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Non-targeted effects (NTE) of ionizing radiation may initiate myeloid neoplasms (MN). Here, protein mediators (I) in irradiated human mesenchymal stromal cells (MSC) as the NTE source, (II) in MSC conditioned supernatant and (III) in human bone marrow CD34+ cells undergoing genotoxic NTE were investigated. Healthy sublethal irradiated MSC showed significantly increased levels of reactive oxygen species. These cells responded by increasing intracellular abundance of proteins involved in proteasomal degradation, protein translation, cytoskeleton dynamics, nucleocytoplasmic shuttling, and those with antioxidant activity. Among the increased proteins were THY1 and GNA11/14, which are signaling proteins with hitherto unknown functions in the radiation response and NTE. In the corresponding MSC conditioned medium, the three chaperones GRP78, CALR, and PDIA3 were increased. Together with GPI, these were the only four altered proteins, which were associated with the observed genotoxic NTE. Healthy CD34+ cells cultured in MSC conditioned medium suffered from more than a six-fold increase in γH2AX focal staining, indicative for DNA double-strand breaks, as well as numerical and structural chromosomal aberrations within three days. At this stage, five proteins were altered, among them IQGAP1, HMGB1, and PA2G4, which are involved in malign development. In summary, our data provide novel insights into three sequential steps of genotoxic signaling from irradiated MSC to CD34+ cells, implicating that induced NTE might initiate the development of MN.

Radiation-induced bystander effects (RIBE) in human hematopoietic stem and progenitor cells may initiate myeloid neoplasms (MN). Here, the occurrence of RIBE caused by genotoxic signaling from irradiated human mesenchymal stromal cells (MSC) on human bone marrow CD34+ cells was investigated. For this purpose, healthy MSC were irradiated in order to generate conditioned medium containing potential genotoxic signaling factors. Afterwards, healthy CD34+ cells from the same donors were grown in conditioned medium and RIBE were analyzed. Increased DNA damage and chromosomal instability were detected in CD34+ cells grown in MSC conditioned medium when compared to CD34+ cells grown in control medium. Furthermore, reactive oxygen species and distinct proteome alterations, e.g., heat-shock protein GRP78, that might be secreted into the extracellular medium, were identified as potential RIBE mediators. In summary, our data provide evidence that irradiated MSC induce genetic instability in human CD34+ cells potentially resulting in the initiation of MN. Furthermore, the identification of key bystander signals, such as GRP78, may lay the framework for the development of next-generation anti-leukemic drugs.

Background Availability of patient-specific image data, gathered from preoperatively conducted studies, like computed tomography scans and magnetic resonance imaging studies, during a surgical procedure is a key factor for surgical success and patient safety. Several alternative input methods, including recognition of hand gestures, have been proposed for surgeons to interact with medical image viewers during an operation. Previous studies pointed out the need for usability evaluation of these systems. Objectives We describe the accuracy and usability of a novel software system, which integrates gesture recognition via machine learning into an established image viewer. Methods This pilot study is a prospective, observational trial, which asked surgeons to interact with software to perform two standardized tasks in a sterile environment, modeled closely to a real-life situation in an operating room. To assess usability, the validated “System Usability Scale” (SUS) was used. On a technical level, we also evaluated the accuracy of the underlying neural network. Results The neural network reached 98.94% accuracy while predicting the gestures during validation. Eight surgeons with an average of 6.5 years of experience participated in the usability study. The system was rated on average with 80.25 points on the SUS. Conclusion The system showed good overall usability; however, additional areas of potential improvement were identified and further usability studies are needed. Because the system uses standard PC hardware, it made for easy integration into the operating room.

To evaluate the usefulness of a novel MRI sequence strategy in the assessment of the periprosthetic anatomical structures after primary total knee arthroplasty. Two MR sequences were retrospectively compared for the imaging of 15 patients with implanted cruciate-retaining/fixed-bearing TKAs (DePuy, PFC Sigma): a slice encoding sequence for metal artifact correction (SEMAC) and a standard sequence. Images were acquired on a 1.5-T system. The degree of artifact reduction was assessed using several qualitative (Likert-type scale) (artifact size, distorsion, blur, image quality, periprosthetic bone, posterior cruciate ligament, lateral collateral ligament, medial collateral ligament, patella tendon, popliteal vessels) and quantitative (artifact volume, Insall–Salvati index, length of patella/tendon, prosthesis dimensions) parameters by blinded reads performed by four investigators. The SEMAC sequences were statistically compared with the standard sequence using Wilcoxon test. Additionally, the intraclass correlation coefficient (ICC) for interobserver agreement was calculated. Higher levels of blurring were found with SEMAC compared to standard sequences (p

