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Routine ABO blood group typing of apparently healthy individuals sporadically uncovers unexplained mixed-field reactions. Such blood group discrepancies can either result from a haematopoiesis-confined or body-wide dispersed chimerism or mosaicism. Taking the distinct clinical consequences of these four different possibilities into account, we explored the responsible cause in nine affected individuals. Genotype analyses revealed that more than three-quarters were chimaeras (two same-sex females, four same-sex males, one sex-mismatched male), while two were mosaics. Short tandem repeat analyses of buccal swab, hair root and nail DNA suggested a body-wide involvement in all instances. Moreover, genome-wide array analyses unveiled that in both mosaic cases the causative genetic defect was a unique copy-neutral loss of heterozygosity encompassing the entire long arm of chromosome 9. The practical transfusion- or transplantation-associated consequences of such incidental discoveries are well known and therefore easily manageable. Far less appreciated is the fact that such findings also call attention to potential problems that directly ensue from their specific genetic make-up. In case of chimerism, these are the appearance of seemingly implausible family relationships and pitfalls in forensic testing. In case of mosaicism, they concern with the necessity to delineate innocuous pre-existent or age-related from disease-predisposing and disease-indicating cell clones., (© 2024 The Author(s). British Journal of Haematology published by British Society for Haematology and John Wiley & Sons Ltd.)

BackgroundThe exact epidemiology of tick-borne encephalitis virus (TBEV) infections is unknown because many TBEV infections have an influenza-like or asymptomatic course. Surveillance data are based on patients with any (predominantly neurological) symptoms that prompted diagnostic testing. Infection- and vaccine-induced antibodies against TBEV can be distinguished using an NS1 IgG ELISA.AimIn a seroprevalence study we aimed to investigate TBEV antibody prevalence, incidences, manifestation indices and potential protection rates in a highly endemic district in south-western Germany.MethodsWe analysed 2,220 samples from healthy blood donors collected between May and September 2021. The reported number of TBEV infections was provided on a sub-district level by the local public health authorities. Blood samples were first screened using a TBEV IgG ELISA. In a second step, all positive samples were further analysed with a recently established NS1 IgG ELISA. The presence of specific antibodies against TBEV (excluding cross-reacting antibodies against other flaviviruses) was confirmed by testing screening-positive samples with a microneutralisation assay.ResultsOf 2,220 included samples, 1,257 (57%) tested positive by TBEV IgG ELISA and 125 tested positive for infection-induced TBEV NS1 antibodies, resulting in a TBEV NS1 IgG seroprevalence at 5.6% in our population. The yearly incidence based on the NS1 ELISA findings resulted in 283 cases per 100,000 inhabitants.ConclusionUsing the TBEV NS1 IgG assay, we confirmed a manifestation index of ca 2% and a high incidence of predominantly silent TBEV infections (> 250/100,000/year), which exceeds the incidence of notified cases (4.7/100,000/year) considerably.

Background and Objectives: This study aimed to describe motives as well as donation experiences and the intention to return for further donations of German whole blood donors who donated at the beginning of the COVID-19 pandemic., Materials and Methods: To describe motives and donor experiences, a retrospective survey was conducted among whole blood donors that had a donation appointment at the German Red Cross Blood Donation Service in the first 4 weeks of the pandemic. A donor questionnaire including 17 retrospective questions was sent to 7500 donors. Donor motivation and donor experiences were compared for different donor groups using chi-square statistics. Finally, in an ordinal logistic regression model predictors for the intention to return were identified., Results: More than half of the participating donors (56.9%) wanted to contribute to the fight against the pandemic by donating blood. Most of the donors were satisfied with their last donation experience and felt safe during the blood donor appointment. However, some donors would have liked more information on how to deal with the pandemic (20.3%). Intention to return for further donations was strongly associated with overall satisfaction (OR: 1.67, CI: 1.47-1.90) and the feeling of being safe during blood donation (OR: 1.33, CI: 1.05-1.68)., Conclusion: Donor satisfaction with the last donation was high and the vast majority of donors felt very safe. However, those donors who felt unsafe expressed a low intention to return and blood donation services should therefore carefully monitor donor satisfaction., (© 2021 The Authors. Vox Sanguinis published by John Wiley & Sons Ltd on behalf of International Society of Blood Transfusion.)

Regulatory CD4 + T cells (Treg) prevent tumor clearance by conventional T cells (Tconv) comprising a major obstacle of cancer immune-surveillance. Hitherto, the mechanisms of Treg repertoire formation in human cancers remain largely unclear. Here, we analyze Treg clonal origin in breast cancer patients using T-Cell Receptor and single-cell transcriptome sequencing. While Treg in peripheral blood and breast tumors are clonally distinct, Tconv clones, including tumor-antigen reactive effectors (Teff), are detected in both compartments. Tumor-infiltrating CD4 + cells accumulate into distinct transcriptome clusters, including early activated Tconv, uncommitted Teff, Th1 Teff, suppressive Treg and pro-tumorigenic Treg. Trajectory analysis suggests early activated Tconv differentiation either into Th1 Teff or into suppressive and pro-tumorigenic Treg. Importantly, Tconv, activated Tconv and Treg share highly-expanded clones contributing up to 65% of intratumoral Treg. Here we show that Treg in human breast cancer may considerably stem from antigen-experienced Tconv converting into secondary induced Treg through intratumoral activation.

