Recruiting refugees and migrants as potential hematopoietic stem cell donors to serve patients of comparable ethnicities with rare human leucocyte antigen patterns - The BluStar.NRW project in North Western Germany.

Currently, approximately 19 million people with a migration background live in Germany. The majority of those descend from regions where the population has a genetically different distribution of HLA antigens when compared to the HLA frequencies usually found in North Western Europe. In case of severe haematological disorders of these individuals, allogeneic stem cell transplantation may be the treatment of choice. However, finding appropriate histocompatible hematopoietic stem cell donors continues to be a major challenge. If no matching sibling donors are available, there are only few suitable donors with a similar genetic background available in international blood stem cell donor registries. The "BluStar.NRW" project aimed to recruit new blood and hematopoietic stem cell donors with a migration background and to noticeably increase the number of suitable donors for patients within this group. Since December 2017, a total number of 9100 blood and stem cell donors with a migration background were recruited and typed for this project. HLA typing for HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1 was performed by Next Generation Sequencing. We assessed the proportion of rare alleles according to HLA frequency tables, as defined by a frequency of <1:1000. The rare HLA allele frequencies according to HLA frequency tables of the BluStar.NRW cohort were compared with a matched control donor cohort: Rare HLA-A, -B, -C, -DRB1 and -DQB1 alleles occurred three times more frequent than in the control group, but rare HLA-DPB1 alleles occurred more frequently in the control cohort. This difference was highly significant for all HLA alleles (p < 0.0001 for HLA-A, -B, -C, -DRB1, -DPB1; p = 0.0002 for HLA-DQB1). In addition, the distribution of rare alleles differed between the two groups. To date, 29 work-ups were initiated, 12 PBSC, one BM and three DLI were collected so far out of the BluStar.NRW cohort. The apheresis probability is twofold higher (0.18% vs. 0.07%) compared to the control group which clearly shows a serious medical need. However, 13 work-ups were cancelled in the BluStar.NRW donor cohort which represents an almost twice as higher cancellation rate (45% vs. 25%). This single registry analysis with a large sample cohort clearly indicates that hematopoietic stem cell donors with a migration background represent an adequate donor pool to serve patients of comparable ethnicity.

Pushing the limits of in vivo diffusion MRI for the Human Connectome Project.

Perhaps more than any other "-omics" endeavor, the accuracy and level of detail obtained from mapping the major connection pathways in the living human brain with diffusion MRI depend on the capabilities of the imaging technology used. The current tools are remarkable; allowing the formation of an "image" of the water diffusion probability distribution in regions of complex crossing fibers at each of half a million voxels in the brain. Nonetheless our ability to map the connection pathways is limited by the image sensitivity and resolution, and also the contrast and resolution in encoding of the diffusion probability distribution. The goal of our Human Connectome Project (HCP) is to address these limiting factors by re-engineering the scanner from the ground up to optimize the high b-value, high angular resolution diffusion imaging needed for sensitive and accurate mapping of the brain's structural connections. Our efforts were directed based on the relative contributions of each scanner component. The gradient subsection was a major focus since gradient amplitude is central to determining the diffusion contrast, the amount of T2 signal loss, and the blurring of the water PDF over the course of the diffusion time. By implementing a novel 4-port drive geometry and optimizing size and linearity for the brain, we demonstrate a whole-body sized scanner with G(max) = 300 mT/m on each axis capable of the sustained duty cycle needed for diffusion imaging. The system is capable of slewing the gradient at a rate of 200 T/m/s as needed for the EPI image encoding. In order to enhance the efficiency of the diffusion sequence we implemented a FOV shifting approach to Simultaneous MultiSlice (SMS) EPI capable of unaliasing 3 slices excited simultaneously with a modest g-factor penalty allowing us to diffusion encode whole brain volumes with low TR and TE. Finally we combine the multi-slice approach with a compressive sampling reconstruction to sufficiently undersample q-space to achieve a DSI scan in less than 5 min. To augment this accelerated imaging approach we developed a 64-channel, tight-fitting brain array coil and show its performance benefit compared to a commercial 32-channel coil at all locations in the brain for these accelerated acquisitions. The technical challenges of developing the over-all system are discussed as well as results from SNR comparisons, ODF metrics and fiber tracking comparisons. The ultra-high gradients yielded substantial and immediate gains in the sensitivity through reduction of TE and improved signal detection and increased efficiency of the DSI or HARDI acquisition, accuracy and resolution of diffusion tractography, as defined by identification of known structure and fiber crossing.

