[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.

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.

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.

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.

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.

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.

Does histocompatibility affect homograft valve function after the Ross procedure?

BACKGROUND: Homograft valves have been shown to be immunogenic, but it is unknown whether this affects valve function. Therefore, we prospectively studied the degree of histoincompatibility (defined as the number of human leukocyte antigen [HLA] mismatches between valve donor and recipient) and the response of the recipient (measured by antibodies against HLA) in relation to echocardiographic parameters of homograft valve function after the Ross procedure. METHODS AND RESULTS: Twenty-six patients (mean age 41+/-14 years; 20 males, 6 females) and the cryopreserved pulmonary homograft valves that were implanted during a Ross procedure were typed for HLA-A, HLA-B, and HLA-DR. After a mean follow-up of 15+/-6 months, 14 (54%) of the patients were anti-HLA class I antibody positive. In all but 1 patient, these antibodies were shown to be donor specific. During follow-up, there was a significant increase of the maximal (+6.2+/-7.1 mm Hg) and mean (+3.2+/-4.3 mm Hg) transhomograft pressure gradients but not of homograft regurgitation. Neither the number of HLA mismatches nor antibody status was found to have significant impact on homograft valve function. In a multivariate analysis, smaller homograft size (P=0.001) and younger recipient age (P=0.044) were shown to be significantly associated with increased transhomograft pressure gradients. CONCLUSIONS: Implantation of a cryopreserved pulmonary homograft during the Ross procedure can induce a specific humoral response. We observed a significant increase of the transhomograft pressure gradients within 15+/-6 months after surgery. For this period, we were unable to demonstrate a relationship between this increase and the degree of histoincompatibility.

Ex vivo induction of cytokine mRNA expression in human blood samples.

The interest in the quantitative analysis of cytokine mRNA profiles has increased substantially in recent years. This is based on the potential use of basal cytokine mRNA expression as sensitive markers for in vivo lymphocyte activation in a variety of clinical settings. However, it is less well known to what extent differences in blood collection and preparation techniques may cause ex vivo alteration of quantitative cytokine mRNA levels. We therefore evaluated the effect of blood sampling and the impact of cell separation on interleukin (IL)-2, IL-4, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha mRNA expression in an intraindividual study design (n=8). Two different blood sampling procedures were applied. A whole blood sample 1 was collected by constant moderate blood flow into a blood collection tube containing lithium-heparin. Moreover, a second sample from the same donor was collected by a 5-fold acceleration of blood flow. Furthermore, peripheral blood mononuclear cell (PBMC) were isolated from the first whole blood sample by density separation over Ficoll-Hypaque. The quantification of cytokine mRNA expression was performed by real-time PCR in native whole blood/PBMC samples or unstimulated cultures. We found a significant increase of IL-2, IL-4 and TNF-alpha mRNA expression (P=0.018, P=0.028, P=0.018) in whole blood samples collected by rapid sampling. The isolation of PBMC by density gradient separation prompted on upregulation of the mRNA levels of IL-2, IL-4 and TNF-alpha 5-9-fold (P=0.018, P=0.018, P=0.018). In contrast, IFN-gamma mRNA expression was not significantly influenced by differences in blood sample preparation. Our data clearly demonstrate that differences in the blood sampling technique or cell separation should be considered as important factors for non-physiological ex vivo induction of cytokine mRNA expression. The current data emphasize the need for data on the impact of ex vivo variation in order to extract reliable and consistent information, particularly when cytokine mRNA expression data from healthy blood donors are included in clinical studies.

Effects of amantadine treatment on in vitro production of interleukin-2 in de-novo patients with idiopathic Parkinson's disease.

An involvement of immunological events in the process of neurodegeneration has frequently been reported. We investigated the cytokine producing capacity for interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and interleukin-10 (IL-10) in whole blood cultures of de-novo patients with idiopathic Parkinson's disease (PD) at the time of first diagnosis and after oral amantadine treatment. Before treatment, productions of IL-2 and IFN-gamma were markedly decreased in PD patients compared to patients with major depressive disorder and healthy controls. After amantadine treatment, the in vitro IL-2 secretion defect was corrected to normal levels in half of the patients, and the increase in IL-2 production was correlated with an increase in IFN-gamma secretion. Our findings suggest that immunological abnormalities occur in the course of PD and that a formerly unappreciated therapeutic potential of amantadine may arise from its immunomodulatory effects on altered T cell function in patients with PD.

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.

Quantification of platelet-associated IgG for differential diagnosis of patients with thrombocytopenia.

Immune thrombocytopenia is due to platelet destruction by circulating glycoprotein-specific antibodies and is found in various disorders. Methods for the detection of platelet-associated IgG (PAIgG) are generally sensitive but unspecific, whereas glycoprotein-specific assays are highly specific but less sensitive. Usefully, a sensitive screening method for PAIgG detection would also provide information for differential diagnosis. We developed a quantitative direct Platelet Immunofluorescence Test (PIFT) by flow cytometry and studied 79 thrombocytopenic patients with immune thrombocytopenia and other disorders. The sensitivity of the assay was 94%, its specificity 66% for the detection of a clinically obvious immune thrombocytopenia. PAIgG levels of patients with immune thrombocytopenia differed significantly from those of other patients with low platelet counts (p <0.001). The quantitative PIFT proved to be a sensitive method for PAIgG detection and should therefore be used as a screening method. In addition, it could be helpful for differential diagnosis in marked thrombocytopenia where a MAIPA is not feasible.

