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Abstract Background Complex regional pain syndrome (CRPS) may occur after trauma, usually to one limb, and is characterized by pain and disturbed blood flow, temperature regulation and motor control. Approximately 25% of cases develop fixed dystonia. Involvement of dysfunctional GABAergic interneurons has been suggested, however the mechanisms that underpin fixed dystonia are still unknown. We hypothesized that dystonia could be the result of aberrant proprioceptive reflex strengths of position, velocity or force feedback. Methods We systematically characterized the pattern of dystonia in 85 CRPS-patients with dystonia according to the posture held at each joint of the affected limb. We compared the patterns with a neuromuscular computer model simulating aberrations of proprioceptive reflexes. The computer model consists of an antagonistic muscle pair with explicit contributions of the musculotendinous system and reflex pathways originating from muscle spindles and Golgi tendon organs, with time delays reflective of neural latencies. Three scenarios were simulated with the model: (i) increased reflex sensitivity (increased sensitivity of the agonistic and antagonistic reflex loops); (ii) imbalanced reflex sensitivity (increased sensitivity of the agonistic reflex loop); (iii) imbalanced reflex offset (an offset to the reflex output of the agonistic proprioceptors). Results For the arm, fixed postures were present in 123 arms of 77 patients. The dominant pattern involved flexion of the fingers (116/123), the wrists (41/123) and elbows (38/123). For the leg, fixed postures were present in 114 legs of 77 patients. The dominant pattern was plantar flexion of the toes (55/114 legs), plantar flexion and inversion of the ankle (73/114) and flexion of the knee (55/114). Only the computer simulations of imbalanced reflex sensitivity to muscle force from Golgi tendon organs caused patterns that closely resembled the observed patient characteristics. In parallel experiments using robot manipulators we have shown that patients with dystonia were less able to adapt their force feedback strength. Conclusions Findings derived from a neuromuscular model suggest that aberrant force feedback regulation from Golgi tendon organs involving an inhibitory interneuron may underpin the typical fixed flexion postures in CRPS patients with dystonia.

Higher body mass index (BMI) is associated with osteoarthritis (OA) in both weight-bearing and non-weight-bearing joints, suggesting a link between OA and poor metabolic health beyond mechanical loading. This risk may be influenced by systemic factors accompanying BMI. Fluctuations in concentrations of metabolites may mark or even contribute to development of OA. This study explores the association of metabolites with radiographic knee/hip OA prevalence and progression. A 1 H-NMR-metabolomics assay was performed on plasma samples of 1564 cases for prevalent OA and 2,125 controls collected from the Rotterdam Study, CHECK, GARP/NORREF and LUMC-arthroplasty cohorts. OA prevalence and 5 to 10 year progression was assessed by means of Kellgren-Lawrence (KL) score and the OARSI-atlas. End-stage knee/hip OA (TJA) was defined as indication for arthroplasty surgery. Controls did not have OA at baseline or follow-up. Principal component analysis of 227 metabolites demonstrated 23 factors, of which 19 remained interpretable after quality-control. Associations of factor scores with OA definitions were investigated with logistic regression. Fatty acids chain length (FALen), which was included in two factors which associated with TJA, was individually associated with both overall OA as well as TJA. Increased Fatty Acid chain Length is associated with OA.

Background: Total knee replacement (TKR) is increasingly performed in short term hospital stay, making same day mobilization an important issue is after surgery. This implies little joint effusion by reducing intra-articular blood loss, which will enhance knee range of motion. The application of a topical fibrin sealant on the intraoperative bare bone and synovial tissue may contribute to better early full mobilization and thus improved functional outcomes. Since ambulation with a fully extended knee is less strenuous, we hypothesized that patients who received fibrin sealant would demonstrate improved early knee extension after six weeks compared to patients who received standard care., Methods: A multicenter randomized controlled trial in a consecutive series of osteoarthritis patients scheduled for TKR surgery. Participants were randomized to receive fibrin sealant or not before closing the knee joint capsule. Primary outcome was change in knee extension angle(°) at short term (2 weeks) follow-up (cExt). Secondary outcomes were 6-week extension angle, knee flexion angle, hemoglobin loss, blood transfusion rates, complication rates, the Knee Society Score, and the KOOS and EQ5D questionnaires., Results: When data on primary outcome became available from 250 patients, an interim analysis was performed by an independent Data Safety Monitoring Board for safety and effectivity assessment. This analysis showed that sufficient patients were included to detect a cExt of 10° between both groups. Inclusion was stopped however, all in the meantime included patients were treated according to their randomization. A total of 466 were available for analysis. Both groups were comparable in terms of baseline characteristics. The estimated mean cExt difference was 0.2° (95%CI -0.5 to 0.9). No differences in secondary outcomes were found., Conclusions: No beneficial effects or side effects were found of a topically applied fibrin sealant during TKR surgery. These results discourage the clinical use of a fibrin sealant in TKR., Trial Registration: Dutch Trial Register, NTR2500., Competing Interests: The authors have declared that no competing interests exist.