Effect of ABO incompatibility on T-cell flow cytometry cross-match results prior to living donor kidney transplantation.

BACKGROUND: Due to its high sensitivity, the flow cytometry cross-match (FCXM) has been described as valuable tool for identifying an optimal donor. We here focused on the impact of ABO incompatibility on FCXM results. METHODS: We analyzed 29 ABO incompatible and 89 ABO compatible donor-recipient pairs (73 and 175 datasets, respectively) prior to living donor kidney transplantation. In all patients, lymphocytotoxic cross-matches for B and T cells were negative. RESULTS: Recipients with blood group O (A to O and B to O) displayed significantly (P < 0.05) higher T-FCXM results than those with blood group A and B (A to B, B to A and AB to A), respectively. Donor-specific T-FCXM responses (ΔMFI values) were significantly higher (P < 0.05) in ABO incompatible vs. compatible pairs (ABO incompatible recipients with blood group O: 32 ± 6; with blood group A: 19 ± 7; with blood group B: 7 ± 4; recipients with ABO compatibility: 3 ± 2, respectively, data represent mean ± SEM). Consistent with the T-FCXM results donor-specific isohemagglutinins (IgG titers) were significantly higher in recipients with blood group O vs. A, both prior to rituximab treatment and plasmapheresis/immune adsorption (P = 0.004) and immediately prior to transplantation, i.e., after rituximab and antibody-depleting therapies (P = 0.04). CONCLUSIONS: ABO incompatibility was associated with higher T-FCXM responses, especially in recipients with blood group O. This finding has major impact on the interpretation of flow cross-match results. Current cut-off values need to be reassessed in the ABO incompatible setting. © 2016 International Clinical Cytometry Society.

Posterior cervical haemangiopericytoma with intracranial and skull base extension. Diagnostic and therapeutic challenge of a rare hypervascular neoplasm.

Haemangiopericytomas are rare hypervascular tumors arising from pericytes. They may occur anywhere in the body, but posterior cervical location is rather uncommon. A case of posterior cervical haemangiopericytoma with posterior fossa and temporal bone extension is reported. Although the patient had undergone preoperative endovascular embolization and surgical resection on three separate occasions, control of the skull base extension was not successful. Following endovascular embolization combined with radiotherapy, the patient has been asymptomatic for 48 months. Angiographic features may help in differentiating haemangiopericytomas from other hypervascular lesions. Preoperative endovascular embolization is recommended due to the pronounced tendency for haemorrhage throughout biopsy and surgical procedures.

[Epidermoid cyst of the fourth ventricle: four case reports]. / Les kystes épidermoïdes du quatrième ventricule: à propos de quatre cas.

Patients with epidermoid cyst of the fourth ventricle usually present with headaches and/or disequilibrium. These cysts are characterized by a focal lesion that is nearly isodense to CSF at CT and nearly isointense to CSF on T1W and T2W MR images. MRI using FLAIR and diffusion weighted images as well as 3D CISS acquisitions is useful to better characterize the lesions and their relation with the vermis, foramen magnum and CP angle cisterns. DWI images are useful for postsurgical evaluation of residual tumor. Extension of the cyst into the CP angle cisterns usually precludes complete surgical resection.

[Value of MRI findings in Gayet-Wernicke encephalopathy]. / Apport de l'IRM dans l'exploration de l'encéphalopathie de Gayet-Wernicke.

Wernicke encephalopathy (Wernicke-Korsakoff encephalopathy) is related to thiamine deficiency. We report the MRI findings in four patients with visualization of bilateral and symmetrical hyperintense foci on T2W and FLAIR images involving the periaqueductal gray matter, the mamillary bodies and around the third ventricle. Diffusion weighted images obtained in two patients demonstrated mild hypersignal in the same areas. Contrast enhancement within the mamillary bodies was noted in one patient. Follow-up MRI obtained in three patients showed rapid regression of signal abnormalities without correlation with good clinical outcome.

[Siderosis of the brain and spinal cord. Report of two cases]. / Sidérose cérébrale et médullaire. A propos de deux observations.

