Stefanie Zimmermann

MYH9-related disease: a novel prognostic model to predict the clinical evolution of the disease based on genotype-phenotype correlations.

MYH9‐related disease (MYH9‐RD) is a rare autosomal‐dominant disorder caused by mutations in the gene for nonmuscle myosin heavy chain IIA (NMMHC‐IIA). MYH9‐RD is characterized by a considerable variability in clinical evolution: patients present at birth with only thrombocytopenia, but some of them subsequently develop sensorineural deafness, cataract, and/or nephropathy often leading to end‐stage renal disease (ESRD). We searched for genotype–phenotype correlations in the largest series of consecutive MYH9‐RD patients collected so far (255 cases from 121 families). Association of genotypes with noncongenital features was assessed by a generalized linear regression model. The analysis defined disease evolution associated to seven different MYH9 genotypes that are responsible for 85% of MYH9‐RD cases. Mutations hitting residue R702 demonstrated a complete penetrance for early‐onset ESRD and deafness. The p.D1424H substitution associated with high risk of developing all the noncongenital manifestations of disease. Mutations hitting a distinct hydrophobic seam in the NMMHC‐IIA head domain or substitutions at R1165 associated with high risk of deafness but low risk of nephropathy or cataract. Patients with p.E1841K, p.D1424N, and C‐terminal deletions had low risk of noncongenital defects. These findings are essential to patients clinical management and genetic counseling and are discussed in view of molecular pathogenesis of MYH9‐RD.

IL-2-driven regulation of NK cell receptors with regard to the distribution of CD16+ and CD16- subpopulations and in vivo influence after haploidentical NK cell infusion

To characterize natural killer (NK) cell subpopulations during activation, we analyzed the NK cell receptor repertoire and functionality of purified clinical scale CD56+CD3− donor NK cells during stimulation with 1000u2009U/mL interleukin (IL)-2 for up to 14 days. In a phase I/II trial, we investigated the efficacy and feasibility of nonidentical NK cell infusion in patients with neuroblastoma after haploidentical stem cell transplantation. After IL-2 stimulation, large differences in the distribution of CD16negative and CD16positive subpopulations were found in 12 donors. Thereby, surface expression for all natural cytotoxicity receptors (NCRs) and NKG2D increased. In addition, killer cell immunoglobulin-like receptor (KIR)+ NK cells were overgrown by KIR− proportion and the homing receptor CD62L was lost during stimulation. NK cell cytotoxicity against K562 and neuroblastoma cells increased and significantly higher cytokine secretion (eg, interferon-γ, tumor necrosis factor-β, macrophage inflammatory protein-1α, macrophage inflammatory protein-1β) was observed after IL-2 stimulation compared with freshly isolated NK cells. However, NK cells of donors showing an initially enhanced cytotoxicity combined with NCRbright and CD69 expression, seemed to be exhausted and did not favor a stimulation period over 9 days. When IL-2–stimulated NK cells were given to transplant recipients, they induced a decrease of peripheral blood NK, in particular of CD56bright-NK cells. Our data indicate that IL-2 stimulation increases the expression of activating receptors and emphasizes mechanisms beside KIR/human leukocyte antigen. Furthermore, the results suggest that the expansion period of purified NK cells has to be individualized to optimize NK cell immunotherapy.

Imbalance in distribution of functional autologous regulatory T cells in rheumatoid arthritis