Background: In the recent past, the discrepancy between blood supply and future demand may have been overestimated. As medical progress develops rapidly, it will be essential to monitor ongoing demographic changes in the donor population regularly and to re-evaluate retention and recruiting strategies. The aim of the current study was to compare first-time donor (FTD) characteristics and their return rates. We therefore compared whole blood (WB) donations in total and the annual donation frequencies in 2010 and in 2015/2016. Furthermore, we evaluated whether over the same observation period, medical reasons for deferral underwent a change (2010 vs. 2015)., Methods: The return probability of FTD within 12 months was analysed in 2010 and 2015 with respect to successful donation versus deferral and with regard to age. The total number of WB donations was investigated, and age distribution was compared between 2010, 2013 and 2016. WB donation frequencies were calculated with respect to age and gender in 2010 and 2016. In a second analysis, medical reasons for deferral were differentiated into 14 categories and a possible impact of time (2010 vs. 2015) on the respective percentage was studied., Results: We observed a significant decline of the FTD return rate from 42.5% to 38.8% in donors that successfully donated WB while the rate remained unchanged in deferred FTD. At the same time the mean FTD age decreased from 29.1 ± 11.6 to 28.5 ± 11.7 years in 2016. Analysis of total WB donations revealed an increase of all donations from donors ≥60 years, a constant percentage from donors <30 years but a declining proportion of donors aged 30-59 years from 2010 to 2013 to 2016. In parallel, annual mean WB donation frequencies decreased over time. Deferrals due to travel history increased significantly from 2010 to 2015 both in FTD and repeat donors., Conclusion: There is ongoing demographic change in our WB donor population. Our data prove a need for a re-evaluation of retention and recruitment strategies since previous marketing campaigns seem to have neglected the age group 30-59 years. This must be addressed in further studies as this age group will be highly relevant for assuring future blood supplies since donor recruitment from adolescents will be limited due to declining birth rates. Furthermore, deferral due to travel history is increasing significantly. Thus we will require further studies on the possible impact on donor retention.

Background/aims: The aim of this study was to compare donor return patterns of non-compensated and compensated German first-time donors to assess the effect of monetary reward on donor return., Methods: We conducted a retrospective analysis of a donor survey of 3,077 non-compensated and 738 compensated German first-time donors. Survey data were pooled and linked with blood donor return rates within the 1st, 2nd, and 3rd year. Logistic regression models were used to estimate differences in the probability of donor return between non-compensated and compensated donors., Results: In the first 2 years following the initial donation, compensated donors were more likely to return with the odds of giving at least one further donation 1.86 (1st year) and 1.32 (2nd year) times higher for compensated donors than for non-compensated donors. In the 3rd year, there were no significant differences in donor return., Conclusion: This report, which was based on two non-randomized donor samples, suggests that monetary compensation may increase the likelihood of donors returning in the first months after the initial donation. Monetary reward may therefore be used as a short-term strategy to recruit new donors. The long-term commitment, however, seems not to be affected by monetary reward, and complementary donor retention strategies are needed.

Background: The aim of this study was to describe donor satisfaction regarding different aspects of the new German blood donor questionnaire (BDQ) and to assess whether donor satisfaction is associated with the intention to return for further donations., Methods: A random number of 6,600 blood donors, donating at the German Red Cross Blood Service Baden-Wuerttemberg - Hessen, were asked to rate their satisfaction with four different aspects of the BDQ. Chi-square statistics was used to test for associations between satisfaction and the intention to return., Results: Most of the donors were satisfied with format and layout (72.7%) and the clarity of the questions (72.5%). However, only 39.5% of the donors were satisfied with the scope of the BDQ and 44.3% with the questions about sexual risk behavior. The lowest satisfaction seemed to be among experienced donors and among donors from small municipalities. Among experienced and very experienced donors, a significant association between the satisfaction with the different aspects and the intention to return became apparent., Conclusion: When considering the implementation of the BDQ, Blood Donor Services have to weigh up the advantages of increased deferral rates among donors with high-risk behavior against the potential drop-out of dissatisfied blood 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., (© 2011 The Author(s). Vox Sanguinis © 2011 International Society of Blood Transfusion.)

BACKGROUND: The aim of the study was to identify characteristics of lapsed donors 4 years after the initial donation as well as self-reported barriers to return for further blood donations. METHODS: A random number of 8,000 blood donors, donating for the German Red Cross Blood Service Baden-Württemberg - Hessen, were asked to fill in a self-administered questionnaire. The response rate was 38.5%. Donors were categorized as 'lapsed' if they had not donated within the last 24 months. The odds of being a lapsed donor were determined in a multivariate logistic regression. RESULTS: Multivariate analysis showed that lapsed donors were more likely to be female, between 26 and 33 years old, not employed, have moved, and were dissatisfied with the last donation experience. Furthermore, lapsed donors were less likely to have family members or friends who also donate blood. Medical reasons and having moved to another city were the most frequently named reasons preventing lapsed donors from continuing to donate blood. CONCLUSION: The importance of medical reasons and having moved was rated much higher than in previous studies. We conclude that barriers to return may vary considerably between countries and blood services. Therefore, donor surveys are required to guide reactivation campaigns.

BACKGROUND: The implementation of a new national German blood donor questionnaire was proposed to improve donor and recipient safety. METHODS: We compared deferral/exclusion rates of whole blood donors before (May 2010, n = 64,735) and after (May 2011, n = 71,687) the implementation of a new blood donor questionnaire. Considering seasonal variations, analysis was performed with respect to collection site (mobile vs. fixed), sex, donor status (first-time vs. repeat), age, and the frequencies of sexual risk behavior and other reasons for deferral. RESULTS: We observed a statistically significant increase (p < 0.001) of the overall deferral/exclusion rate from 6.2 to 8.1%, irrespective of type of collection site (fixed: from 6.0 to 8.5%; mobile: from 6.2 to 8.0%), sex (females: from 7.5 to 9.9%; males: from 5.1 to 6.6%), donor status (first-time donors: from 19.7 to 24.7%; repeat donors: from 4.6 to 6.3%) or age (18-29 years: from 9.1 to 11.7%; 60-71 years: from 5.1 to 6.6%). Confidential self-exclusion increased from 0.08 to 0.14% (p < 0.001). Besides risk behavior, various medical reasons could be identified that explain this increase. CONCLUSIONS: The new blood donor questionnaire resulted in an increased deferral/exclusion of all donor groups. Thus the impact on future blood supply must be considered carefully, and long-term studies and investigation of donor acceptance will be needed.