Pathogen inactivation of platelet concentrates reduces the risk for blood-borne infections. However, its effect on platelet function and hemostatic efficacy of transfusion is unclear. We conducted a randomized noninferiority trial comparing the efficacy of pathogen-inactivated platelets using riboflavin and UV B illumination technology (intervention) compared with standard plasma-stored platelets (control) for the prevention of bleeding in patients with hematologic malignancies and thrombocytopenia. The primary outcome parameter was the proportion of transfusion-treatment periods in which the patient had grade 2 or higher bleeding, as defined by World Health Organization criteria. Between November 2010 and April 2016, 469 unique patients were randomized to 567 transfusion-treatment periods (283 in the control arm, 284 in the intervention arm). There was a 3% absolute difference in grade 2 or higher bleeding in the intention-to-treat analysis: 51% of the transfusion-treatment periods in the control arm and 54% in the intervention arm (95% confidence interval [CI], -6 to 11; P = .012 for noninferiority). However, in the per-protocol analysis, the difference in grade 2 or higher bleeding was 8%: 44% in the control arm and 52% in the intervention arm (95% CI -2 to 18; P = .19 for noninferiority). Transfusion increment parameters were ∼50% lower in the intervention arm. There was no difference in the proportion of patients developing HLA class I alloantibodies. In conclusion, the noninferiority criterion for pathogen-inactivated platelets was met in the intention-to-treat analysis. This finding was not demonstrated in the per-protocol analysis. This trial was registered at The Netherlands National Trial Registry as #NTR2106 and at www.clinicaltrials.gov as #NCT02783313., (© 2018 by The American Society of Hematology.)

Introduction: Patients with chemotherapy-induced thrombocytopaenia frequently experience minor and sometimes severe bleeding complications. Unrestrictive availability of safe and effective blood products is presumed by treating physicians as well as patients. Pathogen reduction technology potentially offers the opportunity to enhance safety by reducing bacterial and viral contamination of platelet products along with a potential reduction of alloimmunisation in patients receiving multiple platelet transfusions., Methods and Analysis: To test efficacy, a randomised, single-blinded, multicentre controlled trial was designed to evaluate clinical non-inferiority of pathogen-reduced platelet concentrates treated by the Mirasol system, compared with standard plasma-stored platelet concentrates using the percentage of patients with WHO grade ≥ 2 bleeding complications as the primary endpoint. The upper limit of the 95% CI of the non-inferiority margin was chosen to be a ≤ 12.5% increase in this percentage. Bleeding symptoms are actively monitored on a daily basis. The adjudication of the bleeding grade is performed by 3 adjudicators, blinded to the platelet product randomisation as well as by an automated computer algorithm. Interim analyses evaluating bleeding complications as well as serious adverse events are performed after each batch of 60 patients. The study started in 2010 and patients will be enrolled up to a maximum of 618 patients, depending on the results of consecutive interim analyses. A flexible stopping rule was designed allowing stopping for non-inferiority or futility. Besides analysing effects of pathogen reduction on clinical efficacy, the Pathogen Reduction Evaluation and Predictive Analytical Rating Score (PREPAReS) is designed to answer several other pending questions and translational issues related to bleeding and alloimmunisation, formulated as secondary and tertiary endpoints., Ethics and Dissemination: Ethics approval was obtained in all 3 participating countries. Results of the main trial and each of the secondary endpoints will be submitted for publication in a peer-reviewed journal., Trial Registration Number: NTR2106; Pre-results., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Background: Reduction of blood transfusion in cardiac surgery is an important target. The aim of this study was to investigate the cost-effectiveness of the use of CryoSeal®, an allogeneic single-donor fibrin sealant, in patients undergoing coronary artery bypass grafting (CABG)., Methods: This randomized clinical study involved seven cardiac surgery centres in the Netherlands. Patients undergoing elective isolated CABG with the use of at least one internal thoracic artery (ITA) graft were assigned randomly to receive either CryoSeal® (5 ml per ITA bed) or no CryoSeal®. Primary efficacy endpoints were units of transfused red blood cells, fresh frozen plasma and platelet concentrates, and duration of intensive care unit stay. Secondary efficacy endpoints were 48-h blood loss, reoperation for bleeding, mediastinitis, 30-day mortality and duration of hospital stay., Results: Between March 2009 and January 2012, 1445 patients were randomized. The intention-to-treat (ITT) population comprised 1436 patients; the per-protocol (PP) population 1292. In both the ITT and the PP analysis, no significant difference between the treatment groups was observed for any of the primary and secondary efficacy endpoints. In addition, no significant difference between the groups was seen in the proportion of transfused patients. Estimated CryoSeal® costs were €822 (95 per cent c.i. €808 to €836) per patient, which translated to €72,000 per avoided transfusion (unbounded 95 per cent c.i.)., Conclusion: The use of the fibrin sealant CryoSeal® did not result in health benefits. Combined with the high cost per avoided transfusion, this study does not support the implementation of routine CryoSeal® use in elective isolated CABG., Registration Number: NTR1386 ( http://www.trialregister.nl)., (© 2015 BJS Society Ltd Published by John Wiley & Sons Ltd.)