Recent data suggest diminished post-operative quadriceps muscle strength after tourniquet application during total knee arthroplasty (TKA). The metabolic effects of the commonly utilized intraoperative tourniquet with consecutive ischaemia on the skeletal muscle cells were unknown. Ubiquitin proteasome system represents one of the main pathways involved in muscle protein breakdown contributing to muscle atrophy. Therefore, the purpose of the present study was to quantify the acute effects of the tourniquet application during TKA on the (1) concentrations of free/conjugated ubiquitin, (2) total ubiquitin-protein ligase activity, (3) proteasome-dependent and (4) proteasome-independent peptidase activities in the cells of vastus medialis. The randomized, controlled, monocentric trial included 34 patients scheduled to undergo primary TKA. Each patient was randomly assigned to the tourniquet (n = 17) or non-tourniquet group (n = 17) after receiving a written consent. Muscle biopsies of (5 × 5 × 5 mm) 125 mm3 were obtained from vastus medialis immediately after performing the surgical approach and exactly 60 min later. After preparation of the muscle tissue specimen, the concentrations of the free/conjugated ubiquitin (Ub) were measured by western blot analyses. The ubiquitination was determined as biotinylated Ub incorporated into the sum of the cytosolic proteins and expressed as total ubiquitin-protein ligase activity (tUbPL). The quantification of the proteasome-dependent and proteasome-independent peptidase activities was performed with peptidase assays. Tourniquet application did not influence the concentration of the free/conjugated Ub. There were no differences in tUbPL activities between groups and time points. Tourniquet-induced ischaemia resulted in statistically significant higher proteasome-dependent (caspase-like p = 0.0034; chymotryptic-like p = 0.0013; tryptic-like p = 0.0036) and proteasome-independent (caspase-like p = 0.03; chymotryptic-like p = 0.0001; tryptic-like p = 0.0062) peptidase activities. Tourniquet application did not affect the free/conjugated Ub as well as tUbPL significantly, emphasizing the sophisticated regulation of ubiquitination. The proteasome-dependent peptidase activities were significantly upregulated during tourniquet application, suggesting an increase in protein degradation, which in turn might explain the skeletal muscle atrophy occurring after TKA. These findings add further knowledge and should raise the awareness of surgeons about the effects of tourniquet-induced ischaemia at the molecular level. Additional high-quality research may be warranted to examine the short- and long-term clinical significance of the present data. I.

SummaryMyelodysplastic syndromes (MDSs) are a heterogeneous group of myeloid neoplasms with defects in hematopoietic stem and progenitor cells (HSPCs) and possibly the HSPC niche. Here, we show that patient-derived mesenchymal stromal cells (MDS MSCs) display a disturbed differentiation program and are essential for the propagation of MDS-initiating Lin−CD34+CD38− stem cells in orthotopic xenografts. Overproduction of niche factors such as CDH2 (N-Cadherin), IGFBP2, VEGFA, and LIF is associated with the ability of MDS MSCs to enhance MDS expansion. These factors represent putative therapeutic targets in order to disrupt critical hematopoietic-stromal interactions in MDS. Finally, healthy MSCs adopt MDS MSC-like molecular features when exposed to hematopoietic MDS cells, indicative of an instructive remodeling of the microenvironment. Therefore, this patient-derived xenograft model provides functional and molecular evidence that MDS is a complex disease that involves both the hematopoietic and stromal compartments. The resulting deregulated expression of niche factors may well also be a feature of other hematopoietic malignancies.

The hypothesis of our study is that a routine tibial cut during cruciate retaining TKA may result in a partial or a total removal of the PCL footprint. Therefore providing a reliable landmark is essential to estimate the probability of PCL damage with a tibial cut and to enable the surgeon to decide pre-operatively whether a cruciate retaining implant design is suitable.In a case series of 175 cruciate retaining TKA, the routinely made standing postoperative AP-view radiographs were evaluated to determine the distance between fibula head and tibial cutting plane. In a second case series knee MRI of 223 subjects were consecutively used to measure the vertical distance between tibial attachment of PCL and fibula head. The probability of partial or total PCL damage was calculated for different vertical distances between tibial cut and fibula head.The vertical distance between the tibial cut and the most proximal point of the fibula head averaged 6.1 mm ±4.8 mm. The mean vertical distance from fibula head to proximal and to distal PCL footprint revealed to be 11.4 mm ±3.7 mm and 5.4 mm ±2.9 mm, respectively. The location of the insertion was not significantly different between subgroups such as age (50 or50 years), gender and side. Based on our results 11 (7%) knees were considered at high risk of an entire PCL removal after implantation of a cruciate retaining TKA design.Currently available routine tibial preparation techniques result in partial or total posterior cruciate ligament detachment. Fibula head as a landmark aids to predict the PCL location and to estimate its disruption pre- and postoperatively on AP-view radiographs.