Kidney transplant recipients with the interleukin-6 (IL-6) GGG/GGG promoter (-597/-572/-174)genotype were shown to have a better long-term outcome. Further, the same (-597/-572/-174)genotype was found to be associated with less IL-6 production in healthy control subjects. To verify this observation in potential kidney transplant recipients, IL-6 production was analyzed in 85/142 hemodialysis patients. We could not confirm an impaired IL-6 secretion in carriers of the GGG/GGG (-597/-572/-174)genotype and propose a significantly lower IL-6 production in hemodialysis patients versus healthy control subjects to explain this. However, we suggest subsequent studies of IL-6 production in kidney allograft recipients to further elucidate the pathophysiological relevance of IL-6 for transplant outcome.

SUMMARY: BACKGROUND AND METHODS: As a source of hematopoietic stem cells, cord blood (CB) is an alternative to bone marrow or peripheral blood stem cells (PBSC). The Mannheim Cord Blood Bank has currently stored about 1,750 allogeneic CB units. Here we report our experiences and discuss future perspectives of CB banking. We analyzed CB units for nucleated cell (NC), mononucleated cell (MNC) and CD34+ cell count, volume, colony-forming units (CFU-GM) as well as ethnic background of the donor. Transplanted CB units were analyzed for patient and transplant characteristics and compared to stored CB units. RESULTS: Only 25% of all collected CB units met storage criteria. Main reasons for exclusion were: i) insufficient volume (57.7%), ii) delayed arrival at the processing site (19.2%) and iii) little cell count (7.2%). Up to now 36 CB units have been released for transplantation mainly to children (62%). Transplant indications were hematological diseases, immune deficiencies and metabolic diseases. Transplanted CB units showed significantly higher cell counts compared to stored units (NC: 12.5 vs. 7.2 x 10(8), MNC: 4.7 vs. 2.9 x 10(8), CD34+ cells: 3.3 vs. 1.8 x 10(6), mean; p < 0.001 each) and were found more often in ethnic minority groups (36 vs. 20%; p = 0.04). CONCLUSIONS: Even though cell count and volume are key parameters for the eligibility of CB units, our data indicate that the ethnic background of the donor also plays a major role. Collection and processing of CB should be optimized in order to gain maximum volume and cell counts.

Background: Interleukin-6 (IL-6) promoter polymorphisms at positions -597(G-->A), -572(G-->C) and -174(G-->C) were shown to have a clinical impact on different major diseases. At present PCR-SSP protocols for IL-6 -597/-572/-174haplotyping are elaborate and require large amounts of genomic DNA., Findings: We describe an improved typing technique requiring a decreased number of PCR-reactions and a reduced PCR-runtime due to optimized PCR-conditions., Conclusion: This enables a fast and efficient determination of IL-6 -597/-572/-174haplotypes in clinical diagnosis and further evaluation of IL-6 promoter polymorphisms in larger patient cohorts.

Background: White blood cell (WBC)-associated antibodies can lead to severe pulmonary transfusion reactions (transfusion-related acute lung injury [TRALI]). Investigation of a large number of blood donor samples using the standard granulocyte immunofluorescence test (GIFT) and granulocyte agglutination test (GAT) proved to be difficult to perform due to the time-consuming process and the large quantity of test cells required. This study describes the novel flow cytometric GIFT (Flow-GIFT) method for a rapid detection of granulocyte antibodies by flow cytometric analysis., Study Design and Methods: A total of 141 sera were analyzed for the presence of granulocyte antibodies that were previously associated with suspected TRALI. As test cells whole blood samples from human neutrophil antigen (HNA)-typed donors were isolated using cell sedimentation in a ficoll density gradient. WBCs were incubated with the respective serum and binding of antibodies to the test cells was detected using fluorescein isothiocyanate-conjugated anti-human antibody. Standard GIFT and GAT were performed as reference methods., Results: Seven sera containing anti-HNA-3a, CD16, and HLA Class I were negative in the standard GIFT and eight sera containing anti-HNA-2a, anti-CD16, and anti-HLA Class I were not detected in the GAT. The novel Flow-GIFT was able to detect all granulocyte antibodies, which were only detectable in a combination of standard GIFT and GAT. In serial dilution tests, the Flow-GIFT detected the antibodies at higher dilutions than the reference methods GIFT and GAT., Conclusion: The Flow-GIFT method permits rapid detection of granulocyte antibodies requiring fewer donor test cells. This method is ideal for automation and will potentially open the way for screening of granulocyte antibodies in a large donor population.

Background and Aim of the Study: Rejection is a plausible cause of failure of allograft valves, but has not been demonstrated unequivocally in humans. A cross-sectional study has been conducted on the frequency of anti-human leukocyte antigen (HLA) antibodies in order to identify any correlation with allograft function in adult patients, following the Ross procedure., Methods: Anti-HLA antibodies were determined during regular follow up (median 1.1 years postoperatively) in a random sample of 197 patients (151 males, 46 females; mean age 46 +/- 13 years). Panel-reactive antibodies (PRA) were determined by cytotoxicity testing; anti-HLA class 2 antibodies (HLA2AB) were determined by ELISA in a subgroup of 94 patients. Echocardiographic examinations were performed during the first visit and at a median of 6.8 years postoperatively., Results: The prevalence of positive antibody tests was 47% for PRA and 52% for HLA2AB. A slight deterioration of allograft valve function occurred between the two examinations (median maximal pressure gradient increased from 9 mmHg to 13 mmHg, p < 0.001; median degree of regurgitation increased from zero to trivial, p = 0.020). The degree of regurgitation was slightly, but significantly, higher in PRA-positive patients at both examinations (p = 0.008 and p = 0.038). No relationship was observed between PRA status and pressure gradients, nor between HLA2AB status and allograft valve function. Neither were any other risk factors for allograft valve deterioration identified., Conclusion: Subtle, clinically irrelevant and temporally stable differences with regard to regurgitation across the allograft were observed between PRA-positive and -negative patients. These findings neither proved nor disproved rejection, but rather suggested that a slow deterioration of allograft valve function was complex, and most likely multifactorial.

