Cytokine release in febrile non-haemolytic red cell transfusion reactions

BACKGROUND AND OBJECTIVES: The aim of this study was to elucidate the role and identity of cytokines involved in febrile non-haemolytic red cell transfusion reactions (FNHTRs). MATERIALS AND METHODS: Eighty-one patients experiencing transfusion reactions after receiving packed red blood cells (RBCs) were divided into three groups, as follows, based on the reaction experienced: FNHTRs (n = 60); chills without fever (n = 8); and allergic reaction with urticaria (n = 13). The concentrations of interleukin (IL)-1beta, IL-6, IL-8 and tumour necrosis factor (TNF)-alpha were measured in the packed transfused unit and patients' plasma by using enzyme immunoassays. Wilcoxon's matched-pairs signed test was used to compare the difference in cytokine levels in patients' plasma before and after transfusion. The Kruskal-Wallis test was used first, followed by the Mann-Whitney test, to compare the pretransfusion cytokine levels in patients' plasma between groups and to compare the cytokine levels in packed RBCs transfused to each group of patients. RESULTS: The age of the implicated packed RBC was 11.5 +/- 5.7 days. Significant increases were observed in IL-6 (P < 0.001) and IL-8 (P < 0.001) patients' plasma levels, but not in IL-1beta or TNF-alpha levels, in those patients exhibiting FNHTR. No changes were observed in the patients' plasma samples of the other groups. Cytokine levels in the RBC concentrate supernatants were not appreciably elevated. CONCLUSIONS: Transfusion of packed RBCs may significantly increase intravascular levels of IL-6 and IL-8 in patients with FNHTRs.     

Extended storage of whole blood before the preparation of blood components: in vitro effects on red blood cells in Erythro-Sol environment

Background and Objectives  Routine procedures for extended storage of whole blood (WB) before the preparation of blood components are of interest primarily for logistical reasons. We stored red cell units in either Erythro-Sol 2 (E-Sol 2, test units, 150 ml added) or in saline-adenine-glucose–mannitol (SAG-M) (reference units, 100 ml added) that were prepared after storage of WB at room temperature for 8, 12, 16 or 19 h after blood collection.
Study Design and Methods  Red blood cells were stored for 42 days. We measured pH, glucose, lactate, haemolysis, red blood cell adenosine triphosphate and 2,3-diphosphoglycerate on days 1, 7, 14, 21, 28, 35 and 42.
Results  Haematocrits were significantly lower in E-Sol 2 than in SAG-M due to the higher volume of E-Sol 2 added compared to SAG-M. Significantly reduced levels were found in E-Sol 2 of extracellular pH (throughout storage after 8-h hold and initially after 12-, 16- or 19-h hold), of lactate (initially after 8-h hold and throughout storage after 12-, 16- or 19-h hold), and of haemolysis from day 35 in the 8-h and on day 42 in the 12-h hold group. Sigificantly increased levels of adenosine triphosphate were seen in E-Sol 2 after 8-h hold (from day 14) and after 12-h hold (at days 21, 35 and 42) compared to SAG-M. Significantly higher concentrations of 2,3-diphosphoglycerate were noticed primarily after 8-h hold of WB.
Conclusion  The use of E-Sol 2 as a replacement for SAG-M does not significantly improve in vitro data after extended storage of WB at room temperature before preparation of blood components. However, after 8-h hold in vitro characteristics similar to or better than in fresh blood will be maintained for several weeks in E-Sol 2, a situation that makes E-Sol 2 superior to SAG-M when storage of WB is limited to 8 h. Some improvement was noted after 12-h hold as well.     

Humoral immune response to autologous blood transfusion in hip surgery: whole blood versus packed red cells and plasma.

BACKGROUND AND OBJECTIVES: The immune response to the transfused autologous buffy coat content in whole blood has, to date, not been studied in detail. SUBJECTS AND METHODS: Patients undergoing hip arthroplasty were studied according to whether they received autologous whole blood (WB) (n = 30), autologous fresh-frozen plasma and buffy coat-poor red cells (RC) (n = 40), or no transfusion (NT) (n = 27). Plasma levels of tumour necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and complement SC5b-9 were analysed by enzyme-linked immunosorbent assay (ELISA) 7 days after surgery. RESULTS: There were no significant between-group differences regarding the time course of TNF-alpha, IL-6 and complement SC5b-9 plasma level changes, the infection rate, or the length of hospital stay. CONCLUSION: In comparison to the impact of surgery on cytokine and complement levels, the transfusion of autologous buffy coat is not relevant.     

