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.

Elevated IL-6 levels in the synovial fluid of osteoarthritis patients stem from plasma cells.

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.

The impact of interleukin-6 promoter -597/-572/-174genotype on interleukin-6 production after lipopolysaccharide stimulation.

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.

Immunosuppressive activity of the immunophilin-binding drug Sanglifehrin A in human whole blood: potent inhibition of interleukin-6 produced by lymphocytes and monocytes.

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.

Cytokine responses correlate differentially with age in infancy and early childhood.

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.

Evidence for de novo synthesis of cytokines and chemokines in platelet concentrates.

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.

A 5-year follow-up on antibody response after diphtheria and tetanus vaccination in hemodialysis patients.

Chronic renal failure is associated with a T-cell-dependent immune defect. In the past, various studies have focused on the insufficient immune response to vaccination of hemodialysis patients. An impaired vaccination response rate has been reported for vaccines against hepatitis B, influenza, tetanus, diphtheria, and others. However, no data exist on the long-term success of vaccination against tetanus and diphtheria in these patients. The aim of the present study is to investigate seroresponse to tetanus and diphtheria vaccination over a 5-year period. Antibody levels were determined by enzyme immunoassay. Antidiphtheria antibody levels of 31 hemodialysis patients were determined 5 years after vaccination. After 5 years, 10 of 31 patients (32%) had a protective antibody level against diphtheria (>/=0.1 IU/mL), whereas 12 months after vaccination, 26 of 71 patients (37%) were protected. Also, mean antibody levels significantly decreased. Furthermore, antitetanus antibody levels of 21 patients simultaneously vaccinated against tetanus and diphtheria were investigated. After 5 years, 15 of 21 patients (71%) were protected compared with 46 of 71 patients (65%) in the hemodialysis collective studied after 12 months. In the interval between 1 and 5 years after vaccination, significantly more patients in the initial nonresponder group had died than in the responder group; therefore, the overall protection rate observed in vaccinated patients increased. Our results provide evidence that during a 5-year period, antibody persistence against tetanus toxoid is better than that against diphtheria toxoid. Therefore, detection of individual antibody concentrations may be indicated to decide on revaccination.

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.

Production of monokines in patients under polysulphone haemodiafiltration is influenced by the ultrafiltration flow rate.

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.

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.

Prospective evaluation of the systemic immune response following abdominal, vaginal, and laparoscopically assisted vaginal hysterectomy.

BACKGROUND: Alterations in serum levels of cytokine interleukin-6 (IL-6) and acute-phase protein C-reactive protein (CRP) correlate directly with extent of tissue damage and inflammatory reaction. We therefore prospectively compared the postoperative levels of IL-6 and CRP following abdominal (AH), vaginal (VH), and laparoscopically assisted vaginal hysterectomy (LAVH). METHODS: A total of 29 patients were included in the study (10 VH, 10 LAVH, 9 AH). Nine blood samples were taken from each patient at various time points before, during, and after surgery. CRP and IL-6 were measured under standardized conditions using ELISA and turbidometry. RESULTS: Preoperative levels of IL-6 and CRP were low in all three patient groups. There was a significant increase in the IL-6 level in patients undergoing AH at the time of peritoneal closure that reached a maximum 2 h postoperatively and remained significantly elevated for 12 h postoperatively when compared to the IL-6 levels of patients undergoing VH or LAVH (p < 0.05). The levels of the IL-6 time courses differed significantly among the three operative procedures (p = 0.013). In contrast, the levels of the CRP time courses did not differ significantly (p = 0.066); however, CRP expression was elevated 36 h postoperatively in patients undergoing AH, as compared with those undergoing VH. CONCLUSION: Elevated IL-6 levels subsequent to AH may reflect significantly greater tissue damage in these patients than in patients who undergo VH or LAVH. LAVH should therefore be considered in cases that cannot be managed by the vaginal route alone.

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.

Transfusion of platelet concentrates from pooled buffy-coats: comparison of bedside vs. prestorage leukofiltration.

