Human mesenchymal stromal cells inhibit platelet activation and aggregation involving CD73-converted adenosine.

BACKGROUND: Mesenchymal stromal cells (MSCs) are promising cell therapy candidates. Clinical application is considered safe. However, minor side effects have included thromboembolism and instant blood-mediated inflammatory reactions suggesting an effect of MSC infusion on hemostasis. Previous studies focusing on plasmatic coagulation as a secondary hemostasis step detected both procoagulatory and anticoagulatory activities of MSCs. We now focus on primary hemostasis and analyzed whether MSCs can promote or inhibit platelet activation. METHODS: Effects of MSCs and MSC supernatant on platelet activation and function were studied using flow cytometry and further platelet function analyses. MSCs from bone marrow (BM), lipoaspirate (LA) and cord blood (CB) were compared to human umbilical vein endothelial cells or HeLa tumor cells as inhibitory or activating cells, respectively. RESULTS: BM-MSCs and LA-MSCs inhibited activation and aggregation of stimulated platelets independent of the agonist used. This inhibitory effect was confirmed in diagnostic point-of-care platelet function analyses in platelet-rich plasma and whole blood. Using inhibitors of the CD39-CD73-adenosine axis, we showed that adenosine produced by CD73 ectonucleotidase activity was largely responsible for the LA-MSC and BM-MSC platelet inhibitory action. With CB-MSCs, batch-dependent responses were obvious, with some batches exerting inhibition and others lacking this effect. CONCLUSIONS: Studies focusing on plasmatic coagulation suggested both procoagulatory and anticoagulatory activities of MSCs. We now show that MSCs can, dependent on their tissue origin, inhibit platelet activation involving adenosine converted from adenosine monophosphate by CD73 ectonucleotidase activity. These data may have strong implications for safety and risk/benefit assessment regarding MSCs from different tissue sources and may help to explain the tissue protective mode of action of MSCs. The adenosinergic pathway emerges as a key mechanism by which MSCs exert hemostatic and immunomodulatory functions.

[Cross-sectional field 14 pain medicine. Implementation of the German Pain Society (DGSS) core curriculum in the model study course MaReCuM]. / Querschnittsbereich 14 Schmerzmedizin. Umsetzung des Kerncurriculums der Deutschen Schmerzgesellschaft im Modellstudiengang MaReCuM.

BACKGROUND: The recent introduction of amendments to the medical licensure laws led to the introduction of the field of pain medicine into the study program "Human Medicine". The implementation has to be completed by all medical faculties before 2016. MATERIAL AND METHODS: Pain medicine was implemented into the model study course"MaReCuM" at the medical faculty in Manheim as a compulsory subject in the year 2010. It is structured into five sections in a longitudinal manner. The core section is the "pain awareness week" in the fifth academic year of the medical studies. The content and structure is based on the German Pain Society (DGSS) curriculum. For the purpose of this study the examination results and the student evaluation forms from the academic years 2010/2011 and 2011/2012 were analyzed. RESULTS: The students regarded pain medicine as being highly relevant concerning its impact on the professional activities. The competence to develop a specific and individual therapy was of special interest. A good coordination of the contents of teaching between preclinical and clinical teaching was considered to be of major importance. CONCLUSIONS: The DGSS curriculum is a useful tool for the implementation of pain medicine in a study program. In order to improve access to basic pain medicine in general, a combined teaching program consisting of pain medicine and general medicine could be helpful. Pain medicine could be used as a guide for teaching contents of outpatient medicine.

[Autologous fat grafts and supportive enrichment with adipose tissue stromal cells]. / Autologe Fetttransplantation in der Brustchirurgie - Klinische und Experimentelle Konzepte: vom Lipofilling bis zum Fettgewebe Tissue Engineering.

Liposuction is a most common surgical procedure in aesthetic surgery that aims at the local fat reduction. The obtained adipose tissue is currently used as a biocompatible filler. Autologous fat transplantation, also known as lipofilling, has become an attractive treatment method in the field of aesthetic facial surgery and scar tissue reconstruction. Lipofilling may also offer an alternative method to prosthetic breast surgery. Nevertheless, postoperative fat tissue resorption is still a limitiation to lipofilling in breast reconstruction leading to multiple revisions in order to reach the requested clinical outcome. The therapeutic effect of autologous fat grafts does not solely lie in its role as a filler material, but also in its wound healing and angiogenetic properties. The latter is not attributed to the mature adipocytes, but rather to the undifferentiated adipose derived stromal cells (ASC). Thus enrichment of the fat graft with autologous ASC, known as cell-assisted lipotransfer (CAL) may lead to further optimisation of lipofilling concerning fat graft survival. Still aiming to establish the application of autologous fat grafts and ASC in breast reconstruction, there is a necessity for systematic analyses in order to resolve questions regarding the operational technique and qualitative aspects of the ASC manufacturing in accordance with pharmaceutical guidelines and regulations in Germany. Besides, some open questions need to be addressed regarding the ASC differentiation potential in vivo.