Clostridial sepsis with massive intravascular hemolysis: rapid diagnosis and successful treatment.

A 61-year-old man developed a pyrescia accompanied by a massive intravascular hemolysis after abdominal surgery (Whipple's operation) of a pancreatic adenocarcinoma. Abdominal ultrasound and the abdominal CT-scan showed marked aerobilia and multiple liver abscesses. Laboratory tests demonstrated the presence of the Thomsen-Friedenreich cryptantigen (TCA) on the membranes of the patient's erythrocytes. The enzymatic cleavage of N-acetyl-neuraminic acid usually covering the TCA may lead to a life threatening intravascular hemolysis. Since Clostridial bacteriae typically synthesize neuraminidase, the presumptive diagnosis of Clostridial sepsis complicated by massive hemolysis was made. Immediate antibiotic therapy including penicillin G and metronidazole stopped hemolysis within a few hours and the patient servived. On the following day, microbiological examination identified Clostridium perfringens in the patient's blood cultures. Clostrial sepsis should be suspected in patients with underlying infections and/or malignant diseases, particularly of the gastrointestinal or genitourinary tract, who present with septic shock and acute intravascular hemolysis. Whereas microbiological specification of the organism is time consuming, the relatively simple agglutination test with anti-TCA peanut lectin can provide a rapid presumptive diagnosis. The immediate onset of an appropriate antimicrobial therapy is of central importance and might be life-saving.

Anti-HLA class I antibodies and pulmonary homograft function after the Ross procedure.

BACKGROUND: The Ross procedure provides excellent long-term results in the majority of patients. However, degeneration of the pulmonary homograft in some patients remains an unresolved problem that may be related to immunologic factors. Therefore, we studied the prevalence of antihuman leukocyte antigen (HLA) class I antibodies and echocardiographic results of homograft function at rest. METHODS: Forty-seven patients (37 men, 10 women; 47 +/- 15 years) were seen for echocardiography 1.1 to 63.9 months (median, 27 months) postoperatively. The presence of anti-HLA antibodies was tested against a panel of lymphocytes of 50 donors. RESULTS: Twenty-seven (57%) of the patients produced anti-HLA class I antibodies. No difference in the maximal or mean transhomograft pressure gradient, or in the frequency of homograft regurgitation according to the presence or absence of anti-HLA antibodies was found. However, the right ventricle was slightly but significantly larger in antibody-positive patients (26.3 +/- 4.2 versus 30.7 +/- 3.5 mm; p = 0.001). CONCLUSIONS: In the first years after the Ross procedure, we could not detect significant evidence of an association between anti-HLA class I antibodies and echocardiographic results of homograft function at rest in adults.

Defective production of interleukin-2 in patients with idiopathic Parkinson's disease.

The pathogenesis of Parkinson's disease (PD) is largely unknown. Recently, several studies have presented evidence of an immunological dysfunction in patients suffering from PD. We studied the immune responsiveness of patients with idiopathic PD (n = 20) by investigation of the ability of peripheral blood mononuclear cells to produce cytokines after mitogenic stimulation in a whole blood assay. A group of age-related healthy blood donors served as control (n = 19). Additionally, white blood count, leukocyte differentiation and lymphocyte subtyping were performed. PD patients had a significantly higher neutrophil count, but analysis of T-cell subsets showed no difference between the two groups. In peripheral blood, secretion of interleukin-2 (IL-2) after mitogenic stimulation was significantly diminished in the patients' group (p < 0.01), whereas values of IFN-alpha 2, IL-6, IFN-gamma and sIL-2R were comparable in both groups. IL-2 production correlated negatively with the mean annual dose of levodopa treatment and correlated significantly (p < 0.002) with amantadine uptake. Analysis of sex, age, duration of illness and other drug intake revealed no correlation with cytokine release. Our findings support the view that there is a selective abnormality in the immune repertoire of peripheral blood lymphocytes in patients suffering from PD, the reasons for which need to be explored.

Autologous concentrated platelet-rich plasma (cPRP) for local application in bone regeneration.

Platelets are rich in growth factors that may contribute to an accelerated tissue regeneration process. The therapeutic osteogenic effect of local platelet administration probably depends on the amount of growth factors delivered within. To improve platelet-derived factor preparations, the platelets have to be concentrated without loss of the granular growth factor load. An autologous procedure according to the Good Manufacture Practice (GMP) guidelines to prepare a high concentrate from platelet-rich plasma (cPRP) for clinical application in bone regeneration is presented. Platelet-rich plasma (PRP) was generated from the centrifugation supernatant of 250 ml anticoagulated autologous blood. The PRP was then further centrifuged at the day of operation to generate concentrated PRP (cPRP) in 8 ml plasma. The data from 34 patients (9 m, 25 f), ageing from 21 to 71 years (median 50.4) were evaluated. Both, the platelet counts as well as the TGF-beta1 concentrations in the cPRP were found to be 12-fold increased compared to the PRP values. Overall, a 17-fold increase in platelet concentrations was achieved compared to the patients' whole bloods and platelet counts correlated with increased levels of TGF-beta1 within the cPRP. These results give rise to quality control specifications for a safe and cost effective preparation of cPRP. The method has proven to be suitable for blood services to support the needs for treatment in oral and maxillofacial surgery.