Background: Patient blood management is introduced as a new concept that involves the combined use of transfusion alternatives. In elective adult total hip- or knee-replacement surgery patients, the authors conducted a large randomized study on the integrated use of erythropoietin, cell saver, and/or postoperative drain reinfusion devices (DRAIN) to evaluate allogeneic erythrocyte use, while applying a restrictive transfusion threshold. Patients with a preoperative hemoglobin level greater than 13 g/dl were ineligible for erythropoietin and evaluated for the effect of autologous blood reinfusion., Methods: Patients were randomized between autologous reinfusion by cell saver or DRAIN or no blood salvage device. Primary outcomes were mean intra- and postoperative erythrocyte use and proportion of transfused patients (transfusion rate). Secondary outcome was cost-effectiveness., Results: In 1,759 evaluated total hip- and knee-replacement surgery patients, the mean erythrocyte use was 0.19 (SD, 0.9) erythrocyte units/patient in the autologous group (n = 1,061) and 0.22 (0.9) erythrocyte units/patient in the control group (n = 698) (P = 0.64). The transfusion rate was 7.7% in the autologous group compared with 8.3% in the control group (P = 0.19). No difference in erythrocyte use was found between cell saver and DRAIN groups. Costs were increased by €298 per patient (95% CI, 76 to 520)., Conclusion: In patients with preoperative hemoglobin levels greater than 13 g/dl, autologous intra- and postoperative blood salvage devices were not effective as transfusion alternatives: use of these devices did not reduce erythrocyte use and increased costs.

Background: Patient blood management combines the use of several transfusion alternatives. Integrated use of erythropoietin, cell saver, and/or postoperative drain reinfusion devices on allogeneic erythrocyte use was evaluated using a restrictive transfusion threshold., Methods: In a factorial design, adult elective hip- and knee-surgery patients with hemoglobin levels 10 to 13 g/dl (n = 683) were randomized for erythropoietin or not, and subsequently for autologous reinfusion by cell saver or postoperative drain reinfusion devices or for no blood salvage device. Primary outcomes were mean allogeneic intra- and postoperative erythrocyte use and proportion of transfused patients (transfusion rate). Secondary outcome was cost-effectiveness., Results: With erythropoietin (n = 339), mean erythrocyte use was 0.50 units (U)/patient and transfusion rate 16% while without (n = 344), these were 0.71 U/patient and 26%, respectively. Consequently, erythropoietin resulted in a nonsignificant 29% mean erythrocyte reduction (ratio, 0.71; 95% CI, 0.42 to 1.13) and 50% reduction of transfused patients (odds ratio, 0.5; 95% CI, 0.35 to 0.75). Erythropoietin increased costs by €785 per patient (95% CI, 262 to 1,309), that is, €7,300 per avoided transfusion (95% CI, 1,900 to 24,000). With autologous reinfusion, mean erythrocyte use was 0.65 U/patient and transfusion rate was 19% with erythropoietin (n = 214) and 0.76 U/patient and 29% without (n = 206). Compared with controls, autologous blood reinfusion did not result in erythrocyte reduction and increased costs by €537 per patient (95% CI, 45 to 1,030)., Conclusions: In hip- and knee-replacement patients (hemoglobin level, 10 to 13 g/dl), even with a restrictive transfusion trigger, erythropoietin significantly avoids transfusion, however, at unacceptably high costs. Autologous blood salvage devices were not effective.