Prestorage leucocyte filtration of platelet concentrates (PC) has been considered to be superior to bedside filtration with regard to the incidence of nonhaemolytic transfusion reactions (NHTR) or HLA-alloimmunization. It is currently a matter of debate whether prestorage leucocyte filtration has an impact on the storability of platelets and on the transfusion results of PC. In a clinical retrospective study we investigated the transfusion results of PC from pooled buffy-coats (PC-BC) in haematological patients without known refractoriness to platelets, and compared bedside filtration (n = 228/36 patients) vs. prestorage filtration (n = 271/25 patients). Leukocyte and platelet content of the PC, duration of storage, platelet count of the patient before transfusion and 20 h after transfusion were determined and platelet increment and corrected count increment (CCI) 20 h after transfusion were calculated. The mean leucocyte content of the bedside filtered PC was 66 +/- 50 x 106 and < 0.1 x 106 for the prestorage filtered PC (P < 0.001). Mean platelet content of the PC (2.6 x 1011 vs. 2.7 x 1011) and the duration of PC storage (2.7 vs. 2.6 days) were almost identical in both groups. The platelet increment after 20 h (14.6 x 109 L-1 vs. 14.9 x 109 L-1) was equal for both groups, but CCI values were significantly higher for the bedside filtered PC (14.1 +/- 9.5 vs. 11.4 +/- 6.8) (P = 0.008). Correlation with the storage time revealed that CCI levels were higher for bedside filtered PC after short-term storage (< 36 h) (P = 0.014), but declined more rapidly compared with prestorage filtered PC. A patient-based analysis including fewer cases revealed superior but nonsignificant results for bedside filtration. In conclusion, bedside filtered PC-BC resulted in better CCI results after short-term storage, but values equalized with prestorage filtered PC-BC after longer storage intervals. Prestorage-leucocyte filtration did not improve platelet recovery in vivo, but CCI values decreased only moderately throughout storage. Both preparations showed excellent transfusion results even after a 5-day storage interval.

Monitoring of anti-HLA class I and II antibodies by flow cytometry in patients after first cadaveric kidney transplantation.

While the relevance of pre-formed anti-human leukocyte antigen (HLA) antibodies has been studied extensively, the role of anti-HLA class I and II antibodies produced after cadaveric kidney transplantation is still a matter of discussion. As it has been proposed that they are involved in a considerable number of cases, it should be investigated whether a post-transplant monitoring is a sensitive parameter for the early diagnosis of acute rejection episodes. Additionally, it has been suggested that antibodies are a major cause for chronic rejection; thus, it would be of interest to correlate antibody detection and graft survival. We retrospectively investigated 59 patients after a first cadaveric kidney transplantation without known anti-HLA antibodies (complement-dependent cytotoxicity [CDC] testing). The panel reactivity was determined with a new highly sensitive and specific flow-cytometric technique (Flow-PRA Screening Test, One Lambda, Canoga Park, USA) in sequentially collected serum samples pre- and post-transplant. In patients with acute rejection episodes during the clinical course, the last sample prior to rejection, and in patients without rejection, the last sample prior to discharge, was analyzed. Furthermore, we analyzed 3-yr graft survival and several clinical parameters such as cold ischemia time (CIT). Twenty-four of 59 patients (41%) experienced acute rejections during the clinical course. Five of 59 died with a functioning graft within the first 3 yr. Seven of 54 patients, still alive after 3 yr, lost their graft. Anti-HLA antibodies were detectable in only 7/59 patients and a correlation between antibody positivity and acute rejections (p = 0.32 and 0.54 for anti-HLA class I and II, respectively) could not be identified (sensitivity 12.5 and 8.3%). However, we found a significant correlation between the detection of anti-HLA class II and graft loss within 3 yr (p = 0.005, specificity 97.9%). Additionally, anti-HLA class II positive patients had significantly longer CIT (p = 0.003). Whether the detection of anti-HLA class II antibodies in the early post-transplant phase is of great value for the identification of patients at high risk for early graft loss needs additional investigation. However, we found that anti-HLA antibodies are detectable only in a minority of unsensitized patients and we conclude that flow-cytometric monitoring with Flow PRA is not a sensitive parameter for the early diagnosis of acute rejection episodes in patients after first cadaveric kidney transplantation.