Regenerative therapies in neonatology: clinical perspectives.

Regenerative therapy based on stem cells is applied as standard therapy in pediatric oncology. Furthermore, they are frequently used to treat immunodeficiency disorders of infants. For severe neonatal diseases, e. g. hypoxic-ischemic encephalopathy in term neonates or bronchopulmonary dysplasia in preterm infants, animal models have been established. According to some first preclinical results stem cell administration appears as a promising tool to improve the clinical outcome in high-risk infants. Provided the benefit of regenerative therapies can further be evaluated in appropriate preclinical neonate models, carefully controlled clinical trials to assess the significance of regenerative therapies, such as autologous stem cell administration, are indicated.

Safety and frequency of whole blood donations from elderly donors.

BACKGROUND: Within the coming decades, a steadily growing demand for blood products will face a shrinking blood donor population in many countries. After increasing the donor age of repeat donors for whole blood donation (WB) from 68 to 70 years in 2009 in our Blood Service, we investigated whether this is sufficient as a safe and effective strategy to sustain future blood supply. MATERIALS AND METHODS: Between 1 March 2009 and 28 February 2011, WB donations from donors aged between 69 and 70 and their proportion of total donations in 2010 were determined. We analysed adverse reaction rates in donors with respect to sex and age and calculated mean annual donation frequencies. RESULTS: Of all invited donors, 32·5% responded and contributed 0·98% (men) and 0·56% (women) to all WB units collected in 2010. The overall and systemic adverse reaction rate per 1·000 WB donations declined by age [men: 1·10 (95%CI: 0·84-1·35) vs. 0 (0-0·8), P < 0·0001; 0·99 (0·75-1·23) vs. 0 (0-0·8), P < 0·0001 and women: 1·80 (1·46-2·14) vs. 1·12 (0·1-2·66), P < 0·0001; 1·47 (1·17-1·78) vs. 1·12 (-0·43-2·66), P = 0·0004]. Mean donation frequencies were strongly correlated with increasing age (men: r = 0·953, P < 0·0001; women: r = 0·913, P < 0·0001) with peak values for 70-year-old male: 2·53 ± 1·37 vs. 1·79 ± 1·05, P < 0·0001 and female donors: 2·15 ± 1·06 vs. 1·52 ± 0·78, P < 0·0001. CONCLUSIONS: Elderly donors have very low adverse reaction frequencies and are highly committed to donate blood. Thus, we consider donations from repeat donors aged 69-70 safe and suggest it a powerful short- to midterm strategy to, at least partially, overcome the challenges of the demographic change.

A spatial regression analysis of German community characteristics associated with voluntary non-remunerated blood donor rates.

BACKGROUND AND OBJECTIVES: Previous studies have shown substantial geographical variation in blood donation within developed countries. To understand this issue better, we identified community characteristics associated with blood donor rates in German municipalities in an ecological analysis. MATERIALS AND METHODS: We calculated an aggregated rate of voluntary blood donors from each of 1533 municipalities in south-west Germany in 2007 from a database of the German Red Cross Blood Service. A multiple linear regression model estimated the association between the municipality-specific donor rate and several community characteristics. Finally, a spatial lag regression model was used to control for spatial autocorrelation that occurs when neighbouring units are related to each other. RESULTS: The spatial lag regression model showed that a relatively larger population, a higher percentage of inhabitants older than 30 years, a higher percentage of non-German citizens and a higher percentage of unemployed persons were associated with lower municipality-specific donor rates. Conversely, a higher donor rate was correlated with higher voter turnout, a higher percentage of inhabitants between 18 and 24 years and more frequent mobile donation sites. CONCLUSIONS: Blood donation appears to be a highly clustered regional phenomenon, suggesting the need for regionally targeted recruiting efforts and careful consideration of the value of mobile donation sites. Our model further suggests that municipalities with a decreasing percentage of 18- to 24-year-olds and an increasing percentage of older inhabitants may experience substantial declines in future blood donations.

Detection of granulocyte antibodies using simultaneous analysis of specific granulocyte antibodies assay (SASGA).