Objectives: Anti-inflammatory effects of tranexamic acid and aprotinin, used to abate perioperative blood loss, are reported and might be of substantial clinical relevance. The study of messenger ribonucleic acid synthesis provides a valuable asset in evaluating the inflammatory pathways involved., Methods: Whole-blood messenger ribonucleic acid expression of 114 inflammatory genes was compared pre- and postoperatively in 35 patients randomized to receive either placebo, tranexamic acid, or aprotinin. These results were further confirmed by reverse transcription-polymerase chain reaction., Results: Of the 23 genes exhibiting independently altered postoperative gene expression levels, 8 were restricted to the aprotinin group only (growth differentiation factor 3, interleukin 19, interleukin 1 family member 7, transforming growth factor α, tumor necrosis factor superfamily 10, tumor necrosis factor superfamily 12, tumor necrosis factor superfamily 13B, vascular endothelial growth factor α), whereas both aprotinin and tranexamic acid altered gene expression of 3 genes as compared with placebo (FMS-related tyrosine kinase 3 ligand, growth differentiation factor 5, interferon-α8). In general, less upregulation of pro-inflammatory, and more upregulation of anti-inflammatory, genes was observed for patients treated with antifibrinolytics. Gene expression affected by aprotinin coded mostly for proteins that function through serine proteases., Conclusions: This study demonstrates that the use of tranexamic acid and aprotinin results in altered inflammatory pathways on the genomic expression level. We further demonstrate that the use of aprotinin leads to significant attenuation of the immune response, with several inhibitory effects restricted to the use of aprotinin only. The results aid in a better understanding of the targets of these drugs, and add to the discussion on which antifibrinolytic can best be used in the cardiac surgical patient., (Copyright © 2013 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.)

Background: The reported percentage of haemato-oncological patients experiencing bleeding complications is highly variable, ranging from 5 to 70%, posing a major problem for comparison of clinical platelet transfusion trials using bleeding complications as a primary endpoint. In a pilot study we assessed the impact of the design of scoring of bleeding on the percentage of patients with WHO grade 2 or higher bleeding grades., Study Design and Methods: We performed a prospective, observational study using a rigorous bleeding observation system in thrombocytopenic patients with haemato-oncological disorders. Endpoints of the study were the percentage of patients and days with bleeding WHO grade ≥ 2 comparing designs in which skin bleeding represent a continuation of a previous bleed or a new bleed., Results: In four participating hospitals 64 patients suffering 870 evaluable thrombocytopenic days (platelet count < 80 × 10(9) L(-1)) were included. At least one episode of bleeding grade ≥ 2 occurred in 36 patients (56%). Most grade 2 bleeding complications occurred mucocutaneously. The percentage of days with bleeding of grade ≥ 2 was 16% but decreases to 8% when only newly developed skin bleeding was included., Conclusion: Rigorous daily observation results in a bleeding incidence that is comparable to recent reportings applying the same method. The results of this study show that censoring for stable skin bleeding has a profound effect on bleeding incidence per day. The clinical relevance of rigorous or clinically judged bleeding scores as an endpoint remains to be defined., (© 2012 The Authors. Transfusion Medicine © 2012 British Blood Transfusion Society.)