Variable adhesion of different red blood cell products to activated vascular endothelium under flow conditions

Red blood cells (RBCs) that have been stored prior to transfusion show increased adherence to vascular endothelium in vitro, which suggests a potential for stored blood transfusion to impede blood flow in some patients. Transfusion is often required in patients with sepsis or inflammation; however, whether activation of endothelium affects stored RBC-endothelial cell (EC) interactions is unknown. We investigated whether storage time and leukocyte content of RBC products influences the adhesion of RBCs to activated ECs. RBCs from nonleukocyte-reduced (S-RBCs), buffy-coat-poor (BCP-RBCs), and leukocyte-filtered (LF-RBCs) products and cultured EC layers were pretreated with endotoxin, tumor necrosis factor-alpha (TNF-alpha), or medium alone prior to perfusion of the RBCs across the EC layer in a continuous flow microchamber. After a single day of RBC storage, the number of adherent RBCs was increased in the endotoxin and TNF-alpha pretreated groups compared to the unactivated-control group. These differences were statistically significant for S-RBCs and LF-RBC products (P < 0.05). In contrast, there was no significant difference in RBC adherence to activated and unactivated endothelium at other time-points of RBC product storage. The strength of adhesion of stored RBCs from S-RBC products to activated ECs was not altered following treatment; however, endotoxin significantly increased the adhesive strength of LF-RBCs to endothelium. These results demonstrate that while fresh RBCs show increased adhesion to activated endothelium, storage of RBCs did not promote increased adhesion to activated endothelium. However, inflammatory conditions promote stronger adhesion of stored RBCs to ECs, which may contribute to impaired tissue perfusion in some transfusion recipients.     

The in vitro quality of washed, prestorage leucocyte-depleted red blood cell concentrates

BACKGROUND AND OBJECTIVES: No data are currently available on the quality of washed prestorage leucocyte-depleted red blood cell concentrates (RCCs). MATERIALS AND METHODS: Five groups of RCCs stored in additive solution (SAG-M) were washed. The groups differed in the age of RCCs (2-5 days or 11-15 days), the temperature during the washing procedure and a 6-h storage period (4 degrees C or room temperature) and the washing solution (saline, SAG-M or 5% albumin). We measured ATP, 2,3-diphosphoglycerate (2,3-DPG), haemolysis, blood cell count, Na(+), K(+), pH, pO(2), pCO(2) and lactate, before and after the washing procedure and hourly during the 6-h postwash storage period. RESULTS: The erythrocyte ATP content increased by 2-13%, relative to the baseline value, during the washing procedure. The 2,3-DPG level decreased by 15-35% in 2-6-day-old RCCs and by 30-40% in 11-15-day-old RCCs (relative to baseline values) during the washing procedure. In RCCs that were washed and stored at room temperature, and in 2-week-old RCCs, a further decrease in 2,3-DPG of up to 40%, relative to the baseline value, was observed during the 6-h postwash time-period. CONCLUSIONS: Washing of RCCs stored in SAG-M results in a considerable, significant loss of erythrocyte 2,3-DPG, especially in older RCCs. This loss increases in during a 6-h storage period postwash, even at 4 degrees C. This loss of erythrocyte quality might well outweigh the benefits of washed SAG-M RCCs during massive transfusion in neonates.     

