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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.

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: 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.

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.

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 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.

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.

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.

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: 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.

Background: Chronic haemodialysis patients show various clinical signs of immunodeficiency and there is growing evidence that a dysregulated monocyte cytokine production is heavily involved in this deficiency. The production of monokines in vitro has been proposed to correlate closely with the in vivo immune status and to be of high clinical relevance in cuprophane haemodialysis. Even though it is well known that the biocompatibility of dialyser membranes has a significant impact on immune functions, little is known about the influence of the ultrafiltration flow rate (UFR). The aim of this study was to investigate the potential long-term effects of UFR on the production of interleukin-10 (IL-10), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6) in an intra-individual study design., Methods: In 11 patients previously treated with polysulphone haemodiafiltration, UFR was reduced from 40-46 ml/min to 24-28 ml/min, then to 7-10 ml/min before it was reinstated at 40-46 ml/min for periods of 4 weeks each. Monokine secretion into culture supernatants and mRNA expression (assessed using a novel Taqman PCR technique), were determined in a whole blood assay after lipopolysaccharide stimulation., Results: Reduction of UFR led to a significant increase in IL-10 secretion and mRNA expression (P=0.012, P=0.001). Conversely, a substantial (but not complete) decrease was observed when UFR returned to initial levels. In contrast, supernatant concentrations of IL-1beta (P=0.04) and IL-6 (P=0.003), and mRNA expression of both monokines (P<0.001, P<0.001) decreased significantly when UFR was reduced. Calculation of the IL-1beta/IL-10 ratio also revealed a decrease when UFR was reduced, with an increase again being observed when the initial degree of UFR was reinstated (P<0.001)., Conclusions: These results indicate a significant impact of UFR on the production of monokines at both the transcriptional and the protein level. We suggest that middle molecule removal has to be considered as a possible pathophysiological mechanism to explain our findings. Since monokine production in vitro was shown to be closely correlated with the in vivo immune status in patients on cuprophane haemodialysis, further investigations are necessary to clarify the impact of UFR on the immunocompetence of patients under polysulphone haemodiafiltration.

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.

Despite recent advances in DNA-based genotyping, the microcytotoxicity test is still broadly used for the determination of human leukocyte class I antigens in patients as well as organ donors and also for the detection of HLA antibodies. Excellent purity and viability of peripheral blood mononuclear cells (PBMC) are essential for reliable HLA typing results. Background staining and cell loss can contribute to impaired typing results or even cause misinterpretations. A novel isolation procedure using cell preparation tubes (CPT) with prefilled Ficoll was compared with the standard Ficoll gradient. We determined the recovery, purity, and viability of the PBMC after several periods of storage. Finally, the isolated cells were used for HLA class I typing, and background reactivities were scored. By using the CPT method, the recovery of PBMC was significantly higher than recovery with the standard technique (P