Aim: To determine the effect of red blood cell (RBC) transfusions during cardiac surgery on cytokine gene expression (GE) in relation to multiple organ failure (MOF) development after systemic inflammatory response syndrome (SIRS)., Background: RBC transfusion in cardiac surgery patients is dose-dependently associated with post-operative MOF, possibly acting as a second hit after cardiopulmonary bypass., Methods: For this observational study, 29 patients divided into four groups of cardiac surgery patients were selected from a randomised controlled trial (RCT). Group 1: no-RBC, no-MOF (N = 8); group 2: MOF, no-RBC (N = 7); group 3: RBC, no-MOF (N = 6); group 4: RBC and MOF (N = 8). Selection was based on age, gender, number of (leukocyte-depleted) RBC transfusions, type and duration of surgery. A 114 cytokine GE array was applied to blood samples withdrawn before and 24 h after surgery. Expression of selected genes was confirmed with reverse transcriptase real time-polymerase chain reaction (RT-PCR)., Results: Nineteen of the 39 detectable genes showed a significant change in GE after surgery. Confirmed by RT-PCR, transfused MOF patients exhibit significantly less downregulation of CD40 ligand than control patients. Patients who would develop MOF show significantly larger increases in GE of transforming growth factor-α (TGF-α), tumour necrosis factor (TNF)-superfamily members 10 and 13B (TNFsf10/13B)., Conclusions: When tested at 24 h after surgery, cytokine GE in peripheral blood leucocytes showed no significant differences between those transfused and those not transfused. Some alterations were seen in those developing MOF compared to those who did not, but the findings offer no role of leukocyte depleted (LD) RBC transfusion in the development of MOF., (© 2011 The Authors. Transfusion Medicine © 2011 British Blood Transfusion Society.)

Background: It has been suggested that transfusion information from scientific sources (vs. popular sources) is seen as more trustworthy and that interventions should consider using scientific styles. Before such suggestions can be implemented, it is necessary to know if this science source-trust link is observed across different sociodemographic groups and psychological characteristics. A large-scale field-based study examining the importance of sociodemographics and psychological characteristics on the source-trust link was conducted., Study Design and Methods: A large field-based experiment (the Euro Blood Substitutes Project) was conducted on four different samples (the general public, blood donors, patients, and health experts) in the UK and The Netherlands (total n = 3935). Questions examined levels of trust about sources of transfusion medicine, various aspects of knowledge, and demographic data., Results: People differentiated between scientific and popular sources, with scientific sources perceived as more trustworthy. General trust in transfusion medicine was higher for those who believe that they or scientists were knowledgeable about transfusion medicine or genetic modification (GM). This suggests that people do not differentiate in their subjective knowledge between GM and transfusion medicine. This science trust-source relationship was moderated by a variety of demographic (e.g., younger people were more likely to trust scientific sources) and psychological (e.g., those who rate science as knowledgeable were more trusting of scientific sources) factors., Conclusion: The trust-source link is not stable and communications should be targeted to the specific population samples for which they will be most effective; scientifically styled information will be particularly effective for communicating information within certain populations.

Background: Perioperative red blood cell (RBC) transfusion may be associated with a poor prognosis in cancer surgery. Allogeneic leucocytes are assumed to play a causal role. This study evaluated the long-term effect of transfusion with leucocyte-depleted (LD) blood in patients with gastrointestinal cancer., Methods: The Transfusion Associated Complications = Transfusion Induced Complications? (TACTIC) study is a multicentre randomized controlled trial evaluating the short-term benefits of LD versus non-LD RBC transfusions. The present study evaluated 5-year survival and cancer recurrence among 512 patients with gastrointestinal cancer included in the TACTIC study., Results: Some 89.2 per cent of patients had a primary tumour and 79.7 per cent underwent surgery with curative intent; 243 patients received perioperative RBC transfusion (median 3 units). The 5-year overall survival rate of patients with any type of gastrointestinal cancer was 50.8 per cent in the LD group and 45.8 per cent in the non-LD group (P = 0.191). Corresponding 5-year disease-free survival rates were 60.0 and 56.6 per cent (P = 0.482), and recurrence rates 32.9 and 34.3 per cent (P = 0.864)., Conclusion: Leucocyte depletion is not associated with better long-term survival and lower recurrence rates in patients with gastrointestinal cancer., (Copyright 2009 British Journal of Surgery Society Ltd.)