Objectives: Regulatory T cells (Tregs) exert their anti-inflammatory activity predominantly by cell contact-dependent mechanisms. A study was undertaken to investigate the regulatory capacity of autologous peripheral blood Tregs in contact with synovial tissue cell cultures, and to evaluate their presence in peripheral blood, synovial tissue and synovial fluid of patients with rheumatoid arthritis (RA). Methods: 44 patients with RA and 5 with osteoarthritis were included in the study. The frequency of interferon (IFN)γ-secreting cells was quantified in synovial tissue cell cultures, CD3-depleted synovial tissue cell cultures, synovial tissue cultures co-cultured with autologous CD4+ and with CD4+CD25+ peripheral blood T cells by ELISPOT. Total CD3+, Th1 polarised and Tregs were quantified by real-time PCR for CD3ε, T-bet and FoxP3 mRNA, and by immunohistochemistry for FoxP3 protein. Results: RA synovial tissue cell cultures exhibited spontaneous expression of IFNγ which was abrogated by depletion of CD3+ T cells and specifically reduced by co-culture with autologous peripheral blood Treg. The presence of Treg in RA synovitis was indicated by FoxP3 mRNA expression and confirmed by immunohistochemistry. The amount of FoxP3 transcripts, however, was lower in the synovial membrane than in peripheral blood or synovial fluid. The T-bet/FoxP3 ratio correlated with both a higher grade of synovial tissue lymphocyte infiltration and higher disease activity. Conclusion: This study has shown, for the first time in human RA, the efficacy of autologous Tregs in reducing the inflammatory activity of synovial tissue cell cultures ex vivo, while in the synovium FoxP3+ Tregs of patients with RA are reduced compared with peripheral blood and synovial fluid. This local imbalance of Th1 and Treg may be responsible for repeated rheumatic flares and thus will be of interest as a target for future treatments.

Age-matched lymphocyte subpopulation reference values in childhood and adolescence: application of exponential regression analysis.

Background:u2002 Normal values of lymphocyte subpopulations for healthy children and adults have been published in defined age groups exclusively, which results in difficult data interpretation for patients close to the limit of contiguous age group ranges. In addition, normal values for a number of lymphocyte subpopulations have not been established to date.

Clinical Grade Purification and Expansion of NK Cell Products for an Optimized Manufacturing Protocol

Allogeneic natural killer (NK) cells are used for adoptive immunotherapy after stem cell transplantation. In order to overcome technical limitations in NK cell purification and activation, the following study investigates the impact of different variables on NK cell recovery, cytotoxicity, and T-cell depletion during good manufacturing practice (GMP)-grade NK cell selection. Forty NK cell products were derived from 54 unstimulated donor leukaphereses using immunomagnetic CD3 T-cell depletion, followed by a CD56 cell enrichment step. For T-cell depletion, either the depletion 2.1 program in single or double procedure (D2.11depl, nu2009=u200918; D2.12depl, nu2009=u200913) or the faster depletion 3.1 (D3.1, nu2009=u20099) was used on the CliniMACS instrument. Seventeen purified NK cell products were activated in vitro by IL-2 for 12u2009days. The whole process resulted in a median number of 7.59u2009×u2009108 CD56+CD3− cells with both purity and viability of 94%, respectively. The T-cell depletion was significantly better using D2.11depl/2depl compared to D3.1 (log 4.6/log 4.9 vs. log 3.7; pu2009<u20090.01) and double procedure in two stages led always to residual T cells below 0.1%. In contrast D3.1 was superior to D2.11depl/2depl with regard to recovery of CD56+CD3− NK cells (68% vs. 41%/38%). Concomitant monocytes and especially IL-2 activation led to increased NK cell activity against malignant target cells compared to unstimulated NK cells, which correlated with both up-regulation of natural cytotoxicity receptors and intracellular signaling. Overall, wide variations in the NK cell expansion rate and the distribution of NK cell subpopulations were found. In conclusion, our results indicate that GMP-grade purification of NK cells might be improved by a sequential processing of T-cell depletion program D2.1 and D3.1. In addition NK cell expansion protocols need to be further optimized.

Childhood cancer predisposition syndromes—A concise review and recommendations by the Cancer Predisposition Working Group of the Society for Pediatric Oncology and Hematology

Heritable predisposition is an important cause of cancer in children and adolescents. Although a large number of cancer predisposition genes and their associated syndromes and malignancies have already been described, it appears likely that there are more pediatric cancer patients in whom heritable cancer predisposition syndromes have yet to be recognized. In a consensus meeting in the beginning of 2016, we convened experts in Human Genetics and Pediatric Hematology/Oncology to review the available data, to categorize the large amount of information, and to develop recommendations regarding when a cancer predisposition syndrome should be suspected in a young oncology patient. This review summarizes the current knowledge of cancer predisposition syndromes in pediatric oncology and provides essential information on clinical situations in which a childhood cancer predisposition syndrome should be suspected.