Prevention of Cytokine Accumulation in Platelets Obtained with the COBE Spectra Apheresis System

Background and Objectives: Febrile nonhemolytic transfusion reactions frequently accompany platelet trasfusions and may be due to accumulation of cytokines mediating inflammation during storage of platelet concentrates (PCs). We wished to determine whether PCs collected using the COBE® spectra? Apheresis System (Version4) were sufficiently leukocyte reduced (LR) to limit cytokine accumulation during storage. Materials and Methods: Cytokine accumulation-interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor-α (TNF-α)- and release of platelet α-granule - P-selectin, transforming growth factor-β1 (TGF-β1), platelet-derived growth factor AB (PDGF-AB), von Willebrand factor (vWf)- or dense granule (serotonin) markers were investigated during a 7-day storage period comparing apheresis-collected, LR PCs (LR PCs) and random donor platelets prepared from whole blood (WB). Results: Leukocyte counts were reduced 99.95% comparing LR PCs (5.7×10⁵/1) and WB PCs (1.09×10⁹/1). Little or no accumulation of leukocyte-derived cytoknies was observed in LR PCs during storage in contrast to WB PCs. A reduction in the release of platelet α-granule proteins, such as P-selectin, TGF-β1 and PDGF-AB, was observed on day 0 for LR PCs compared to WB PCs with little or no difference observed from day 3 to 7. Plasma vWf levels were higher in LR PCs compared to WB PCs on days 0–7. Conclusion: Leukocyte levels in PCs collected with the COBE Spectra Apheresis System are sufficiently low to limit cytokine production during 7 days of storage.     

Modified formulation of CPDA for storage of whole blood, and of SAGM for storage of red blood cells, to maintain the concentration of 2,3-diphosphoglycerate

BACKGROUND AND OBJECTIVES: A dramatic decrease in the level of 2,3-diphosphoglycerate (2,3-DPG) takes place during the storage of whole blood (WB) in CPDA (citrate-phosphate-dextrose-adenine) and a similar decrease occurs during the storage of red blood cells (RBCs) in SAGM (saline-adenine-glucose-mannitol). The aim of the present study was to prevent this decrease by modifying CPDA and SAGM. MATERIALS AND METHODS: The pH of WB anticoagulant or RBC preservative solution was maintained at 7.6 by autoclaving the dextrose solution separately, by incorporating ascorbic acid and nicotinic acid into both CPDA and SAGM (to produce modified CPDA and SAGM solutions), and by reducing the concentration of adenine and adding citrate to the modified SAGM solution. The concentration of 2,3-DPG in WB after 28 days of storage in modified CPDA, and in RBCs stored in modified SAGM, was compared with that in WB or RBCs stored in unmodified solutions. RESULTS: The initial 2,3-DPG levels were maintained after 28 days in the modified formulations [10.63 +/- 2.58 microM/g of haemoglobin (Hb) in the case of modified CPDA and 12.07 +/- 1.47 microM/g of Hb in the case of modified SAGM], whereas in standard CPDA and SAGM solutions, the concentration of 2,3-DPG decreased to very low levels (0.86 +/- 0.97 microM/g Hb for CPDA and 0.12 +/- 0.008 for SAGM). CONCLUSIONS: Our modification in the formulation of CPDA or SAGM is effective in arresting the dramatic decrease in the level of 2,3-DPG that occurs during storage of WB and RBCs in unmodified solutions.     

Impact of Storage at 22°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°C, citrate phosphate dextrose (CPD) anticoagulation. Secretion of the cytokines interleukin-1β (IL-1β), IL-2, IL-6 and interferon-γ (IFN-γ) 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-γ and IFN-α. Cytokine values were compared with respect to incubation temperature (22 vs. 37°C) and anticoagulant (CPD vs. lithium-heparin). Results: PBMC stored at 22°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°C were significantly lower (p < 0.001) compared with 37°C for both anticoagulants. Conclusion: Storage of PBMC at 22°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°C.     