Reconstruction of the joint line is crucial in total knee arthroplasty (TKA). A routine height of tibial cut to maintain the natural joint line may compromise the preservation of the PCL. Since the PCL footprint is not accessible prior to tibial osteotomy, it seems beneficial to identify a reliable extraarticular anatomic landmark for predicting the PCL footprint and being visible within standard TKA approach. The fibula head predicts reliably the location of PCL footprint; however, it is not accessible during TKA. The aim of this study now was to analyze whether the tibial tuberosity can serve as a reliable referencing landmark to estimate the PCL footprint height prior to tibial cut.The first consecutive case series included 216 CR TKA. Standing postoperative lateral view radiographs were utilized to measure the vertical distance between tibial tuberosity and tibial osteotomy plane. In the second case series, 223 knee MRIs were consecutively analyzed to measure the vertical distance between tibial tuberosity and PCL footprint. The probability of partial or total PCL removal was calculated for different vertical distances between tibial tuberosity and tibial cutting surface.The vertical distance between the tibial tuberosity and tibial cut averaged 24.7 ± 4 mm. The average vertical distance from tibial tuberosity to proximal and to distal PCL footprint was found to be 22 ± 4.4 and 16 ± 4.4 mm, respectively. Five knees were considered at 50% risk of an entire PCL removal after CR TKA.Current surgical techniques of tibial preparation may result in partial or total PCL damage. Tibial tuberosity is a useful anatomical landmark to locate the PCL footprint and to predict the probability of its detachment pre-, intra-, and postoperatively. This knowledge might be useful to predict and avoid instability, consecutive pain, and dissatisfaction after TKA related to PCL insufficiency.III.

Ultrasonic velocity and attenuation of 29 MHz waves in molten (K,Li)NO3, (K,Na)NO3, (K,Rb)NO3, (Cs,Li)NO3, (Cs,Na)NO3, (Cs,K)NO3, (Cs,Rb)NO3, and (Rb,Ag)NO3 are measured as a function of composition and temperature. An improved pulse transmission device is used together with a new measuring cell allowing a temperature accuracy of better than 1 K over the cell-vertical. The temperature dependence of the velocity is linear up to 675 K, the absorption is governed by the activation energy. The velocity and absorption (referred to the square of frequency) values are given in plots and analytical expressions. A reasonable general dependence on composition cannot be given. The isentropic compressibility is listed. The bulk viscosity of the eight investigated systems exceeding the shear viscosity by more than one order of magnitude is given together with an analytical expression for the ratio bulk/shear viscosity based on the Eyring theory.

Abstract 1694 Introduction: Myelodysplastic syndromes are a heterogeneous group of malignant clonal hematologic disorders characterized by ineffective hematopoiesis, peripheral cytopenias and dysplastic bone marrow cells, with frequent progression to acute myeloid leukemia. Because of its heterogeneous nature, modeling of this disease has proven to be very difficult in cell culture systems as well as mice. In addition, attempts to generate a xenotransplant model in immuno-compromised mice have only achieved very low levels of engraftment that are often transient, making it very difficult to study the biology of this disease in vivo. Recent studies in mice have shown that conditional impairment of the small RNA processing enzyme Dicer in mouse osteolineages induced a stromal niche that promoted myelodysplasia, leading to the hypothesis that abnormal bone marrow stromal cells might provide a “fertile soil“ for the expansion of the malignant clone. Patients and Methods: To the date of writing, a total of 12 primary hematopoietic stem cell- and mesenchymal stroma cell (MSCs) samples selected from patients with MDS have been isolated and xenotransplanted into NOD.Cg-Prkdscid Il2rgtm1Wjl/Szj (NSG) mice: MDS 5q- (n=7), MDS RCMD (n=3), MDS RAEB I (n=1), MDS-U (n=1). Engraftment was monitored by FACS using human specific antibodies to CD45, CD34 and CD38. In addition cell cycle behavior was analyzed by Ki67/Hoechst staining. Mesenchymal stromal cells were characterized using previously described stromal markers: CD105, CD271, CD73, CD166, CD90, CD146 and CD44. To isolate genomic DNA and RNA for molecular analyses, MDS xenografts were flow sorted based on human CD45 expression. Molecular characterization of primary MDS samples and xenotransplants was carried out by serial copy number analysis using Affymetrix SNP 6.0 Arrays, metaphase cytogenetics and direct sequencing of known mutations in the transplanted MDS samples. Results: We show, that the concomitant transplantation of MDS-derived mesenchymal stromal cells with the corresponding hematopoietic patient stem/progenitor cells leads to significant and long-term engraftment (0.1 – 15% for up to 23 weeks) of cells isolated from IPSS low and intermediate risk MDS patients. In addition to the bone marrow, MDS hematopoietic cells also infiltrate other hematopoietic compartments of the mouse including the spleen. Significant engraftment of cells with progenitor (CD34+CD38+) as well as stem cell phenotype (CD34+CD38-) was observed, which is consistent with engraftment of an MDS stem cell that sustains long-term hematopoiesis. SNP array analysis confirmed the clonal origin of the engrafted cells as MDS xenografts harboring the identical genomic lesions as present in the patient disease. Conclusion: We present a robust MDS xenograft model of low risk MDS entities based on the concomitant transplantation of primary MDS hematopoietic cells with MSCs from the same patients. This model does not only allow to study the biology of this disease in vivo but also the molecular and cellular interactions between MSCs and hematopoietic MDS cells. In addition it provides a useful platform to study the effects of new experimental therapeutic agents for the treatment of MDS in molecularly defined MDS cells. Disclosures: No relevant conflicts of interest to declare.