Increased concentrations of interleukin 1-beta in whole blood cultures supernatants after 12 weeks of moderate endurance exercise.

The aim of the study was to investigate whether a regular moderate endurance exercise programme influenced the in vitro cytokine synthesis by stimulated whole blood cultures. To this end, eight healthy subjects exercised moderately by running for 3-5 h a week over a period of 12 weeks, whilst seven other healthy subjects served as the control group. The intensity of the exercise was determined by lactic acid concentrations in the blood which were maintained between 1.8 and 2.5 mmol x l(-1). Over the period of training the running velocity producing the 4 mmol x l(-1) lactic acid threshold increased from 2.86 (SD 0.83) m x s(-1) to 3.06+/-0.79 m x s(-1) (P < or = 0.008). Blood samples were taken at rest before and after the training programme. The following blood parameters were determined: leucocyte count, differential leucocyte count, lymphocyte subpopulations [CD14 positive (+)/CD45+, CD4+/ CD25+, CD8+, CD16+/CD122+]. Whole blood cultures were stimulated with lipopolysaccarides [interleukin (IL)-1 beta and IL-6] and staphylococcal enterotoxin B [IL-2, soluble interleukin 2 receptor (sIL2-R) and interferon (IFN)-gamma]. Cytokine concentrations in the supernatants were measured using an enzyme-linked immunosorbent assay. The white blood cell count, differential leucocyte count, lymphocyte subset distribution and the expression of the CD25 and CD122 antigen on lymphocytes were unchanged by training. After the training programme the IL-1 beta production changed significantly [1496 (SD 264) pg ml(-1) before, compared to 2127 (SD 672) pg ml(-1) after training, P < or = 0.008]. In the control group these parameters remained unchanged. With respect to changes in the values in both groups the syntheses of IL-1 beta (P < or = 0.023) and IL-6 (P < or = 0.021) were significantly higher after regular training. The syntheses of IL-2, sIL-2 and INF-gamma were not significantly influenced. Regular endurance exercise influenced the in vitro production of monocyte derived cytokines, while the effect of exercise on the cytokines synthesized by T-cells appeared to be of lesser importance.

Evaluation of a novel mononuclear cell isolation procedure for serological HLA typing.

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

Impact of storage at 22 degrees C and citrate anticoagulation on the cytokine secretion of mononuclear leukocytes.

BACKGROUND AND OBJECTIVES: Peripheral blood mononuclear cells (PBMC) and cytokines accumulating in platelet concentrates (PC) during storage have been implicated in causing non-hemolytic transfusion reactions, in particular after prolonged PC storage. We investigated the impact of major storage parameters, such as storage time, anticoagulation and temperature, on the capability of PBMC to secrete cytokines. MATERIALS AND METHODS: In a first study, PBMC from whole blood donations (n = 16) were exposed to standard PC storage conditions: 22 degrees C, citrate phosphate dextrose (CPD) anticoagulation. Secretion of the cytokines interleukin-1 beta (IL-1 beta), IL-2, IL-6 and interferon-gamma (IFN-gamma) on mitogenic stimulation was measured directly after blood donation and after 1, 3 and 5 days of storage. In a second study, paired whole blood samples (n = 24) were investigated for the mitogenic induction of IL-2, IL-6, IFN-gamma and IFN-alpha. Cytokine values were compared with respect to incubation temperature (22 vs. 37 degrees C) and anticoagulant (CPD vs. lithium-heparin). RESULTS: PBMC stored at 22 degrees C with CPD anticoagulation showed an altered cytokine secretion pattern in comparison to freshly donated cells. After storage for 3 days, IL-2 release was decreased (p < 0.05) and after 5 days secretion of all cytokines was significantly decreased, though still detectable (p < 0.01). In the second study, CPD anticoagulation resulted in a significantly impaired cytokine secretion compared with lithium-heparin. Cytokine secretion at 22 degrees C were significantly lower (p < 0.001) compared with 37 degrees C for both anticoagulants. CONCLUSION: Storage of PBMC at 22 degrees C with CPD leads to an impaired cytokine response, but PBMC are still capable of secreting cytokines after 5 days of storage. PBMC remain a potential source of cytokines even after 5 days of storage in CPD at 22 degrees C.