OBJECTIVE: Granulocyte-associated antibodies can cause several clinical granulocytopenic disorders. The monoclonal-antibody-specific immobilization of granulocyte antigens (MAIGA) is currently used as the standard assay to specify these antibodies. Here we describe an assay for specific analysis of granulocyte antibodies (SASGA) which is able to simultaneously detect and specify granulocyte IgG- and IgM-antibodies using flow cytometry. METHODS: Bead populations with distinct fluorescence intensities were used as solid phase for immobilization of mAb. Typed granulocytes were incubated with human sera and a mix of three distinct mouse monoclonal antibodies against specific granulocyte antigens (for example CD16, CD11a, HLA class I). After cell lysis and incubation of lysate with beads, goat antibodies against human IgG and IgM antibodies were added. Seventy-one frozen sera of donors and patients previously implicated in transfusion reactions and various underlying disorders were analysed for specific granulocyte-binding antibodies using MAIGA and SASGA. RESULTS: The SASGA assay was able to simultaneously detect granulocyte-specific antibodies for different glycoproteins. Overall, the results of MAIGA and SASGA were concordant in 92·9%. 5 sera containing anti-HNA-1b (n=2) and -HLA class I (n=3) were not detected by MAIGA, but were recognized by the SASGA. In serial dilution tests with sera containing anti-HNA-1a, -1b, -2a and HLA class I, the SASGA assay detected the antibodies at higher dilutions than MAIGA. CONCLUSION: The SASGA assay permits reliable detection of specific granulocyte antibodies. Six distinct antibodies can be simultaneously determined. This method will potentially open the way to investigations on additional specific antibodies as it facilitates laboratory diagnosis.

Pathogen reduction technology (Mirasol) treated single-donor platelets resuspended in a mixture of autologous plasma and PAS.

BACKGROUND AND OBJECTIVES: Mirasol pathogen reduction technology (PRT) for platelet concentrates uses riboflavin and ultraviolet light. Previously, we described increased metabolism and activation for PRT platelets stored in 100% plasma. To improve platelet quality, we resuspended platelets in a mixture of plasma and platelet additive solution (PAS). MATERIALS AND METHODS: Single-donor platelets were resuspended in plasma and split into an untreated control and a PRT-treated single product. One hundred and fifty millilitre PAS (SSP+) was added to both. Over 7 days, we assayed pH, glucose consumption-, lactate production rate and CD62p with and without TRAP. RESULTS: On day 5, PRT units showed a significantly lower pH (7.087 +/- 0.105 vs. 7.288 +/- 0.200) accompanied by a higher lactate production (0.104 +/- 0014 vs. 0.063 +/- 0.017 mmol/10(12)/h) and glucose consumption rate (0.039 +/- 0005 vs. 0.028 +/- 0.009 mmol/10(12) platelets/h). CD62p expression was higher in treated units (44.5 +/- 13.0 vs. 16.5 +/- 7.6%). CONCLUSION: In comparison to PRT platelets resuspended in 100% plasma, a mixture of plasma and PAS improves pH and platelet metabolism but not platelet activation. Prolonged shelf-life for up to 7 days may be possible

Differentiation of monocyte-derived dendritic cells under the influence of platelets.

BACKGROUND: Monocytapheresis has been established to collect a sufficient number of monocytes (MO) for differentiation to dendritic cells (DC) as a cancer vaccine. Platelets (Plt) are invariably found as a contaminant in the final monocytapheresis product. The aim of this study was to investigate DC differentiation under the influence of Plt with regard to their function and phenotype. METHODS: MO were isolated and co-cultured with autologous Plt at different MO:Plt ratios (1:1.7, 1:5, 1:15, 1:45 and 1:135) in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). IL-12p70 release after ligation of CD40L was determined in the supernatant by enzyme-linked immunosorbent assay (ELISA). For T-cell stimulation, tetanus toxoid was added to immature DC and maturation was induced by adding cytokines (IL-1beta, IL-6, tumor necrosis factor-alpha and prostaglandin E(2)). Stimulated T cells were analyzed for activation and proliferation as well as for intracellular cytokines by flow cytometry. RESULTS: All DC cultures were strongly positive for CD83. At a contaminating concentration of 5 Plt/MO, matured DC showed the highest expression of HLA-DR, CD80 and CD86, inducing a strong T-cell proliferation with high production of IL-4 and interferon-gamma. The highest level of IL-12p70 production was observed by the same DC group. DISCUSSION: Plt did not negatively influence DC maturation but enhanced the expression of co-stimulatory molecules and the release of IL-12. Functionally this was reflected by a strong T-cell response that involved T-helper 1 (Th1)- as well as Th2-biased T cells. Our findings show that controlling the Plt concentration may provide important advantages for the generation of DC for use in immunotherapy.

