contrast in the binding activity of the two different separated phases were then quantified using microfluorometry. DNP conjugated to fluorescein-labeled bovine serum albumin (BSA) showed virtually no binding to the lipid regions, but gave a ratio of bound DNP-BSA to Fab'-lipid of greater than 50% in the protein-rich domains proving that the Fab'-moiety retained its biological activity. This demonstrates that the technique presented here is well suited to modify different solid surfaces with a pattern of a given biological function. The optional control of lateral packing and orientation of the components in the monolayer makes it a general tool for the reconstitution of supported lipid-protein membranes and might also open new ways for the two-dimensional crystallization of proteins at membranes.