Previous investigations have shown the immunosuppressive activity of the immunophilin-binding macrolide Sanglifehrin A (SFA). In adults, SFA also exerts anti-inflammatory properties in lipopolysaccharide (LPS)-stimulated whole blood cultures. It was the aim of this study to investigate whether the unique properties of SFA are also present in the neonatal immune system, as neonates are susceptible to serious infection due to an immaturity of immune responses. We used a whole blood assay to investigate the impact of SFA on T-cell proliferation and secretion of T-cell cytokines upon Anti-CD3/Anti-CD28 costimulation. In addition, interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha) secretion was assessed in whole blood monocytes after LPS stimulation. Furthermore, the influence of SFA on LPS-induced signal transduction pathways, specifically the activity of p42/44, p38 and Ap-1, was assessed in neonatal PBMCs. Neonatal cord blood lymphocytes were found to have a diminished IL-4, IL-6, TNF-alpha and interferon-gamma (IFN-gamma) production upon Anti-CD3/Anti-CD28 costimulation compared to adult whole blood lymphocytes. In contrast, no significant differences were noted for either IL-2 production or proliferation of CD4+ cells. Upon addition of 1000nM SFA to neonatal whole blood cultures, a significant inhibition of both, T-cell cytokine secretion and proliferation was demonstrated. In line with data from adult whole blood cultures, SFA proved to be a strong inhibitor of LPS-induced expression of IL-6 and TNF-alpha in the neonatal whole blood system. In signal transduction studies of the LPS pathway, a potent inhibition of the protein kinase p42/44 was demonstrated. SFA was also shown to block nuclear translocation of the transcription factor Ap-1. SFA was proved to have inhibitory effects on innate and acquired immune response of neonatal whole blood cells. The protein kinase p42/44 and the transcription factor Ap-1 were demonstrated to be potential key molecules for the anti-inflammatory effect of SFA.

Sirolimus (SRL) has become an important alternative to calcineurin inhibitors due to its unique mechanism of action. Since rejection and poor graft outcome are still frequent problems despite therapeutic-range blood concentrations, pharmacodynamic measurements of its immunosuppressive effects would be of great clinical value to optimize treatment in individual patients. We performed a human whole blood assay using real time cytokine RT-PCR for the pharmacodynamic assessment of SRL. IL-2, IL-4 and IL-6 mRNA levels were quantitatively determined upon T-cell-specific stimulation in healthy individuals (n=11; in vitro) and in kidney-transplant patients (n=3; ex vivo). Furthermore, IL-2 protein secretion and T-cell proliferation was measured. After 24h incubation we observed a stronger suppression of IL-2 and IL-4 mRNA expression upon SRL addition (p<0.005; p<0.005) versus 4h (p<0.05; p<0.05). SRL effects displayed a remarkable interindividual variation, which proved to be independent of the concentration applied. Notably, 3/11 and 2/11 individuals had unaffected IL-2 and IL-4 mRNA expression after 4h incubation with SRL, respectively. In contrast, a general suppression of IL-2 protein secretion and T-cell proliferation was induced. Analysis of kidney-transplant patients verified interindividual variation and proved comparability of in vitro and ex vivo effects. We describe an individual degree of SRL-sensitivity that may correlate with clinical efficacy. Rather than analysis of one single peak, we suggest determination of two absolute cytokine mRNA peak levels for the pharmacodynamic assessment of SRL. However, prospective clinical studies are necessary to determine whether individual degrees of SRL-sensitivity correlate with clinical outcome.

Background: It was the aim of this study to evaluate the effects of the well-known immunosuppressive drugs ciclosporin A (CsA), tacrolimus and sirolimus on the intracytoplasmic cytokine expression of neonatal immune cells., Methods: Immunosuppressive drugs were added to whole blood cultures of neonatal cord blood samples (n = 17) and peripheral blood samples of adults (n = 17) in vitro prior to stimulation of lymphocytes with phorbol 12-myristate 13-acetate (PMA)/ionomycin or monocytes., Results: Upon exposure to ciclosporin A (500 ng/mL) or tacrolimus (25 ng/mL) the number of cytokine expressing T cells was almost completely blocked in neonatal T cells while sirolimus (10 ng/mL) only inhibited intracytoplasmatic tumour necrosis factor alpha (TNF-alpha) expression (mean% positive cells; 4.0 +/- 2.1% vs. 1.09 +/- 0.6%, p = 0.003), but mildly stimulated the intracellular expression of interleukin (IL)-2 (24.4 +/- 6.5% vs. 28.1 +/- 7.1%, p = 0.041). In cord blood lymphocytes, the inhibitory effect of ciclosporin A and tacrolimus was dose-dependent (e.g. IL-2: control, 12.3 +/- 5.33%, ciclosporin A 5 ng/mL, 10.1 +/- 5.5%; 50 ng/mL, 7.1 +/- 4.7%; 500 ng/mL, 1.2 +/- 0.3%; tacrolimus 0.25 ng/mL, 9.3 +/- 4.9%; 2.5 ng/mL, 6.1 +/- 3.3%; 25 ng/mL, 1.0 +/- 0.6%), while the function of adult lymphocytes was only impaired at high doses of both compounds. In contrast, the number of cytokine expressing monocytes was not influenced by ciclosporin A and tacrolimus except for a minor decrease of TNF-alpha producing neonatal monocytes after addition of tacrolimus (17.9% vs. 13.9%, p = 0.031). Interestingly, sirolimus was shown to inhibit intracellular IL-6 production in adults (63.1 +/- 12.7% vs. 52.0 +/- 16.0%, p = 0.005), but in neonatal monocytes intracellular IL-6 expression was stimulated (53.5 +/- 22.0% vs. 64.7 +/- 19.1%, p = 0.041)., Conclusions: The potent dose-dependent inhibitory effect of ciclosporin A and tacrolimus in cord blood lymphocytes provides the basis for further studies on functional immaturity of the neonatal immune system and for future strategies to optimize umbilical cord blood transplantion. Sirolimus was demonstrated to have a distinct effect on neonatal immune cells as shown by increased expression of IL-2 in lymphocytes and IL-6 in monocytes, while only lymphocytic TNF-alpha expression was inhibited.