Introduction of a validation concept for a PCR-based Mycoplasma detection assay.

BACKGROUND: Mycoplasma contamination is amongst the most frequently occurring problems associated with cell cultures. In order to meet the legal requirements (European Pharmacopoeia and FDA) for Mycoplasma testing of cell lines and therapeutics, we have developed a PCR-based method to detect mycoplasms and introduce a validation concept. METHODS: The PCR assay specifically amplifies a 280-bp DNA fragment of the gene coding for the 16S rDNA. Simultaneous amplification of an artificial oligonucleotide containing primer-binding sites allowed control of the efficacy of the PCR. The validation of the PCR assay was performed with two Mycoplasma reference strains, M. orale and M. pneumoniae. The validation concept included (i) cultivation of M. orale and M. pneumoniae in medium with an indicator for bacterial metabolism, (ii) determination of the color-changing units (CCU) in repeated dilution experiments and (iii) correlation of the PCR results with CCU values. RESULTS: The detection range was found to include all Mycoplasma species most commonly found in cell cultures. The analytical sensitivity of the PCR was the CCU equivalent of 100 for M. orale and M. pneumoniae. Probit analysis revealed a detection probability of 9% for a mean concentration of 1222 (935-1844) CCU/mL for M. pneumoniae and 2547 (1584-10,352) CCU/mL for M. orale. DISCUSSION: The validation of the Mycoplasma detection assay supported PCR as an attractive diagnostic tool that will help manage the important issue of Mycoplasma contamination of cell cultures.

[A comparison of the gene expression patterns of human chondrocytes and chondrogen differentiated mesenchymal stem cells for tissue engineering]. / Vergleich der Genexpressionsmuster humaner Chondrozyten und chondrogen differenzierter mesenchymaler Stammzellen für das Tissue-Engineering.

BACKGROUND: Tissue engineering is a promising method for the generation of chondrogenic grafts for reconstructive surgery. In cultured chondrocytes, the dedifferentiation of cells seems unavoidable for multiplication. METHODS: In this study, we investigated the expression of distinct markers during the dedifferentiation of human chondrocytes (HC) harvested during septoplasty and human mesenchymal stem cells (hMSC) from cartilage biopsies in cell culture using the microarray technique. RESULTS: The genes for collagen 1alpha1, 2alpha1, 3alpha1, 4alpha1, 11alpha1, biglycan, fibromodulin and lumican were activated during the dedifferentiation of the HCs, collagen 9alpha2, 9alpha3, 10alpha1 and chondroadherin were inactivated. During chondrogenic differentiation of hMSCs, the genes for collagen 3alpha1, 9alpha2, 9alpha3, 10alpha1, 11alpha1 were activated, collagen 4alpha1 and fibromodulin inactivated and the genes for Col 1alpha1, biglycan und chondroadherin constantly expressed. CONCLUSION: The genetic profile for the investigated markers in human chondrocytes generated from hMSCs resembles the profile in differentiated chondrocytes. Collagen 2alpha1, 9alpha2, 9alpha3, 10alpha1 could represent markers for the differentiation of chondrocytes, Col 1alpha1, 3alpha1 und 4alpha1, biglycan, fibromodulin and lumican markers for the dedifferentiation into a more fibroblastoid cell type.

Effect of the psoralen-based photochemical pathogen inactivation on mitochondrial DNA in platelets.

Photochemical treatment (PCT) of platelet concentrates, using amotosalen HCl and UVA-light, inactivates pathogens by forming adducts between amotosalen and nucleic acids. The impact of the photochemical treatment on pathogens and leukocytes has been studied extensively. Yet little is known about the effect of PCT on nucleic acids in platelets. Platelets contain viable mitochondria and mitochondrial DNA (mtDNA) and this study aimed at evaluating the amotosalen modifications on platelet mtDNA. We applied two independent but complementary molecular assays to investigate qualitative as well as quantitative aspects of the psoralen-mediated DNA modifications in platelet mtDNA. The amotosalen-DNA modification density was measured using (14)C-labeled amotosalen. Amotosalen (150 microM) yielded 4.0 +/- 1.2 psoralen adducts per 1,000 bp in mtDNA after irradiation with 3 J/cm(2) UVA. Furthermore, we tested if the PCT-induced DNA modifications could be detected by a PCR assay. On the basis of PCR inhibition due to amotosalen-DNA adducts, mtDNA-specific PCR assays were developed and tested for their specificity and sensitivity. Our data revealed that mtDNA in platelets is substantially modified by PCT and that these modifications can be documented by a PCR inhibition system.