Two cases of superficial siderosis of the brain and spinal cord with cochleovestibular and cerebellar symptoms are diagnosed on brain and spinal MRI scans. Low signal intensity lines are noted on the surface of the brainstem, cerebellum, spinal cord and within the interhemispheric and sylvian fissures. In one case, no brain or vascular malformation is identified; in the second case, two cavernous angiomas are noted on the MRI study. 3D CISS may visualize thickening of the cochleovestibular nerve.

[Brain MR imaging of chronic alcoholism]. / Imagerie par résonance magnétique (IRM) des complications encéphaliques de l'alcoolisme.

Brain complications from chronic alcoholism (Wernicke encephalopathy, central pontine myelinolysis, Marchiafava-Bignami disease, Korsakoff's syndrome, hepatic encephalopathy, cerebellar atrophy, hemorrhagic and ischemic brain lesions) may be diagnosed by MR imaging.

[Scientific basis for impression taking with silicone elastomers]. / Werkstoffkundliche Grundlagen der Abformens mit Silikon-Elastomeren

Starting from the chemical and physical processes which occur during the cross-linking of silicones, the author represents the material and processing properties of these impression compounds. He reports above all of the results of utilizationoriented studies which evidence the effects of different conditions of processing on the behaviour during impression taking and on the impression itself. Conclusions are drawn as to the appropriate use of silicone impression materials.

Enzyme-assisted semisynthesis of shortened B26-modified insulins.

The role of the invariant residue B26-tyrosine in determining the structural and biological properties of insulin has been extensively investigated by the use of semisynthetic des-(B27-B30)-insulins with modifications of position B26. Apart from the conventional trypsin-catalyzed peptide bond formation between the C-terminal amino acid ArgB22 of des-(B23-B30)-insulin and synthetic tetrapeptides we elaborated a new approach using des-(B26-B30)-insulin as substrate in alpha-chymotrypsin-mediated syntheses. Results obtained from bioassays and CD-spectroscopy underline the importance of position B26 to the association of the native molecule and to the modulation of structural and hormonal properties of shortened insulins.

[Investigations on the conditions of scanning on cell images by means of a microscope-TV-system (author's transl)]. / Untersuchungen der Abtastbedingungen bei Zellbildern mit einem Mikroskop-TV-System.

One common method for determining the spatial resolution of a TV-microscope scanning system is to measure a sharp edge, differentiate the digitized response and calculate the Modulation Transfer Function (MTF) using the Fourier transformation. The MTF provides a measure of the information content of a digitized picture as well as the resolving power of the whole system. Knowledge of this measure is prerequisite for understanding the capabilities and limitations of computer aided techniques for analyzing cellular structures as observed in the light microscope. The information content of a digitized image depends on the MTF of the whole measurement system. Results from this study show that the information content increases almost linearly up to a sampling rate of approximately 20 pixel/micrometer and saturates at 30--35 pixel/micrometer. The MTF and the signal/noise ratio is effected by averaged multiscans. The aliasing error is presented as a function of the sampling rate for the described system. The need for this resolving power of the cell image analysis system is demonstrated by an example in the haematology. Small granules with a diameter of 0.2 to 1.0 micrometer must be registered with a high sampling rate up to 30 pixel/micrometer. The analysis of the small granules is basic for automated detection and differentiation of many leukemic conditions.

Proteolyses of a fluorogenic insulin derivative and native insulin in reversed micelles monitored by fluorescence emission, reversed-phase high-performance liquid chromatography, and capillary zone electrophoresis.

The preparation and substrate properties of the fluorogenic insulin derivative N alpha A1-aminobenzoyl-N epilson B29-Tyr(NO2)- insulin are described. This semisynthetic protein intramolecularly quenched by long-range resonance energy transfer between the donor/acceptor pair 2-aminobenzoic acid and 3-nitrotyrosine was used to prove the activity of serine proteases toward substrates of high molecular weight after incorporation in reversed micelles. The proteases investigated, trypsin and alpha-chymotrypsin, were shown to be hydrolytically active in reversed micellar solvent systems stabilized by cetyltrimethylammonium bromide or sodium-1,2-bis(2-ethylhexylcarbonyl)-1- ethane sulfonate. Apart from fluorometric enzyme assays, methods for monitoring proteolyses in reversed micelles were elaborated using either reversed-phase high-performance liquid chromatography or capillary zone electrophoresis. Enzymatic digestions of native insulin by the specific protease trypsin and the less specific protease alpha-chymotrypsin were performed. In contrast to aqueous solution, high but still variable specificity of alpha-chymotrypsin which was dependent on the micellar environment was observed. The results promise further insight into the influence of interfacial environments on enzyme action and a novel approach to enzyme-mediated protein modifications by the use of microstructured solvent systems.