Objective: To analyse the levels of interleukin-6 (IL-6) in the synovial fluids and sera of patients with osteoarthritis (OA) and to identify the IL-6-secreting cells., Methods: Serum, synovial fluid, synovial tissue, and articular cartilage samples were collected from 49 OA patients with end-stage knee or hip OA who underwent joint replacement surgery. Serum and synovial fluid levels of IL-6 were measured by enzyme-linked immunosorbent assay (ELISA) and IL-6-secreting cells were identified by immunohistochemistry., Results: Eight out of 49 patients (16%) exhibited elevated IL-6 levels in the synovial fluids, averaging at 2022+/-526 pg/mL, while the levels in the rest of the patients averaged at 132+/-19 pg/mL. The sera levels of all patients were comparable in the 10 pg/mL range. Immunohistochemical analyses revealed plasma cells in the synovial lining of the high producers as the source of IL-6., Conclusions: Synovial fluid IL-6 levels may help to classify OA patients and may point to a subgroup with a particular impact from their immune system.

Interleukin (IL)-6 is a pleiotropic cytokine, produced by different cells. There is accumulating evidence that IL-6 promoter polymorphisms impact substantially on various diseases and we identified kidney transplant recipients carrying the IL-6 GGG/GGG (-597/-572/-174)genotype to have superior graft survival. To prove a functional impact on gene expression, we analysed systematically IL-6 production in healthy individuals with respect to the IL-6 (-597/-572/-174)genotype. IL-6 was determined in 100 healthy blood donors at protein and mRNA levels upon specific stimulation in monocytes and T lymphocytes under whole blood conditions. GGG/GGG individuals showed a lower IL-6 secretion upon lipopolysaccharide (LPS)-stimulation versus all others (P = 0.039). This link was even stronger when (-597) and (-174)GG genotypes were reanalysed separately (P = 0.008, P = 0.017). However, we found neither a difference at the mRNA level or percentage of CD14(+) cells nor after T cell stimulation. We found evidence for the IL-6 (-597/-572/-174)genotype to affect IL-6 synthesis, i.e. lower levels of IL-6 protein upon LPS-stimulation in GGG/GGG individuals. Further studies are needed in kidney transplant recipients to investigate the potential link between the GGG/GGG genotype and graft survival. In line with this, determination of the genetic risk profiles might be promising to improve the transplant outcome in the individual patient.

The novel immunosuppressant Sanglifehrin A (SFA) is an immunophilin-binding metabolite with a yet unidentified mechanism of action. Several reports demonstrated the effects of SFA on proliferation and cytokine production of purified T cells with in part different results. However, less is known about the impact of SFA on the regulation of innate immune responses. We used a whole blood assay to investigate the impact of SFA on monocyte responses and T-lymphocyte activity/proliferation upon lipopolysaccharide (LPS) stimulation and anti-CD3/anti-CD28 costimulation, respectively. SFA was found to inhibit interleukin (IL)-2 protein expression of T lymphocytes. Whereas IL-2 mRNA expression was significantly reduced after 4 h of costimulation, the mRNA expression of IL-4 and IL-6 but not tumour necrosis factor (TNF)-alpha was inhibited by SFA both after 4 and 24 h of costimulation. The production of IL-2 and IL-6 protein in T lymphocytes was even strongly affected by SFA than the mRNA expression of the respective cytokine. Unlike other immunophilin-binding immunosuppressants, SFA also inhibited LPS-induced IL-6 and TNF-alpha mRNA and protein expression. At the single cell level, SFA was demonstrated to block the intracellular production of IL-6 in CD14+ monocytes but not the expression of other proinflammatory cytokines such as IL-8 and TNF-alpha. On the basis of these data, we propose that SFA may have a significant effect on the initiation and direction of immune responses. Considering the pleiotropic role of bioactive IL-6 production at the interface of innate and acquired immunity in a variety of disease conditions, it was found that these novel aspects of the unique immunosuppressive action could strongly impact on future clinical application of SFA.

The functional differentiation of immune cells at early age plays a central role in immune physiology, e.g. for the sufficient eradication of pathogens. However, imbalances in effector cell responses may also have an impact in the pathophysiology of childhood diseases such as atopy and autoimmune disorders. As information on immune cell responses in infancy and early childhood is scarce, we conducted an observational, cross-sectional study in healthy newborns (n = 18), infants and young children (n = 54) aged 1-96 months and adult controls (n = 19) to assess cytokine mRNA and protein expression upon phorbol 12-myristate 13-actate/ionomycin stimulation and LPS-induced IL-12 expression in monocytes. The intracellular expression of interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha (R = 0.748, P < 0.0001; R = 0.784, P < 0.0001, respectively) and interleukin (IL)-2 protein expression (R = 0.384, P = 0.008) was demonstrated to increase progressively with age. While a correlation between IL-4 protein expression and age was noted (R = 0.342, P = 0.007), the levels of IL-5 and IL-10 protein expression tended to be regulated on an individual basis during infancy and early childhood. An age correlation was also observed for intracellular IL-12 expression (R = 0.331, P = 0.009) in monocytes. These findings are valuable for further assessment of normal variations and maturation processes in immune cell responses and for the clinical-therapeutic monitoring of immunological status in various childhood diseases.