Impact of buffy coat storage on the generation of inflammatory cytokines and platelet activation.

BACKGROUND: Whole blood-derived buffy coat (BC) has become an alternative source from which to prepare random-donor platelet concentrates. The influence of prolonged storage of BC prior to platelet concentrate preparation is a matter of controversy. The impact of BC storage on cytokine release was evaluated and the platelet activation quantified. STUDY DESIGN AND METHODS: BCs were prepared from whole-blood donations after hard-spin centrifugation. After 1, 3, 6, 12, and 24 hours of storage at 22 degrees C without agitation, samples were withdrawn for cell count and blood gas analysis and measurement of interleukin (IL)-1beta, IL-6, IL-8, tumor necrosis factor alpha and platelet factor 4. Platelet surface markers CD41a, CD42b, CD62P, and CD63 were analyzed by flow cytometry, and the antibody-binding sites were quantified by using microbeads. RESULTS: Inflammatory cytokines IL-1beta, IL-6, and tumor necrosis factor alpha were hardly detectable in stored BCs but levels of IL-8 increased in 25 percent of BCs after 24 hours. A constant increase in platelet factor 4 was observed, which accelerated after 12 hours of storage. Analysis of platelet surface markers showed an initial decrease of platelet activation, followed by an increase after storage for 12 to 24 hours. CONCLUSION: Storage of BCs for up to 12 hours without agitation showed a good preservation of platelets but storage of BCs for 24 hours resulted in increased platelet activation and significantly higher release of platelet factor 4 and IL-8. Stored BCs might well be suitable for platelet preparation, but their storage time should not greatly exceed 12 hours.

Moderate and exhaustive endurance exercise influences the interferon-gamma levels in whole-blood culture supernatants.

The aim of this study was to investigate whether moderate or exhaustive endurance exercise influences cytokine levels in whole-blood culture supernatants after stimulation. Therefore, eight healthy subjects were first exposed to moderate exercise on a cycle ergometer for 30 min at 70% of their 4-mmol/l lactic acid (anaerobic) threshold, and 1 week later to exhaustion (for 90 min) at their anaerobic threshold. Blood samples were taken before, 30 min after and 24 h after each exercise bout. The following lymphocyte subpopulations were determined: CD14-positive(+)/CD45+, CD4+, CD8+, and CD16+. Cytokine levels in the supernatants were measured by enzyme-linked immunosorbent assay (ELISA). Production of interleukin (IL)-1beta, IL-6 and tumour necrosis factor (TNF)-alpha were induced with lipopolysaccharides (LPS), and that of IL-2 and interferon (IFN)-gamma with staphylococcal enterotoxin B (SEB) and phytohaemagglutinin (PHA). Cortisol levels were also determined by ELISA. The lymphocyte subset distribution was observed to be unchanged after moderate exercise. Thirty minutes after exhaustive exercise, the CD16+ count was found to be significantly lower, whereas 24 h later the CD4+ count was significantly higher than pre-exercise counts. Moderate exercise influenced the IFN-gamma production (PHA-stimulated), which increased significantly from 974 (391) pg/ml before exercise to 1450 (498) pg/ml 24 h later. Thirty minutes after exhaustive exercise the IFN-gamma level in the supernatants (SEB-stimulated) was significantly decreased (from 14470 (11840) pg/ml before exercise to 6000 (4950) pg/ml after exercise). The IL-1beta and TNF-alpha production per monocyte was also significantly reduced.