Xeno-Free In Vitro Cultivation and Osteogenic Differentiation of hAD-MSCs on Resorbable 3D Printed RESOMER

The development of alloplastic resorbable materials can revolutionize the field of implantation technology in regenerative medicine. Additional opportunities to colonize the three-dimensionally (3D) printed constructs with the patient’s own cells prior to implantation can improve the regeneration process but requires optimization of cultivation protocols. Human platelet lysate (hPL) has already proven to be a suitable replacement for fetal calf serum (FCS) in 2D and 3D cell cultures. In this study, we investigated the in vitro biocompatibility of the printed RESOMER® Filament LG D1.75 materials as well as the osteogenic differentiation of human mesenchymal stem cells (hMSCs) cultivated on 3D printed constructs under the influence of different medium supplements (FCS, human serum (HS) and hPL). Additionally, the in vitro degradation of the material was studied over six months. We demonstrated that LG D1.75 is biocompatible and has no in vitro cytotoxic effects on hMSCs. Furthermore, hMSCs grown on the constructs could be differentiated into osteoblasts, especially supported by supplementation with hPL. Over six months under physiological in vitro conditions, a distinct degradation was observed, which, however, had no influence on the biocompatibility of the material. Thus, the overall suitability of the material LG D1.75 to produce 3D printed, resorbable bone implants and the promising use of hPL in the xeno-free cultivation of human MSCs on such implants for autologous transplantation have been demonstrated

Comparative Analysis of Mesenchymal Stem Cell Cultivation in Fetal Calf Serum, Human Serum, and Platelet Lysate in 2D and 3D Systems

In vitro two-dimensional (2D) and three-dimensional (3D) cultivation of mammalian cells requires supplementation with serum. Mesenchymal stem cells (MSCs) are widely used in clinical trials for bioregenerative medicine and in most cases, in vitro expansion and differentiation of these cells are required before application. Optimized expansion and differentiation protocols play a key role in the treatment outcome. 3D cell cultivation systems are more comparable to in vivo conditions and can provide both, more physiological MSC expansion and a better understanding of intercellular and cell-matrix interactions. Xeno-free cultivation conditions minimize risks of immune response after implantation. Human platelet lysate (hPL) appears to be a valuable alternative to widely used fetal calf serum (FCS) since no ethical issues are associated with its harvest, it contains a high concentration of growth factors and cytokines and it can be produced from expired platelet concentrate. In this study, we analyzed and compared proliferation, as well as osteogenic and chondrogenic differentiation of human adipose tissue-derived MSCs (hAD-MSC) using three different supplements: FCS, human serum (HS), and hPL in 2D. Furthermore, online monitoring of osteogenic differentiation under the influence of different supplements was performed in 2D. hPL-cultivated MSCs exhibited a higher proliferation and differentiation rate compared to HS- or FCS-cultivated cells. We demonstrated a fast and successful chondrogenic differentiation in the 2D system with the addition of hPL. Additionally, FCS, HS, and hPL were used to formulate Gelatin-methacryloyl (GelMA) hydrogels in order to evaluate the influence of the different supplements on the cell spreading and proliferation of cells growing in 3D culture. In addition, the hydrogel constructs were cultivated in media supplemented with three different supplements. In comparison to FCS and HS, the addition of hPL to GelMA hydrogels during the encapsulation of hAD-MSCs resulted in enhanced cell spreading and proliferation. This effect was promoted even further by cultivating the hydrogel constructs in hPL-supplemented media

Common variants in the CLDN2-MORC4 and PRSS1-PRSS2 loci confer susceptibility to acute pancreatitis