Objective: To compare objective and subjective parameters of surgical stress following laparoscopic and open adnexectomy in patients older than 60 years old., Study Design: Twenty patients with a benign ovarian tumour were prospectively randomized to undergo adnexectomy by a laparoscopic or an open surgical procedure. Measurements included C-reactive protein; interleukin-6 before, during, and after surgery; intensity and duration of postoperative pain; and complications and recovery period. Statistical analysis consisted of analysis of variance and a Mann-Whitney U test., Results: The levels of the interleukin-6 and C-reactive protein differed significantly between the 2 operative procedures (P = .013) in favor of the laparoscopic approach. The laparoscopic approach was associated with a reduction in operative morbidity, postoperative pain, analgesic requirement, and recovery period., Conclusions: Minimally invasive surgery is of particular benefit to elderly patients if there is a plan in place for appropriate staging and treatment by laparotomy for malignancy. It should be the first choice and may help to reduce postoperative complications.

Interleukin-6 (IL-6) is required for the clearance of bacteria in pneumococcal pneumonia. The abundance of endogenous IL-6 production on infectious stimuli is associated with genotypic differences in the -174 promoter region of IL-6 (-174 G-->C), showing increased IL-6 levels in patients carrying the GG genotype. One hundred patients with culturally proven pneumococcal disease were analyzed for distribution of the G-/C-alleles in the IL-6 -174 promoter region in comparison to 50 age-matched controls. Extrapulmonary pneumococcal dissemination, including septic metastasis, endocardial and meningeal infection, was used as parameter for impaired clearance of the bacteria. No significant differences in the allele distribution were observed between patients and controls. Within the patient group, the interleukin-6 GG homozygous carriers were less likely to develop extrapulmonary pneumococcal infection (10.3% versus 30.9%; OR 0.26, 95% CI 0.07-0.94, p=0.04). The IL-6 GG genotype, encoding for enhanced IL-6 secretion on bacterial stimuli, reduces the risk of bacterial spread to extrapulmonary sites in pneumococcal infection, possibly due to a more effective clearance of the pathogen from the blood and the respiratory tract.

The onset and frequency of Epstein-Barr virus (EBV) reactivation after kidney transplantation are unknown. By use of quantitative real-time polymerase chain reaction measurements, evidence of early EBV reactivation, occurring within the first week after the initiation of immunosuppressive therapy (median, 3 days), was observed in 13 of 23 patients, of whom 10 subsequently developed rejection episodes after 2-45 days (median, 5 days). By contrast, rejection was only diagnosed in 1 of 10 patients who did not show signs of viral reactivation. We suggest that EBV reactivation may induce a T cell response that, through the phenomenon of allo-cross-reactivity, could play a critical role in graft rejection.

Recently, we demonstrated an association of the IL-6 promoter polymorphism at position -174 (G-->C) with kidney allograft survival whereby carriers of the -174GG genotype were identified as having superior graft survival. As two additional polymorphisms were discovered in the neighborhood at positions -572 (G-->C) and -597 (G-->A), respectively, and as functional studies revealed a cooperative impact of all three on the IL-6 gene transcription, we investigated whether there is a combined effect on kidney transplant outcome. We determined IL-6 promoter haplotypes -597 (G-->C)/-572 (G-->A)/-174 (G-->C)(-597/-572/-174haplotype) using a PCR system with sequence-specific primers in 158 patients after primary cadaveric kidney transplantation. We here show that the -597 and -174 polymorphism are in tight-linkage disequilibrium and that homozygous carriers of the GGG-597/-572/-174 haplotype (GGG/GGG genotype) have superior 3-year graft survival rates compared with the 8.0-fold increased risk of premature graft loss in all other patients. Interestingly, patients carrying the GGG/GCG genotype had the lowest allograft survival rate. Thus determination of the combined -597/-572/-174 genotype allows for further differentiation of -174GG patients into subgroups and consequently for a more accurate identification of patients at risk. Our results indicate that the three polymorphisms act in a cooperative fashion and we provide evidence for an exceptional clinical impact of the IL-6-597/-572/-174 genotype on the success of kidney transplantation.

Background: To optimize immunosuppressive treatment in individual transplant patients, functional measurements of the effects of tacrolimus (FK 506) are of clinical importance. Previous investigations have demonstrated the occurrence of tacrolimus-resistant production of interleukin-2 (IL-2) in vitro, which may explain in part why rejection episodes are still a frequent problem despite attainment of therapeutic blood concentrations and HLA matching. However, an adequate surrogate marker to define the tacrolimus response in individual patients has not been established., Methods: We investigated the immunosuppressive effects of tacrolimus on anti-CD3/anti-CD28 T-cell costimulation in a human whole-blood assay, analyzing T-cell proliferation, activation marker expression (CD25, CD69), IL-2 protein expression, and cytokine mRNA expression in vitro (n = 11 healthy individuals). We also quantified IL-2 mRNA expression in patients undergoing tacrolimus (n = 4) or cyclosporin A (CsA; n = 4) monotherapy before ex vivo living-donor kidney transplantation., Results: T-cell proliferation; CD25, CD69, and IL-2 concentrations; and IL-4 mRNA were significantly decreased in vitro. In contrast, cytokine mRNA profiles revealed variable tacrolimus sensitivity. Whole-blood samples from 3 of 11 healthy individuals demonstrated marked suppression of IL-2 mRNA expression (>50%) when tacrolimus was administered in vitro. When CsA was added to whole-blood cultures, the influence on IL-2 mRNA expression was comparable to that of tacrolimus in 9 of 11 individuals. Two individuals responded conversely, indicating that differences in the in vitro response to tacrolimus and CsA among individuals may be attributable to potential heterogeneity in the involvement of the CD28 pathway. Kinetic profiles of IL-2 mRNA expression also revealed individually distinct degrees of calcineurin inhibitor sensitivity in patients undergoing tacrolimus or CsA monotherapy before living-donor kidney transplantation., Conclusions: Our results suggest an individual degree of calcineurin inhibitor sensitivity of activated whole-blood lymphocytes based on IL-2 mRNA expression. Our approach is potentially valuable for identifying transplant patients in whom IL-2 mRNA expression is unaffected or even enhanced after initiation of immunosuppressive therapy. Such individuals may be less sensitive to the immunosuppressive agent and therefore at increased risk of transplant rejection. Prospective studies are necessary to determine the correlation of IL-2 mRNA expression with the clinical risk of transplant rejection.