Cellular properties of human erythrocytes preserved in saline-adenine-glucose-mannitol in the presence of L-carnitine

L-Carnitine (LC) in the preservation medium during storage of red blood cells (RBC) can improve the mean 24-hr percent recovery in vivo and increase RBC life-span after reinfusion. The purpose of the study was to investigate the differences in the biochemical properties of RBCs stored in the presence or absence of LC, and the cell-age related responses to storage conditions and to LC. RBC concentrates in saline-adenine-glucose-mannitol (SAG-M) were stored in the presence or absence of 5 mM LC at 4 degrees C for up to 8 weeks. RBC subpopulations of different densities were prepared by centrifugation on Stractan density gradient. Cells were sampled at 0, 3, 6, and 8 weeks, and hematological and cellular properties analyzed (MCV, MCHC, 4.1a/4.1b ratio as a cell age parameter, intracellular Na(+) and K(+)). After 6 weeks, MCV of RBC stored in the presence of LC was lower than that of controls (6 weeks MCV: controls 95.4 +/- 1.8 fl; LC 91.5 +/- 2.0 fl; n = 6; P < 0.005). This was due to swelling of control cells, and affected mainly older RBCs. LC appeared to reduce or retard cell swelling. Among the osmotically active substances whose changes during storage could contribute to cell swelling, only intracellular Na(+) and K(+) differed between stored control RBCs and LC-treated cells. LC reduces the swelling of older cells during storage at 4 degrees C in SAG-M, possibly by acting on the permeability of cell membrane to monovalent cations.     

Inhibitory activity of 1,8-cineol (eucalyptol) on cytokine production in cultured human lymphocytes and monocytes

BACKGROUND: The therapeutic value of secretolytic agents in COPD and asthma is still disputed. For this reason, in a preclinical study we aimed to test the potential anti-inflammatory efficacy of 1,8-cineol (eucalyptol) in inhibiting polyclonal stimulated cytokine production by human unselected lymphocytes and LPS-stimulated monocytes. METHODS: Cytokine production was determined following 20 h of incubation cells with 1,8-cineol simultaneously with the stimuli in culture supernatants by enzyme immunoassay. RESULTS: Therapeutic concentrations of 1,8-cineol (1.5 microg/ml=10(-5)M) inhibited significantly (n=13-19, p=0.0001) cytokine production in lymphocytes of TNF-alpha > IL-1beta> IL-4> IL-5 by 92, 84, 70, and 65%, respectively. Cytokine production in monocytes of TNF-alpha > IL-1beta> IL-6> IL-8 was also significantly (n=7-16, p<0.001) inhibited by 99, 84, 76, and 65%, respectively. In the presence of 1,8-cineol (0.15 microg/ml=10(-6)M) production of TNF-alpha>IL-1beta by monocytes and of IL-1beta> TNF-alpha by lymph-ocytes was significantly inhibited by 77, 61 and by 36, 16%, respectively. 1,8-cineol (10(-6)M) had a larger impact on TNF-alpha and IL-1beta-production in monocytes compared to lymphocytes (p<0.03) and similar effects (p>0.59) at therapeutically relevant concentrations of 1,8-Cineol (10(-5)M). CONCLUSION: These results characterize 1,8-cineol as strong inhibitor of TNF-alpha and IL-1beta and suggest smaller effects on chemotactic cytokines. This is increasing evidence for the role of 1,8-cineol to control airway mucus hypersecretion by cytokine inhibition, suggesting long-term treatment to reduce exacerbations in asthma, sinusitis and COPD.     

Release of proinflammatory cytokines by mitogen-stimulated peripheral blood mononuclear cells from critically ill multiple-trauma victims

This study investigated the alterations in circulating proinflammatory cytokines and cytokine production by peripheral blood mononuclear cells (PBMCs) in response to lipopolysaccharide (LPS) or phytohemagglutinin (PHA) after severe trauma. Plasma and PBMCs were collected from 17 severely injured trauma patients and 10 healthy subjects. Plasma was stored at -80 degrees C and analyzed for cytokines. Isolated PBMCs from each subject were stimulated with LPS or PHA and incubated at 5% CO2 for 24 hours. Supernatants were collected and analyzed for cytokines. There was no significant change in the plasma concentration of free TNF-alpha and IL-1beta between healthy subjects and trauma patients. Plasma IL-6, total TNF-alpha, and total IL-1beta were significantly increased in severely traumatized patients compared with healthy control subjects. PBMCs from trauma patients produced higher levels of TNF-alpha in response to LPS but it showed no significant change in IL-1beta and IL-6 production in response to PHA or LPS in comparison to PBMCs from control subjects. We conclude that severe trauma results in a significant increase in plasma proinflammatory cytokine IL-6. Free TNF-alpha and IL-1beta in plasma remain at levels comparable to those in uninjured controls, while plasma free IL-6 levels in trauma patients remain high. Serious injury is associated with an enhanced production of TNF-alpha by PBMCs stimulated with LPS.     