Background: The objective was to investigate the use of autologous red blood cells (RBCs) derived from umbilical cord blood (UCB), as an alternative for allogeneic transfusions in premature infants admitted to a tertiary neonatal center., Study Design and Methods: UCB collection was performed at deliveries of less than 32 weeks of gestation and processed into autologous RBC products. Premature infants requiring a RBC transfusion were randomly assigned to an autologous or allogeneic product. The primary endpoint was an at least 50 percent reduction in allogeneic transfusion needs., Results: Fifty-seven percent of the collections harvested enough volume (> or =15 mL) for processing. After being processed, autologous products (> or =10 mL/kg) were available for 36 percent of the total study population and for 27 percent of the transfused infants and could cover 58 percent (range, 25%-100%) of the transfusion needs within the 21-day product shelf life. Availability of autologous products depended most on the gestational age. Infants born between 24 and 28 weeks had the lowest availability (17%). All products, however, would be useful in view of their high (87%) transfusion needs. Availability was highest (48%) for the infants born between 28 and 30 weeks. For 42 percent of the infants with transfusion needs in this group, autologous products were available. For the infants born between 30 and 32 weeks, autologous products were available for 36 percent of the infants. Transfusion needs in this group were, however, much lower (19%) compared to the other gestational groups., Conclusion: Autologous RBCs derived from UCB could not replace 50 percent of allogeneic transfusions due to the low UCB volumes collected and subsequent low product availability.

Objective: Determination of factors related to the need for transfusion in premature infants., Design: Descriptive., Method: The need for transfusion in premature infants was determined in 2 academic centres: University Medical Center Utrecht and Leiden University Medical Center, The Netherlands. The data had been acquired in another study. The factors under study were: hospital, pregnancy duration, birth weight, gender, time of clamping of the umbilical cord, total volume of blood sampled for diagnostic purposes, number of days of mechanical ventilation, total duration of admission and duration of the admission to the Neonatal Intensive care unit. Both hospitals followed the national interdisciplinary practice guideline 'Blood transfusion'., Results: The total volume ofsampled blood for diagnosis, the duration of the mechanical ventilation and the admission period were related to a greater need for transfusion. On the other hand, the chance of transfusions diminished with longer pregnancy duration or increased birth weight. The difference in need for blood transfusion between both centres was significant. The total volume of transfused erythrocytes showed a strong correlation with the volume sampled for diagnostic procedures., Conclusion: Anaemia in neonates is strongly related to the amount of blood taken for diagnostic procedures. Alternatives for blood transfusions in premature infants, and consequently for the reduction of the number of donors per child, are to be sought in delayed clamping of the umbilical cord, use of erythropoietin and use ofautologous umbilical cord blood.

The use of A23187 as a biochemical tool is described. A23187 was used to elucidate the mechanisms of intracellular transport and secretion of N-hydroxylated aniline metabolites in rat hepatocyte primary culture. Results indicate a membrane-bound intracellular transport. Also, A23187 induced macrophages in their anti-tumour cytostatic activity using P815 tumour cells in in vitro co-cultures of macrophages and tumour cells. Results indicate the activating role of A23187 in macrophage leukotriene C4 release and enhanced macrophage anti-tumour activity. In conclusion, A23187 proved to be a useful tool in studying calcium dependent metabolic processes.

Protein conformations of the putative cysteinylleukotriene (LT) receptor of macrophages were characterized using anti-idiotypic IgG (AIAb) against an anti-LT monoclonal Ab (LTmAb). The AIAb nature of two rabbit antisera were demonstrated with titers of up to 1:1000 against F(ab')2 from the LTmAb (in an enzyme-linked immunoassay) which also inhibit LTD4 binding to the LTmAb (in a radioimmunoassay), whereas non-immunized rabbit serum was not reactive. The specific reactivity of Fc-purified AIAb towards LTmAb was measured by two fractions obtained after passage over columns of Sepharose either coupled with LTmAb (fraction A, representing immunoglobulins not absorbed to LTmAb) or coupled with homologous immunoglobins (fraction B, representing immunoglobulins not absorbed to homologous IgG). The difference in immunoreactivity between both fractions showed that fraction B contains AIAb against a LT-recognizing domain of the LTmAb (in enzyme-linked immunoassays coated with LTmAb and homologous IgG) and AIAb against the functional LT-binding site of LTmAb (in radioimmunoassay). Using the antisera, Western-blot analysis with peritoneal cell proteins detected signals at 236, 198, 118, 99, 75, 25 and 18 kDa. Dithiothreitol-reduced proteins were detected at 25 kDa and 18 kDa. In general, this suggested recognition of a 236-kDa oligomeric protein composed of subunits with molecular masses of 25 kDa and 18 kDa, including intramolecular disulfide bridges all bearing an epitope similar to the LTmAb. From these conformations, an overlay assay with [3H]LTD4 favoured a 75-kDa protein. Immunohistochemical analysis demonstrated that the recognized proteins may be located at cell membranes, because (a) in an ELISA, enriched plasma membrane preparations from peritoneal cells showed a threefold increase in reactivity to the AIAb, compared to the original cell homogenate; (b) after Western-blot analysis, the membrane-enriched protein fraction exhibited stronger protein signals than the microsomal fraction and the original cell homogenate; (c) regions of AIAb binding on the surface of cultured mouse peritoneal macrophages were detected by indirect immunofluorescence. Taken together, this study demonstrated AIAb binding to macrophage membrane-associated proteins bearing the LTD4-binding site of LTmAb, which may include identification of the putative LT receptor.