Semisynthetic des-(B27-B30)-insulins with modified B26-tyrosine.

Semisynthetic des-(B27-B30)-insulins containing modified B26-tyrosine residues were prepared to refine the understanding of the importance of position B26 with regard to biological and structural properties of the hormone. The following shortened insulin analogues were synthesized by trypsin-catalysed peptide-bond formation between the C-terminal amino acid ArgB22 of des-(B23-B30)-insulin and synthetic tetrapeptides as amino components: des-(B27-B30)-insulin, des-(B27-B30)-insulin-B26-methyl ester, -B26-carboxamide with varying C-terminal hydrophobicity of the B-chain, and [Tyr(NH2)B26]-, [Tyr(NO2)B26]-, [Tyr(I2)B26]-, [D-TyrB26]des-(B27-B30)-insulin-B26-carboxamide containing non-proteinogenic amino acids in position B26. Starting from insulin and an excess of synthetic Gly-Phe-Phe-Tyr-OMe as nucleophile, des-(B27-B30)-insulin-B26-methyl ester--the formal transpeptidation product at ArgB22--was formed in one step. Biological in vitro properties (binding to cultured human IM-9 lymphocytes, relative lipogenic potency in isolated rat adipocytes) of all semisynthetic analogues are reported, ranging from slightly decreased to two-fold receptor affinity and nearly three-fold biopotency relative to insulin. If the C-terminal tetrapeptide B27-B30 is removed, full relative insulin activity is still preserved, while the shortening results in the loss of ability to associate in solution. Only after carboxamidation or methyl esterification of TyrB26 the self-association typical of native insulin can be observed, and the CD-spectral effects in the near UV spectrum related to association and hexamerization of the native hormone are qualitatively reestablished. The results of this investigation underline the importance of position B26 to the modulation of hormonal properties and solution structure of the shortened insulins.

Structure and rotational dynamics of fluorescently labeled insulin in aqueous solution and at the amphiphile-water interface of reversed micelles.

Insulin is a proteohormone with amphipathic three-dimensional structure and the ligand of a receptor, which itself spans the plasma membrane of glucose-metabolizing cells. In this study, the possible impact of amphiphiles on structural and dynamic properties of the hormone was investigated in reversed micelles mimicking the amphipathic nature of biological membranes. To make insulin susceptible to fluorescence measurements, two derivatives labeled with 2-aminobenzoic acid (Abz), N epsilon B29-Abz-insulin and [AbzB1]insulin, were prepared. First, the Abz-labeled insulins were shown by CD spectroscopy to exhibit conformational properties and self-association as well as the T-->R transition similar to the native hormone. By means of time-resolved fluorescence measurements, not only metal-ion induced hexamerization was observable in aqueous solution: The T-->R allosteric transition of the hexamer was shown to be accompanied by a diminution of its hydrodynamic radius. Second, structure and rotational dynamics of the labeled insulins were investigated in reversed micelles. In sodium bis(2-ethylhexyl)sulfosuccinate (AOT) reversed micelles, the main-chain conformation is similar to that in aqueous solution according to CD spectroscopy in the far-UV, whereas the weak circular dichroism in the near-UV is indicative of reduced aromatic contacts as well as of the absence of quaternary structure, and the CD spectra show the same shape as found for proteins in an intermediate state of folding referred to as the "molten globule". Fluorescence anisotropy decay measurements of N epsilon B29-Abz-insulin in reversed micelles of AOT, cetyltrimethylammonium bromide, and alpha-L-1,2-dioctanoylphosphatidylcholine showed that the internal mobility of the solubilizate is reduced compared to that in aqueous solution and that the rotational mobility of the labeled insulin decreases with decreasing micellar size. With respect to the immobilization, insulin interacts in a stronger way with the anionic than with the cationic or zwitterionic amphiphile; an integration into the amphiphile monolayer, however, could be ruled out in all cases. In conclusion, the results reveal an evident influence of amphiphiles on the structure and rotational dynamics of insulin. Further investigations should be focused on this finding also with regard to the possible importance of lipid-insulin interactions in vivo.