Relationship between antipyretic effects and cytokine levels in uncomplicated falciparum malaria during different treatment regimes

We have previously shown that both chloroquine and paracetamol (acetaminophen) have antipyretic activity during treatment of acute uncomplicated Plasmodium falciparum malaria in children 1-4 years old. Here, we studied if this effect was accompanied by changes in plasma cytokine levels. The 104 children were treated with either chloroquine or sulfadoxine/pyrimethamine (SP) alone, SP+chloroquine or SP+paracetamol for 4 days. Cytokine levels were determined days 0, 2 and 3, body temperature every sixth hour until 72h and parasitemia once daily for 4 days. At admission, body temperature correlated with levels of IL-10, IFN-gamma and IL-6, and parasitemia correlated with IL-10 and IL-6. Except for TNF-alpha and IL-1beta, where no significant effect was found, all cytokine levels (IL-10, IFN-gamma, IL-6, IL-12, IL-13, IL-18 and IL-4) decreased up to day 2 (p<0.05). IL-6 levels continued to fall from days 2 to 3 (p<0.05), whereas increased levels were found for several cytokines (IL-12, IL-13, IL-18 and IL-1beta) (p<0.05). The antipyretic effects of chloroquine and paracetamol could not be related to any specific changes in the evaluated cytokine production or in Th1/Th2 or inflammatory/anti-inflammatory cytokine ratios. Alternative mechanisms for antipyretic effects and associations between fever and cytokine levels during uncomplicated P. falciparum malaria are therefore discussed.     

Systemic cytokine levels and the effects of etanercept in TNF receptor-associated periodic syndrome (TRAPS) involving a C33Y mutation in TNFRSF1A

OBJECTIVE: To investigate the levels of the pro-inflammatory cytokines IL-6, TNF-alpha, IL-1beta, IL-8, IL-10 and IL-12p70 in the plasma of patients with TNF receptor-associated periodic syndrome (TRAPS) in relation to CRP levels and treatment with etanercept. METHODS: Cytokine concentrations were measured in sequential plasma samples obtained from eight patients with a C33Y mutation in TNFRSF1A and diagnosed with TRAPS, using cytokine bead array. The TRAPS samples were compared with samples from normal controls and rheumatoid arthritis patients. RESULTS: Levels of IL-6 were significantly elevated in C33Y TRAPS patients and these correlated with CRP levels in some of the patients. IL-8 levels were also significantly elevated in the TRAPS patients. However, neither TNF-alpha nor IL-1beta demonstrated a similar increase. This differed from the patients with rheumatoid arthritis, for whom levels of IL-6, IL-8, TNF-alpha, IL-1beta and IL-10 were significantly elevated. The levels of detectable TNF-alpha in the TRAPS patients' plasma were elevated during etanercept treatment. CONCLUSIONS: The cytokine profile of C33Y TRAPS differs from that of a typical autoimmune inflammatory condition such as rheumatoid arthritis, as only IL-6 and IL-8 were elevated in C33Y TRAPS patients, as distinct from a generalized elevation of pro-inflammatory cytokines. However, only some of the C33Y patients tested showed a relationship between elevated IL-6 and CRP. This is consistent with clinical observations that there is marked heterogeneity between individuals with TRAPS, including those in the same family cohort. Although etanercept has a therapeutic effect in some TRAPS patients, it induces increased plasma concentrations of TNF-alpha, possibly by increasing TNF-alpha stability.     