Cytochrom b-Sequenz-Vergleiche zwischen Rothirsch(Cervus elaphus hippelaphus), Damhirsch(Dama dama dama) und Reh(Capreolus capreolus capreolus)

ZusammenfassungVon Rothirsch(Cervus elaphus hippelaphus), Damhirsch(Dama dama dama) und Reh(Capreolus capreolus capreolus) wurden mit Hilfe der Kapillarelektrophorese die Nukleotid-Sequenzen des Cytochrom b-Gens zum größten Teil bestimmt. Um, ausgehend von diesen Sequenzen, Aussagen über die verwandtschaftlichen Beziehungen dieser drei Tierarten zueinander machen zu können, wurden die Basen- bzw. die Aminosäurensubstitutionsraten ermittelt. Auch das Verhältnis von Transitionen zu Transversionen wurde berechnet. Die Homologien der bestimmten Nukleotid- und Aminosäuren-Sequenzen zwischen Reh, Rot- und Damhirsch sprechen dafür, daß die drei Spezies etwa gleichzeitig entstanden sind.SummaryWith the aid of capillary electrophoresis the nucleotide sequence of the cytochrome b gene for red deer(Cervus elaphus hippelaphus), fallow deer(Dama dama dama) and roe deer(Capreolus capreolus capreolus) were in large part determined. In order to make a statement on the degree of relatedness of these three species based on these sequences, the substitution rates of the bases respectively the amino acids were determined. The ratio of transitions to transversions was also calculated. The homologies of the determined nucleotide and amino acid sequences among roe deer, red deer, and fallow deer indicate that these species evolved at about the same time.RésuméLa plupart des séquences des nucléotides du gène du Cytochrome b ont été déterminées chez le Cerf(Cervus elaphus hippelaphus), le Daim(Dama dama dama) et le Chevreuil(Capreolus capreolus) au moyen de lélectrophorèse par capillaire. Afin de pouvoir définir, au départ de ces séquences, des parentés entre ces trois espèces, les taux de substitution en bases et en acides aminés ont été établis. De même on a calculé le rapport de transitions et de transversions. Les analogies des séquences de nucléotides et des acides aminés sont telles que lon peut considérer que les trois espèces sont apparues un peu près à la même époque.

Cytochrom b-Sequenz-Vergleiche zwischen Wisent(Bison bison bonasus) und Hausrind(Bos primigenius f. taurus)

ZusammenfassungVom Wisent (bison bison bonasus) wurde die Nukleotid-Sequenz des Cytochrom b-Gens bestimmt. Die erstellte Basenfolge und die daraus ermittelte Aminosäuren-Sequenz wurde mit den entsprechenden Sequenzen des Rindes aus der EMBL-Datenbank verglichen. Dabei wurden die Basen- bzw. die Aminosäurensubstitutionsraten sowie das Verhältnis von Transitionen zu Transversionen berechnet. Die Homologien zwischen den verglichenen Nukleotid- und Aminosäuren-Sequenzen von Wisent und Rind entsprachen den aufgrund der verwandtschaftlichen Beziehungen erwarteten Werten.SummaryThe nucleotide sequence of the cytochrome b gene for bison(Bison bison bonasus) was determined. The base sequence and the resulting amino acid sequence were compared to the corresponding sequences of domestic cow as taken from the EMBL data bank. The substitution rates for the bases and amino acids respectively as well as the relationship of transitions to transversions were calculated. Due to the relationship between these species, the homologies between the compared nucleotide and amino acid sequences of bison and cow correspond to the expected values.RésuméLa séquence des nucléotides du gène du Cytochrome b du Bison(Bison bison bonasus) a été déterminée. La séquence des bases et celle, qui en résulte, des acides aminés ont été comparées avec les séquences correspondantes du Bœf, telles quon les trouve dans la banque de données EMBL. Par la même occasion, les taux de substitution des bases et des acides aminés ainsi que le rapport des transitions et des transversions ont été calculés. Les analogies observées entre les séquences de nucléotides et dacides aminés du Bison et du Bœf correspondaient aux valeurs attendues sur base de leur parenté.