BACKGROUND/OBJECTIVES: Acute pancreatitis (AP) is one of the most common gastrointestinal disorders often requiring hospitalization. Frequent aetiologies are gallstones and alcohol abuse. In contrast to chronic pancreatitis (CP) few robust genetic associations have been described. Here we analysed whether common variants in the CLDN2-MORC4 and the PRSS1-PRSS2 locus that increase recurrent AP and CP risk associate with AP. METHODS: We screened 1462 AP patients and 3999 controls with melting curve analysis for SNPs rs10273639 (PRSS1-PRSS2), rs7057398 (RIPPLY), and rs12688220 (MORC4). Calculations were performed for the overall group, aetiology, and gender sub-groups. To examine genotype-phenotype relationships we performed several meta-analyses. RESULTS: Meta-analyses of all AP patients depicted significant (p-value<0.05) associations for rs10273639 (odds ratio (OR) 0.88, 95% confidence interval (CI) 0.81-0.97, p-value 0.01), rs7057398 (OR 1.27, 95% CI 1.07-1.5, p-value 0.005), and rs12688220 (OR 1.32, 95% CI 1.12-1.56, p-value 0.001). For the different aetiology groups a significant association was shown for rs10273639 (OR 0.76, 95% CI 0.63-0.92, p-value 0.005), rs7057398 (OR 1.43, 95% CI 1.07-1.92, p-value 0.02), and rs12688220 (OR 1.44, 95% CI 1.07-1.93, p-value 0.02) in the alcoholic sub-group only. CONCLUSIONS: The association of CP risk variants with different AP aetiologies, which is strongest in the alcoholic AP group, might implicate common pathomechanisms most likely between alcoholic AP and CP

Ceftriaxone induced hemolysis complicated by acute renal failure

Over the last decade, second and third generation cephalosporins have been the most common drugs causing hemolytic anemia (HA). Of these cases, 20% have been attributed to ceftriaxone. The clinical presentation of ceftriaxone-induced HA is usually abrupt with sudden onset of pallor, tachypnea, cardio-respiratory arrest and shock. Acute renal failure (ARF) has been reported in 41% of such cases with a high fatality rate. We report a pediatric patient with ARF complicating ceftriaxone-induced HA who survived. Ceftriaxone is a commonly used drug, and early recognition of HA and institution of supportive care, including dialysis is likely to improve the outcome. Pediatr Blood Cancer 2008;50:139–142. © 2006 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/57397/1/20839_ftp.pd

Novel method for determining when a field-collected donor unit is sufficiently full

Background: Whole blood (WB) collections can occur downrange for immediate administration. An important aspect of these collections is determining when the unit is sufficiently full. This project tested a novel method for determining when a field collection is complete. Methods: The amount of empty space at the top of WB units, destined to become LTOWB or separated into components, that were collected at blood centers or hospitals was measured by holding a WB unit off the ground and placing the top of a piece of string where the donor tubing entered the bag. The string was marked where it intersected the top of the column of blood in the bag and measured from the top. The WB units were also weighed. Results: A total of 15 different bags, two of which were measured in two different filling volumes, from 15 hospitals or blood centers were measured and weighed. The most commonly used blood bag, Terumo Imuflex SP, had a median string length of 9 mm (range: 2–24 mm) and weighed a median of 565.1 g (range: 524.8–636.7 g). Conclusion: Pieces of string can be precut to the appropriate length depending on the type of bag before a mission where field WB collections might be required and a mark placed on the bag before the collection commences to indicate when the unit is full.</p

Genetic and Mechanistic Evaluation for the Mixed-Field Agglutination in B3 Blood Type with IVS3+5G>A ABO Gene Mutation

Background: The ABO blood type B3 is the most common B subtype in the Chinese population with a frequency of 1/900. Although IVS3+5G.A (rs55852701) mutation of B gene has been shown to associate with the development of B3 blood type, genetic and mechanistic evaluation for the unique mixed-field agglutination phenotype has not yet been completely addressed. Methodology/Principal Findings: In this study, we analyzed 16 cases of confirmed B3 individuals and found that IVS3+5G.A attributes to all cases of B3. RT-PCR analyses revealed the presence of at least 7 types of aberrant B3 splicing transcripts with most of the transcripts causing early termination and producing non-functional protein during translation. The splicing transcript without exon 3 that was predicted to generate functional B3 glycosyltransferase lacking 19 amino acids at the N-terminal segment constituted only 0.9 % of the splicing transcripts. Expression of the B3 cDNA with exon 3 deletion in the K562 erythroleukemia cells revealed that the B3 glycosyltransferase had only 40 % of B1 activity in converting H antigen to B antigen. Notably, the typical mixed-field agglutination of B3-RBCs can be mimicked by adding anti-B antibody to the K562-B3 cells. Conclusions/Significance: This study thereby demonstrates that both aberrant splicing of B transcripts and the reduced B3 glycosyltransferase activity contribute to weak B expression and the mixed-field agglutination of B3, adding to th