Introduction Myelodysplastic syndromes (MDS) are clonal hematologic disorders characterized by ineffective hematopoiesis, dysplasia and increased risk of progression to acute myeloid leukemia. The development of targeted therapies for MDS has been lagging behind and remains a key clinical challenge that has been hampered, at least in part, by difficulties to establish in vivo model systems that recapitulate disease heterogeneity and complexity. Attempts to generate a xenograft model of lower risk MDS have only achieved low and often transient levels of engraftment. Recent evidence from mouse studies suggests that MDS is a disease in which both the hematopoietic system and the bone marrow microenvironment might be involved. Thus, we hypothesized that a specific MDS microenvironment might be required for the successful modeling of low risk MDS in mice, proposing a dependency of the “disease propagating cells“ on their corresponding niche cells in human MDS. Methods Our study is based on xenotransplantation of material from 19 MDS patients classified as follows: IPSS low risk (n=6), intermediate-1 risk (n=13), WHO 2008 classification: MDS 5q- (n=7), MDS RCMD (n=7), MDS RAEB I (n=3), MDS-U (n=1), MDS RARS (n=1). MDS CD34+ cells were co-injected with patient-derived mesenchymal stromal cells (MSCs) directly in the bone marrow cavity (i.f) of NOD.Cg-Prkdscid Il2rgtm1Wjl/Szj (NSG) or NSGS (NSG mice expressing human SCF, IL3 and GM-CSF) mice. Molecular tracking of MDS cells was carried out by copy number analysis (Affymetrix SNP 6.0 Arrays), metaphase cytogenetics, interphase FISH, Roche 454 deep sequencing and pyrosequencing of known mutations. Mice were analyzed after a minimum of 16 weeks post transplantation. Results We show that co-injection of MDS CD34+ cells with their corresponding MSCs leads to significant and long-term engraftment of over 77% of the MDS patients analyzed, both in NSG (10/13 patients, range hCD45+= 1-18%) and NSGS mice (7/8 patients, range hCD45+=2.2-74%). In contrast, absence of MSCs or co-injection of healthy age-matched MSCs only gave rise to limited engraftment in NSG mice (2/7 patients (hCD45+=1-3.8%) and 1/2 patients (hCD45+=2%), respectively). Transplanted samples exhibited a clear myeloid bias and significant engraftment of cells with progenitor (CD34+CD38+) and stem cell phenotype (CD34+CD38-) that could be serially transplanted. In addition, presence of morphologically dysplastic cells was readily detectable in NSGS mice. Importantly, molecular analysis of the engrafted cells confirmed their “diseased” origin as they carried identical lesions to the ones present in the original MDS patient. Furthermore, we could demonstrate that disease-propagating stem cells in lower risk MDS exclusively reside within the lin-CD34+CD38- stem cell fraction. Finally, RNA sequencing analysis comparing MDS and age-matched healthy control MSCs revealed altered expression of key genes involved in cellular adhesion, extra-cellular matrix (ECM) remodeling and cellular cross-talk in diseased MSCs, strongly supporting the notion of a complex interplay between MDS hematopoietic cells and their corresponding stroma. In addition, patient MSCs exhibited clear molecular features of fibrosis, a clinical feature often associated with MDS. Conclusion In this study we have identified patient-derived MSCs as a critical functional component of lower risk MDS. Together with MDS stem cells, these patient MSCs form a functional stem cell-niche unit, which allows the propagation of the disease in a xenograft recipient. The striking changed expression in diseased MSCs of genes involved in processes like cytokine-cytokine receptor interaction, cellular adhesion, ECM remodeling as well as hypoxia further suggests that diseased MDS cells might alter the function of the normal HSC niche into one that can support the requirement of MDS cells. Studying the interaction of MDS stem cells and MSCs at the cellular and molecular level will provide a platform for unraveling the molecular basis of clonal dominance in MDS as well as allow the design of targeted strategies aimed to disrupt the MDS stem cell-MSC niche interactions. Disclosures: No relevant conflicts of interest to declare.