Resident peritoneal macrophages incubated with 3.5 x 10(-7) M Calcium ionophore A23187 in tumor cell growth medium (TGM) release large amounts of leukotriene (LT)E4 and an unidentified 5-lipoxygenase product, whereas A23187-stimulated macrophages produce in serum free medium LTD4, predominately. LTC4 and 3H-LTC4 incubated for 20 min at 37 degree C in serum containing TGM, convert into LTE4 and 3H-LTE4, respectively. Thus, LTC4 released from A23187-stimulated macrophages is an intermediate in TGM which rapidly converts into LTE4, probably because of the presence of gamma-glutamyl transpeptidase and cystenylglycinase in TGM. Macrophages express antitumor cytostatic activity towards P815 cells (49-53%) in a cocultured ratio (macrophage: tumor cell) 2:1 when stimulated with 3.5 x 10(-7) M A23187 in TGM. The 5-lipoxygenase inhibitor AA861 reverses the cytostatic activity by 42-58% and it inhibits also the formation of A23187-induced 5-lipoxygenase products from macrophages. Restoration of 38% macrophage- antitumor cytostatic activity by exogenous LTC4 (10(-8) M) indicates that LTC4 is an essential 5-lipoxygenase intermediate in the pathway of required signals underlying A23187-induced macrophage antitumor cytostatic activity. Macrophages not stimulated by A23187 do not express cytostatic activity in the presence of LTC4. This implies that besides LTC4, increased cytosolic [Ca2+] is required for A23187 induction of macrophage cytostatic activity.

A23187-treated murine peritoneal macrophages release increased quantities of the immunoreactive 5-lipoxygenase metabolite leukotriene B4 (LTB4) and the immunoreactive cyclooxygenase products 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha) and thromboxane B2 (TXB2) during a 40 min incubation period. The increase in release of LTB4 was marked already after 5 min of incubation and was maximal after 20 min. The increase in release of 6-keto PGF1 alpha and TXB2 started in most cases after 5 min of incubation and augmented gradually up to 40 min after incubation. The ratio of the increase of LTB4/6-keto PGF1 alpha and of LTB4/TXB2 revealed an increase in favor of LTB4 in the first 5 min of incubation.

A23187-stimulated cytostatic activity of peritoneal macrophages towards P815 tumor cells served as a model for macrophage activation: a macrophage enriched preparation, separated on the basis of cell size in a discontinuous FCS gradient column, expressed cytostatic activity when stimulated by A23187. This was inhibited dose-dependently, by AA-861 but not by nordihydroguaiaretic acid (NDGA). AA-861 inhibited 5-lipoxygenase specifically, NDGA inhibited both 5-lipoxygenase- and cyclooxygenase activity. The ratio cyclooxygenase/lipoxygenase products increased with AA-861 but not with NDGA. These results show that lipoxygenase products are necessary for expression of cytostatic activity of these arachidonic acid metabolite-producing macrophages and that the ratio cyclooxygenase/lipoxygenase metabolites plays an important role in macrophage activation.

The calcium ionophore A23187 stimulated cytostatic activity of peritoneal macrophages towards P815 tumor cells in coculture served as a model for macrophage activation. A macrophage enriched preparation, separated on the basis of cell size in a discontinuous fetal calf serum gradient column, expressed cytostatic activity when stimulated by A23187. This was inhibited dose-dependently, by AA-861 but not by nordihydroguaiaretic acid (NDGA). AA-861 inhibited the 5-lipoxygenase specifically, NDGA inhibited both lipoxygenase- and cyclooxygenase activity: The ratio cyclooxygenase/lipoxygenase products increased with AA-861 but not with NDGA. These results show that lipoxygenase products are necessary for expression of cytostatic activity of these arachidonic acid metabolite producing macrophages and that the ratio of cyclooxygenase/lipoxygenase metabolites plays an important role in macrophage activation.