Serum and ascites fluid cytokine levels in patients with chronic heart failure

OBJECTIVE: Cytokines that are capable of modulating cardiovascular function were reported to be elevated in patients with advanced heart failure. We evaluated the diagnostic importance of cytokines both in the serum and ascites. MATERIAL AND METHODS: We determined serum and ascites fluid TNF-alpha, IL-1beta, IL-6, IL-8, and soluble IL-2 receptor levels in 14 patients with congestive heart failure (group 1) and in 15 patients with chronic liver disease (group 2). RESULTS: Ascites fluid IL-8 and soluble IL-2 receptor levels were found to be significantly elevated in group 1 when compared with group 2 (p = 0.014 and p = 0.005). There were no statistical differences in serum TNF-alpha, IL-1beta, IL-6, IL-8, and soluble IL-2 receptor levels and ascites fluid TNF-alpha, IL-1beta, and IL-6 levels. Ascites fluid/serum IL-1beta and IL-8 ratio was lower in group 1 when compared with group 2 (p = 0.001 and p = 0.005). Ascites fluid/serum IL-2 and IL-6 ratio was higher in group 1 when compared with group 2 (p = 0.035 and p = 0.025). CONCLUSION: Cytokine levels in ascites fluid, but not in serum, are important in congestive heart failure. Ascites fluid/serum cytokine level ratios were detected to be more conclusive and valid in the diagnosis work-up of ascites aetiology.     

Preactivated peripheral blood monocytes in patients with essential hypertension.

The purpose of this study was to investigate the possible involvement of human peripheral blood monocytes in the pathology of hypertensive disease. We determined the in vitro secretion patterns of proinflammatory cytokines obtained from isolated peripheral monocytes from normal controls and from hypertensive patients either after in vitro stimulation with angiotensin II (Ang II) with or without preincubation with an Ang II type 1 receptor antagonist (losartan) or after stimulation with lipopolysaccharide. Blood samples were obtained from 22 patients with essential hypertension (before any drug administration or after interruption of antihypertensive therapy) and from 24 normotensive healthy individuals used as a control group. Peripheral blood monocytes were isolated by density gradient centrifugation and plastic adherence. The state of monocyte activity was determined by the capacity to secrete tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interleukin-6, (IL-6) either spontaneously or after stimulation. Cytokine concentrations were determined in culture supernatants by specific ELISA. Proinflammatory cytokine levels were assessed by semiquantitative reverse transcribed polymerase chain reaction. After stimulation with Ang II, the IL-1beta secretion of peripheral blood monocytes was significantly increased in hypertensive patients versus healthy individuals (P<0.05). In contrast, in monocytes preincubated with losartan before exposure to Ang II, IL-1beta secretion was diminished in both groups to comparable levels. The secretion of IL-1beta and TNF-alpha was significantly increased in peripheral blood monocytes from hypertensive patients versus healthy individuals after stimulation with lipopolysaccharide (TNF-alpha, P<0.02; IL-1beta, P<0.05). Upregulation of IL-1beta and TNF-alpha secretion in peripheral blood monocytes from hypertensive patients was also seen at the RNA level. Our results indicate preactivated peripheral blood monocytes in hypertensive patients. Ang II may be directly involved in the process of monocyte activation.     

Immune activation in patients with irritable bowel syndrome

BACKGROUND AND AIMS: We set out to test the hypothesis that irritable bowel syndrome (IBS) is characterized by an augmented cellular immune response with enhanced production of proinflammatory cytokines. We further aimed to explore whether symptoms and psychiatric comorbidity in IBS are linked to the release of proinflammatory cytokines. METHODS: We characterized basal and Escherichia coli lipopolysaccharide (LPS)-induced cytokine production in peripheral blood mononuclear cells (PBMCs) from 55 IBS patients (18 mixed-, 17 constipation-, 20 diarrhea-predominant) and 36 healthy controls (HCs). PBMCs were isolated by density gradient centrifugation and cultured for 24 hours with or without (1 ng/mL) LPS. Cytokine production (tumor necrosis factor [TNF]-alpha, interleukin [IL]-1beta, and IL-6) was measured by enzyme-linked immunosorbent assay. Abdominal symptoms and psychiatric comorbidities were assessed by using the validated Bowel Disease Questionnaire and the Hospital Anxiety and Depression Scale. RESULTS: IBS patients showed significantly (P < .017) higher baseline TNF-alpha, IL-1beta, IL-6, and LPS-induced IL-6 levels compared with HCs. Analyzing IBS subgroups, all cytokine levels were significantly (P < .05) higher in diarrhea-predominant IBS (D-IBS) patients, whereas constipation-predominant IBS patients showed increased LPS-induced IL-1beta levels compared with HCs. Baseline TNF-alpha and LPS-induced TNF-alpha and IL-6 levels were significantly higher in patients reporting more than 3 bowel movements per day, urgency, watery stools, and pain associated with diarrhea compared with patients without these symptoms (all P < .05). LPS-induced TNF-alpha production was associated significantly (r = 0.59, P < .001) with anxiety in patients with IBS. CONCLUSIONS: Patients with D-IBS display enhanced proinflammatory cytokine release, and this may be associated with symptoms and anxiety.     

