Elevated NET, Calprotectin, and Neopterin Levels Discriminate between Disease Activity in COVID-19, as Evidenced by Need for Hospitalization among Patients in Northern Italy.

Coronavirus disease 2019 (COVID-19) displays clinical heterogeneity, but little information is available for patients with mild or very early disease. We aimed to characterize biomarkers that are useful for discriminating the hospitalization risk in a COVID-19 cohort from Northern Italy during the first pandemic wave. We enrolled and followed for four weeks 76 symptomatic SARS-CoV-2 positive patients and age/sex-matched healthy controls. Patients with mild disease were discharged (n.42), and the remaining patients were hospitalized (n.34). Blood was collected before any anti-inflammatory/immunosuppressive therapy and assessed for soluble C5b-9/C5a, H3-neutrophil extracellular traps (NETs), calprotectin, and DNase plasma levels via ELISA and a panel of proinflammatory cytokines via ELLA. Calprotectin and NET levels discriminate between hospitalized and non-hospitalized patients, while DNase negatively correlates with NET levels; there are positive correlations between calprotectin and both NET and neopterin levels. Neopterin levels increase in patients at the beginning of the disease and do so more in hospitalized than non-hospitalized patients. C5a and sC5b-9, and other acute phase proteins, correlate with neopterin, calprotectin, and DNase. Both NET and neopterin levels negatively correlate with platelet count. We show that calprotectin, NETs, and neopterin are important proinflammatory parameters potentially useful for discriminating between COVID-19 patients at risk of hospitalization.

DNase analysed by a novel competitive assay in patients with complications after ChAdOx1 nCoV-19 vaccination and in normal unvaccinated blood donors.

Increased levels of neutrophil extracellular traps (NETs) have been detected in individuals with vaccine complications after the ChAdOx1 nCov vaccine with a correlation between the severity of vaccine side effects and the level of NETosis. DNases may disrupt NETs by degrading their content of DNA, and a balance has been reported between NETs and DNases. Because of this and since the inflammatory marker NETs may be used as a confirmatory test in diagnosing VITT, it is of interest to monitor levels of DNase in patients with increased NETs levels. The current novel rapid DNase ELISA was tested in blood samples of patients with known increased levels of NETs with or without VITT after ChAdOx1 nCoV-19 vaccination. DNase levels in VITT patients were significantly increased compared with normal unvaccinated blood donors and compared with patients with post-vaccination symptoms but not VITT. However, since EDTA was found to inhibit DNase, serum and not EDTA-plasma samples should be applied for DNase testing. The novel DNase assay may serve as a supplementary test to the NETs test when analysing samples from patients with suspected increased NETs levels.

Effects of storage on quality and function of acid-treated platelets with reduced HLA Class I surface expression.

BACKGROUND: Refractory patients need to be provided with HLA-matched platelets (PLTs), which require time-consuming cross-matching. Treatment of PLTs with citric acid leads to denaturation of the HLA Class I complexes without significant damage to the PLTs. HLA Class I depleted PLTs could alternatively be used to HLA-matched PLTs for transfusion. These PLTs have verified normal function up to 4-6 h after acid treatment. MATERIALS AND METHODS: Buffy coat (BC) PLT concentrates were depleted of HLA Class I complexes by incubation in citric acid. The days after acid-treatment, surface expression of HLA Class I complexes, CD62P and CD63 were determined by flow cytometry, in addition to viability and mitochondrial membrane potential (MMP). Thromboelastography (TEG) tested PLT functionality. RESULTS: Expression of HLA Class I complexes was reduced by 70%-75% in acid-treated PLTs compared to untreated PLTs from day 1 through day 7. Controls and acid-treated PLTs showed insignificant loss of MMP stored for 4 days. Analysis of the residual PLT activation and viability showed no significant differences for 4 days of storage. However, the residual PLT activation potential and viability were significantly decreased in acid-treated PLTs and control PLTs after 7 days of storage. Acid treatment caused a significant decrease in the TEG variable, reaction time (R time), for acid-treated PLTs as compared to control PLTs from days 1 through day 3. CONCLUSION: Our data suggest that extended storage of acid-treated PLTs is possible and will improve flexibility when planning for transfusion of patients with alloimmune PLT refractoriness caused by anti-HLA-antibodies.