Deficient IL-12p70 secretion by dendritic cells based on IL12B promoter genotype.

Interleukin-12 (IL-12), a heterodimeric cytokine, is important in the generation of a Th1-biased immune response. Several polymorphisms have been described in IL12B, the gene encoding the p40 subunit of IL-12. A bi-allelic polymorphism within the IL12B promoter region has been reported to show association with diseases as diverse as severe childhood asthma and fatal cerebral malaria. In order to define the molecular basis for these disease associations, we investigated the secretion of IL-12 by human monocyte-derived dendritic cells. Homozygotes for the IL12B promoter polymorphism showed a 10-fold difference in median p70 secretion in response to CD40 ligation. Remarkably, this difference resulted from the inability of most allele 1 homozygotes to secrete heterodimeric IL-12. In contrast, most of the donors homozygous for allele 2 had detectable secretion. These findings are important for the understanding of the highly complex regulation of IL-12 secretion, and its consequent impact on disease susceptibility, in humans.

Implementation of the INTERCEPT Blood System for Platelets into routine blood bank manufacturing procedures: evaluation of apheresis platelets.

BACKGROUND AND OBJECTIVES: The INTERCEPT Blood System for Platelets utilizes amotosalen-HCl (S-59) in combination with ultraviolet A (UVA) light to inactivate viruses, bacteria, protozoa and leucocytes that may contaminate platelet concentrates (PCs). To facilitate implementation of this technique into routine blood bank manufacturing procedures, this study evaluated the impact of different time settings of photochemical treatment on in vitro platelet function. MATERIALS AND METHODS: Platelets derived from apheresis (6.5-7.0 x 10(11) platelets) were resuspended in 240 ml of autologous plasma and 360 ml of platelet additive solution (PAS III) and split into two equal-sized PC units. Whereas one unit was not treated, the other was treated with 150 microm amotosalen and 3 J/cm2 UVA light followed by a compound adsorption device (CAD) step for reduction of residual amotosalen and photoproducts. In a first series of experiments (arm A, n = 7), PC units were photochemically treated after an overnight storage period of 16-23 h followed by a CAD step of 4 h. In a second series (arm B, n = 8), photochemical treatment occurred after a short storage time of 4 h with a subsequent CAD step of 16 h. Platelet function was evaluated by assaying blood gas analysis, glucose and lactate concentration, lactate dehydrogenase (LDH), hypotonic shock response (HSR) and the expression of CD62p, over a period of 7 days. RESULTS: Neither of the photochemical treatment procedures showed differences for pH, pCO2, pO2, HCO3, glucose consumption or platelet activation until the end of day 7. Increased lactate values detected for the treated units of arm A at the end of the storage period were independent from the PCT time setting. CONCLUSIONS: Photochemical pathogen inactivation with different initial resting periods between 4 and 23 h, and different CAD steps of 4 and 16 h, had no influence on the platelet in vitro function during 7 days of storage.

Optimized sensitivity of allele-specific PCR for prenatal typing of human platelet alloantigen single nucleotide polymorphisms.

PCR using sequence-specific primers (PCR-SSP) is widely employed for the genotyping of single nucleotide polymorphisms (SNPs) in both routine diagnosis and medical research. The human platelet alloantigens (HPAs) represent SNPs in platelet-specific glycoproteins, and HPA-1, -2, -3, and -5 are the most relevant in immunohematology. In most protocols, the respective HPA-SNPs are analyzed in allele-specific reactions, each with at least 100 ng DNA. In many cases, prenatal HPA typing in the diagnosis of neonatal alloimmune thrombocytopenia is often limited by the restricted amounts of fetal DNA that are obtainable. We developed a novel PCR-SSP technique to achieve accurate HPA genotypes using only 1 ng DNA per reaction. The concentration of HPA-specific primers was increased to 1 microM each and exhibited a higher sensitivity compared to a commercial PCR-SSP kit. The modified PCR-SSP technique enabled the identification of fetal HPA genotypes using only 0.5 mL amniotic fluid (from week 16 of gestation) and from a maternal plasma sample (from week 38 of gestation). The principle of the modified PCR-SSP technique may also be applied for the genotyping of other SNPs from limited amounts of DNA.