Background and Aim of the Study: Rejection is thought to contribute to the degeneration of valved homografts. A novel cryopreserved decellularized homograft valve (SynerGraft; CryoLife, Inc.) offers the unique opportunity to gain new insight into the immunology of homograft implantation and its significance for valve function., Methods: Twenty-four patients (group I; mean age 37 +/- 11 years) underwent implantation of a pulmonary SynerGraft and were examined at one and six months postoperatively; 22 patients (group II; mean age 41 +/- 17 years) with conventional homografts served as controls. Temperature, C-reactive protein (CRP) levels and white blood cell count (WBC) were studied perioperatively. Follow up included echocardiography and anti-human leukocyte antigen (HLA) class I antibody determination., Results: Significantly lower temperatures were measured in group I (p = 0.019). CRP level and WBC each increased postoperatively, but did not differ between groups. During follow up, none of the SynerGraft patients became positive for anti-HLA antibodies, compared with 66% of controls (p = 0.011). Homograft diameter and valve orifice area were decreased significantly at one month after surgery in groups I and II (25 +/- 1 versus 18 +/- 3 mm; 25 +/- 1 versus 19 +/- 2 mm, respectively; p <0.001 both groups). Transvalvular pressure gradients significantly increased during follow up., Conclusion: Implantation of the SynerGraft pulmonary homograft appeared safe, and though evidence was found of a reduced immunologic response after SynerGraft implantation this (unexpectedly) did not translate into any hemodynamic advantage. Hence, factors other than rejection appear as the main contributions to the observed functional changes.

Marek's disease virus (MDV) causes a common lymphomatous and neuropathic disease in domestic chickens and, less commonly, turkeys and quail. It is a member of the alpha-herpesviruses and until now was considered to be strongly cell associated. In 1991, MDV was suggested to be the causative infectious agent of multiple sclerosis (MS) in humans. In a previous study, we investigated the leukocytes of 107 well-defined MS patients for the presence of MDV DNA but were unable to confirm a role for MDV in the pathogenesis of MS. A recent report (S. Laurent, E. Esnault, G. Dambrine, A. Goudeau, D. Choudat, and D. Rasschaert, J. Gen. Virol. 82:233-240, 2001) described the detection of MDV DNA in 20% of 202 human serum samples, regardless of whether the individuals were exposed to poultry. The detection of MDV DNA in chicken serum samples was reported as well. The aim of the present study was to investigate whether we can confirm the presence of MDV DNA in chickens and humans if we use plasma as the source for nucleic acid isolation. Leukocytes and plasma specimens from 16 chickens experimentally infected with MDV serotype 1 and plasma specimens from 300 volunteer blood donors were tested for MDV DNA by two different TaqMan PCR assays. MDV DNA was repeatedly found in the leukocytes as well as in the plasma specimens of all 16 animals. All human samples analyzed, however, tested negative by both assays. Accordingly, Marek's disease in chickens can be diagnosed by detection of MDV DNA in plasma as well as in leukocytes. Once again, we found no evidence for the spread of MDV to humans.

The quantitative analysis of cyclosporin A (CsA) effects might be helpful for optimizing immunosuppressive treatment after allogeneic organ transplantation in individual patients, as rejection can occur despite the existence of CsA blood levels within therapeutic ranges. Previous investigations found that costimulation of the CD28 pathway generally mediates CsA-resistant proliferation of T cell receptor (TCR)-activated T lymphocytes. However, here we describe considerable interindividual variation regarding the immunosuppressive effects of CsA (1000 microg/L) on anti-CD3/CD28 T cell costimulation in a human whole blood assay. In the in vitro study, we found a significant reduction of T cell proliferation, activation marker expression (CD25, CD69) on the T cell surface, and interleukin-2 (IL-2) protein expression in whole blood samples of all healthy subjects (n = 11). However, the investigation of cytokine mRNA profiles revealed variable results of in vitro CsA sensitivity. Whole blood samples of 3 of 11 healthy individuals demonstrated a marked suppression of IL-2 mRNA expression (>50%) and a partial inhibition of IL-4, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) mRNA expression on addition of CsA. In contrast, the remaining 8 healthy individuals had cytokine mRNA expression levels that were unaffected or even increased when CsA was administered in vitro. In patients undergoing CsA monotherapy (ex vivo study, n = 9), we found a significant suppression of IL-2 mRNA levels in 4 of 9 patients ex vivo. Thus, we cannot confirm a universal CsA resistance of T cells on anti-CD3/CD28 costimulation. Instead, our results suggest an individual degree of CsA sensitivity that might be more consistent with clinical experience. Prospective studies are necessary to determine if individual degrees of CsA sensitivity correlate with clinical events and are associated with a low or high risk of transplant rejection.