Storage of whole blood overnight in different blood bags preceding preparation of blood components: in vitro effects on red blood cells

Background

Routines for the storage of whole blood (WB) overnight for the preparation of blood components on the following day are of increasing interest primarily for logistic reasons. The present study focuses on in vitro effects during storage for 6 weeks on red blood cells (RBC) prepared in different blood containers after being held overnight.

Study design and methods

Five different blood collection systems were used with either inline leucocyte reduction red cell filters for the preparation of RBC, buffy coat (BC) and plasma or WB filters for the preparation of RBC and plasma. A new container with an integrated WB filter removing leucocytes but not platelets was also included for the preparation of leucocyte-reduced RBC, BC and plasma units. Standard CPD solution (63 or 70 mL) and SAG-M solution (100 or 110 mL) were used for the collection of either 450 or 500 mL blood. All WB units were stored at room temperature, either overnight for 18–24 hours (test groups, n=104) or for up to 8 hours (reference groups, n=20). In addition, five test units were stored overnight under refrigeration.

Results

In test groups (overnight storage at room temperature) we found significantly lower levels of extracellular potassium, 2,3-DPG and pH (up to day 28). During storage, higher levels of ATP (Terumo, CaridianBCT until day 35, Fresenius until day 14, Fenwal throughout storage) were seen in test groups than in reference groups. When WB was stored overnight at 2–6°C before WB filtration, the levels of ATP and haemolysis were higher than in the corresponding reference.

Conclusion

Significant differences in in vitro parameters were observed between RBC prepared within 8 hours and 18–24 hours after blood collection. The results were consistent irrespective of the blood container used. New alkaline solutions may decrease the differences.     

Monocyte-derived dendritic cells from chronic HCV patients are not infected but show an immature phenotype and aberrant cytokine profile.

BACKGROUND: Chronic hepatitis C virus (HCV) infection is characterized by an insufficient immune response, possibly owing to impaired function of antigen-presenting cells such as myeloid dendritic cells (DCs). Therapeutic vaccination with in vitro generated DCs may enhance the immune response. Subsets of DCs can originate from monocytes, but the presence of HCV in monocytes that develop into DCs in vitro may impair DC function. Therefore, we studied the presence of HCV RNA in monocytes and monocyte-derived DCs from chronic HCV patients. METHODS: Monocytes were cultured with granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) for 6 days, and then with GM-CSF, IL-4, tumour necrosis factor-alpha (TNF-alpha), prostaglandin E2, IL-1beta and IL-6 for 2 days to generate mature DCs. HCV RNA was assessed by polymerase chain reaction. Surface molecules were assessed by flow cytometry. Cytokine production was assessed by cytokine bead array. RESULTS: HCV RNA was present in monocytes in 11 of 13 patients, but undetectable in mature DCs in 13 of 13 patients. The morphology of patient DCs was comparable with DCs from healthy controls, but the percentage of cells expressing surface molecules CD83 (P=0.001), CD86 (P=0.023) and human leucocyte antigen-DR (P=0.028) was lower in HCV patients. Compared with control DCs, patient DCs produced enhanced levels of IL-10 (P=0.0079) and IL-8 (P=0.0079), and lower levels of TNF-alpha (P=0.032), IL-6 (P=NS) and IL-1beta (P=0.0079). Patient and control DCs did not produce IL-12. CONCLUSIONS: Monocyte-derived DCs from chronic HCV patients are not infected but show an immature phenotype and aberrant cytokine profile.