Efficacy of UVC-treated, pathogen-reduced platelets versus untreated platelets: a randomized controlled non-inferiority trial

Pathogen reduction (PR) technologies for blood components have been established to reduce the residual risk of known and emerging infectious agents. THERAFLEX UVPlatelets, a novel UVC light-based PR technology for platelet concentrates, works without photoactive substances. This randomized, controlled, double-blind, multicenter, noninferiority trial was designed to compare the efficacy and safety of UVC-treated platelets to that of untreated platelets in thrombocytopenic patients with hematologic-oncologic diseases. Primary objective was to determine non-inferiority of UVC-treated platelets, assessed by the 1-hour corrected count increment (CCI) in up to eight per-protocol platelet transfusion episodes. Analysis of the 171 eligible patients showed that the defined non-inferiority margin of 30% of UVC-treated platelets was narrowly missed as the mean differences in 1-hour CCI between standard platelets versus UVC-treated platelets for intention-to-treat and perprotocol analyses were 18.2% (95% confidence interval [CI]: 6.4%; 30.1) and 18.7% (95% CI: 6.3%; 31.1%), respectively. In comparison to the control, the UVC group had a 19.2% lower mean 24-hour CCI and was treated with an about 25% higher number of platelet units, but the average number of days to next platelet transfusion did not differ significantly between both treatment groups. The frequency of low-grade adverse events was slightly higher in the UVC group and the frequencies of refractoriness to platelet transfusion, platelet alloimmunization, severe bleeding events, and red blood cell transfusions were comparable between groups. Our study suggests that transfusion of pathogen-reduced platelets produced with the UVC technology is safe but non-inferiority was not demonstrated. (The German Clinical Trials Register number: DRKS00011156)

Calculation of Tajima’s D and other neutrality test statistics from low depth next-generation sequencing data

BACKGROUND: A number of different statistics are used for detecting natural selection using DNA sequencing data, including statistics that are summaries of the frequency spectrum, such as Tajima’s D. These statistics are now often being applied in the analysis of Next Generation Sequencing (NGS) data. However, estimates of frequency spectra from NGS data are strongly affected by low sequencing coverage; the inherent technology dependent variation in sequencing depth causes systematic differences in the value of the statistic among genomic regions. RESULTS: We have developed an approach that accommodates the uncertainty of the data when calculating site frequency based neutrality test statistics. A salient feature of this approach is that it implicitly solves the problems of varying sequencing depth, missing data and avoids the need to infer variable sites for the analysis and thereby avoids ascertainment problems introduced by a SNP discovery process. CONCLUSION: Using an empirical Bayes approach for fast computations, we show that this method produces results for low-coverage NGS data comparable to those achieved when the genotypes are known without uncertainty. We also validate the method in an analysis of data from the 1000 genomes project. The method is implemented in a fast framework which enables researchers to perform these neutrality tests on a genome-wide scale

Pathogen reduction/inactivation of products for the treatment of bleeding disorders:what are the processes and what should we say to patients?

Patients with blood disorders (including leukaemia, platelet function disorders and coagulation factor deficiencies) or acute bleeding receive blood-derived products, such as red blood cells, platelet concentrates and plasma-derived products. Although the risk of pathogen contamination of blood products has fallen considerably over the past three decades, contamination is still a topic of concern. In order to counsel patients and obtain informed consent before transfusion, physicians are required to keep up to date with current knowledge on residual risk of pathogen transmission and methods of pathogen removal/inactivation. Here, we describe pathogens relevant to transfusion of blood products and discuss contemporary pathogen removal/inactivation procedures, as well as the potential risks associated with these products: the risk of contamination by infectious agents varies according to blood product/region, and there is a fine line between adequate inactivation and functional impairment of the product. The cost implications of implementing pathogen inactivation technology are also considered