Background: Because cyclosporin A (CsA) and glucocorticoids inhibit the production of interleukin-2 (IL-2) and other cytokines, quantitative analysis of cytokine mRNA might constitute a pharmacodynamic measure for immunosuppressive drug effects. We investigated whether immunosuppressive drugs influence cytokine mRNA expression kinetics during T-cell costimulation., Methods: We used a human whole blood assay to determine basal (unstimulated) IL-2, IL-4, and tumor necrosis factor-alpha (TNF-alpha) mRNA concentrations and expression kinetics after anti-CD3/anti-CD28 monoclonal antibody costimulation in kidney transplant recipients undergoing CsA-based immunosuppressive triple therapy and in healthy controls (ex vivo study I). The effect of CsA on IL-2 mRNA expression kinetics was also determined ex vivo in patients undergoing CsA monotherapy (ex vivo study II) and after in vitro addition of CsA., Results: In ex vivo study I, basal TNF-alpha mRNA but not IL-2 and IL-4 mRNA was decreased in kidney transplant patients. We observed shifts in peak IL-2 and IL-4 (from 8 to 24 h) and TNF-alpha (from 4 to 8 h of costimulation) mRNA expression in kidney transplant patients after T-cell costimulation. In patients undergoing CsA monotherapy (ex vivo study II), the inhibitory effect of CsA was detectable as an individually delayed increase in IL-2 mRNA during costimulation. In vitro addition of CsA also induced a dose-independent displacement of IL-2 mRNA expression kinetics (i.e., a delay)., Conclusions: A delayed increase in cytokine mRNA expression during T-cell costimulation may represent a sensitive effect of immunosuppression. The single analysis of one absolute or peak mRNA value could be misleading. For prospective studies involving measurement of cytokine mRNA, we therefore suggest the parameter "area of cytokine mRNA expression over time", which should include absolute cytokine mRNA values at two different time points of mRNA kinetics.

Background: Th1-dependent effector mechanisms may be responsible for allograft rejection. Recently, interleukin-6 (IL-6) has been shown to antagonize CD4+ T cells to effector Th2 cells and, in the process, differentiate them into Th1 cells., Methods: To assess the role of IL-6 in long-term allograft survival, 158 patients after first cadaveric kidney transplantation were analyzed for the biallelic -174G-->C promoter polymorphism of the IL-6 gene., Results: Carriers of the -174C-allele (genotype GC/CC) had an inferior three-year graft survival (71/104 = 68.3%; P = 0.0059) with a 3.7-fold increased relative risk of graft loss compared to carriers of the -174GG-genotype (48/54 = 88.9%). The -174GC/CC-genotype retained its negative impact on graft survival when other established prognostic factors and further cytokine polymorphisms (-308TNF-alpha, TGF-beta1 codon 10 & 25, -592/-819/-1082IL-10 and +874IFN-gamma) were considered simultaneously., Conclusions: Since the clinical impact on transplant outcome seems as important as matching for histocompatibility antigens, genotyping of the IL-6 -174polymorphism may offer a new method for identifying patients at increased risk of allograft loss.

Background and Objectives: Inflammatory cytokines in platelet concentrates (PC) may cause side-effects such as febrile non-haemolytic transfusion reactions. The maximum white blood cell (WBC) content tolerable to avoid the accumulation of cytokines, and whether these cytokines originate from degranulating leucocytes or de novo synthesis during storage, had not been investigated prior to this study., Material and Methods: We investigated the secretion of interleukin (IL)-1beta, IL-2, IL-6, IL-8, tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) and quantified the appropriate expression of corresponding mRNA in PC with regard to different levels of WBC contamination and storage times. In addition we tested the viability of WBCs during PC storage (by staining with 7-aminoactinomycin D) and their ability to perform de novo cytokine synthesis (by using superantigen stimulation)., Results: We detected a statistically significant increase of IL-1beta, IL-6, IL-8 and TNF-alpha in PC with > or = 108 WBCs. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) showed increasing mRNA expression of the respective cytokines depending on the number of WBC present. On day 5 of storage, WBC viability was > 80% and the leucocytes were still able to produce cytokines de novo., Conclusions: These data show clear evidence for de novo synthesis of cytokines in PC. The cytokine pattern supports the hypothesis that activated monocytes are responsible for this cytokine synthesis. PC with a WBC contamination of > or = 108 contain inflammatory mediators in clinically relevant concentrations.

Background: The introduction of universal WBC filtration of RBCs prior to storage is currently under consideration in many countries, as it is thought to minimize the incidence of transfusion-associated adverse effects. Centrifugation of blood containers with newly developed soft-shell WBC filters is more convenient, and so of great interest., Study Design and Methods: Two different quadruple blood pack systems with integrated soft-shell WBC filters were compared (Sepacell OptiPure RC, Baxter Biotech, vs. LCR 5, Maco Pharma). Buffy coat-depleted RBC units were investigated from whole-blood donations that were held for 2 to 3 hours before centrifugation and subsequent filtration at 22 degrees C (Group 1, OptiPure RC, 450 mL; Group 2, LCR 5, 450 mL; Group 3, OptiPure RC, 500 mL; Group 4, LCR 5, 500, mL, n = 12 per group). Filtration performance was analyzed, and the impact of WBC filtration on hemolysis rate, Hb content, pH, supernatant potassium, ATP, and 2,3 DPG was investigated weekly during storage for 42 days., Results: Filtration reduced the WBC count by 4.4 to 5.1 log. Mean +/- SD Hb content was 44.7 +/- 3.0, 41.2 +/- 3.3, 53.1 +/- 5.0, and 51.5 +/- 6.3 g per unit, respectively, with a corresponding mean RBC recovery after filtration of 71.0 +/- 3.0, 68.3 +/- 3.3, 76.6 +/- 1.7, and 68.9 +/- 4.5 percent. WBC filtration resulted in a significant reduction of Hct (0.10-0.14) in all four groups. Investigation of all RBC storage variables revealed acceptable values throughout the storage for 42 days., Conclusion: WBC filtration with two newly developed soft-shell filters showed acceptable WBC-reduction efficacy without any difference between filter types in buffy coat-depleted RBCs from 450- and 500-mL whole-blood donations. However, the application of both filters resulted in an unacceptably low RBC recovery after filtration, which was particularly evident with the LCR5 filter. Our findings raise concern that WBC reduction with these filters may result in the production of RBCs with an inappropriately low Hb concentration.