Analysis of protein self-association at constant concentration by fluorescence-energy transfer.

Fluorescence-resonance-energy transfer from subunits labelled with a fluorescence donor group to subunits labelled with a fluorescence acceptor group can be used for quantitative analysis of protein self-association. The present approach evaluates fluorescence measurements on mixtures of equimolar solutions of donor-labelled and acceptor-labelled protein composed by systematic variation of the volume ratio. Its attractive feature is that it allows the determination of equilibrium constants at fixed total concentration. Problems encountered by most other methods, which require the equilibria to be followed to high dilution, are avoided. Conditions to be fulfilled are that a reactive site is available on the protein for specific introduction of the labels and that labelling neither affects the conformation nor interferes with the intermolecular interactions. It is desirable that the Forster distance of the donor/acceptor pair complies with its separation. While dimerisation constants can be determined exclusively by fluorescence measurements, the analysis of more complex cases of self-association depends on additional independent information. This communication reports on an application of the approach to the association/dissociation equilibrium between insulin monomers and dimers. Labelling of insulin at the epsilon-amino group of LysB29 does not disturb the conformation nor does it affect dimerisation. 2-Aminobenzoyl and 3-nitrotyrosyl residues served as the donor/acceptor pairs. Because they are less bulky than most other fluorescence labels and are of balanced polarity they do not alter the chemical nature of the protein. Their Forster distance of 29 A matches their 32-A separation in the insulin dimer. Energy transfer was measured as a function of the molar fractions of donor-insulin and acceptor-insulin at constant total concentration. Evaluation of this dependence resulted in a dimerisation constant, K12, of 0.72x10(5) M(-1). Its agreement with values obtained with other methods demonstrates that the present approach is a reliable alternative.

Determination of interspin distances between spin labels attached to insulin: comparison of electron paramagnetic resonance data with the X-ray structure.

A method was developed to determine the interspin distances of two or more nitroxide spin labels attached to specific sites in proteins. This method was applied to different conformations of spin-labeled insulins. The electron paramagnetic resonance (EPR) line broadening due to dipolar interaction is determined by fitting simulated EPR powder spectra to experimental data, measured at temperatures below 200 K to freeze the protein motion. The experimental spectra are composed of species with different relative nitroxide orientations and interspin distances because of the flexibility of the spin label side chain and the variety of conformational substates of proteins in frozen solution. Values for the average interspin distance and for the distance distribution width can be determined from the characteristics of the dipolar broadened line shape. The resulting interspin distances determined for crystallized insulins in the R6 and T6 structure agree nicely with structural data obtained by x-ray crystallography and by modeling of the spin-labeled samples. The EPR experiments reveal slight differences between crystal and frozen solution structures of the B-chain amino termini in the R6 and T6 states of hexameric insulins. The study of interspin distances between attached spin labels can be applied to obtain structural information on proteins under conditions where other methods like two-dimensional nuclear magnetic resonance spectroscopy or x-ray crystallography are not applicable.

Plasma levels of D-dimer and circulating endothelial adhesion molecules in veno-occlusive disease of the liver following allogeneic bone marrow transplantation.

Veno-occlusive disease (VOD) of the liver is a frequent and life-threatening complication of BMT. Recently, successful treatment by t-PA has been reported but has been compromised by fatal bleeding events. Therefore, t-PA application should be restricted to patients with severe VOD. However, moderate and severe forms of VOD are difficult to distinguish in early stages. We analyzed plasma levels of cross-linked fibrin degradation products (D-dimer) and soluble endothelial adhesion molecules such as sE-selectin, sVCAM-1 and sICAM-1 in 10 consecutive patients undergoing allogeneic BMT to evaluate their use in identifying severe forms of VOD. During the observation period, 4 episodes of VOD occurred, 2 of which were fatal due to early onset of multiorgan failure. Concentrations of D-dimer generally increased after transplantation. However, there was an additional significant increase in D-dimer levels during severe VOD. Thus, D-dimer levels above 1000 microg/l were only found in 2 cases with severe VOD and fatal outcome. When compared with bilirubin concentrations substantial increases of D-dimers appeared earlier during the course of severe VOD. In contrast, VOD episodes were not accompanied by significant increases in sE-selectin, sVCAM-1 and sICAM-1 levels. It is concluded that measurement of D-dimer concentrations may aid accuracy to the early diagnosis of severe VOD.