Inflammatory markers calprotectin, NETs, syndecan-1 and neopterin in COVID-19 convalescent blood donors.

Persisting inflammation has been discovered in lungs and other parenchymatous organs of some COVID-19 convalescents. Calprotectin, neutrophil extracellular traps (NETs), syndecan-1 and neopterin are general key inflammatory markers, and systemically enhanced levels of them may remain after the COVID-19 infection. These inflammatory markers were therefore measured in serum samples of 129 COVID-19 convalescent and 27 healthy blood donors or employees at Oslo Blood bank, Norway. Also antibodies against SARS-CoV-2 nucleocapsid antigen were measured, and timing of sampling and severity of infection noted. Whereas neopterin and NETs values remained low and those for syndecan-1 were not raised to statistically significant level, concentrations for calprotectin, as measured by a novel mixed monoclonal assay, were significantly increased in the convalescents. Antibodies against SARS-CoV-2 nucleocapsid antigen were elevated, but did not correlate with levels of inflammatory markers. Difference between the groups in only one biomarker makes evaluation of ongoing or residual inflammation in the convalescents difficult. If there is a low-grade inflammation, it would in that case involve neutrophils.

Elevated NETs and Calprotectin Levels after ChAdOx1 nCoV-19 Vaccination Correlate with the Severity of Side Effects.

ChAdOx1 nCoV-19 vaccination has been associated with the rare side effect; vaccine-induced immune thrombotic thrombocytopenia (VITT). The mechanism of thrombosis in VITT is associated with high levels of neutrophil extracellular traps (NETs). The present study examines whether key markers for NETosis, such as H3-NETs and calprotectin, as well as syndecan-1 for endotheliopathy, can be used as prognostic factors to predict the severity of complications associated with ChAdOx1 vaccination. Five patients with VITT, 10 with prolonged symptoms and cutaneous hemorrhages but without VITT, and 15 with only brief and mild symptoms after the vaccination were examined. Levels of H3-NETs and calprotectin in the vaccinated individuals were markedly increased in VITT patients compared to vaccinees with milder vaccination-associated symptoms, and a strong correlation (r ≥ 0.745, p < 0.001) was found with severity of vaccination side effects. Syndecan-1 levels were also positively correlated (r = 0.590, p < 0.001) in vaccinees to side effects after ChAdOx1 nCoV-19 vaccination. We hypothesize that the inflammatory markers NETs and calprotectin may be used as confirmatory tests in diagnosing VITT.

Proteomic study of apheresis platelets made HLA class I deficient for transfusion of refractory patients.

PURPOSE: Refractoriness can occur after repeated platelet (PLT) transfusions because of alloimmunization to HLA class I antigens on transfused PLTs and generation of anti-HLA antibodies that bind to the foreign PLTs and initiate their destruction. Such refractoriness can be overcome by provision of HLA-matched PLTs from HLA typed donors. However, since the procedure is both expensive and time-consuming, an alternative approach is to deplete PLTs of HLA class I molecules by a brief treatment with citric acid, on ice. This is shown to be feasible without damaging PLT function. We used label free quantitative mass spectrometry (MS)-based proteomics to investigate the effect of acid treatment on apheresis PLTs for combatting immunologic PLT refractoriness. EXPERIMENTAL DESIGN: Proteomic analyses are undertaken using PLTs from seven apheresis concentrates, which were split in two with or without acid treatment. RESULTS: In total 1717 proteins in apheresis PLTs were quantified using proteomics. Data are available via ProteomeXchange with identifier PXD027893 . Of these, the amount of 80 proteins changed significantly after acid treatment, but overall there were not any major differences in proteomes between samples with and without acid treatment. CONCLUSIONS AND CLINICAL RELEVANCE: In general, the changes of PLT proteins after treatment with citric acid were quite small and functionally safe. Hence, this result taken together with our previously published data indicates that acid treated PLTs can be used for treatment of patients with PLT refractoriness and opens up for a clinical trial.

HLA class I depletion by citric acid, and irradiation of apheresis platelets for transfusion of refractory patients.

BACKGROUND: Patients can form antibodies to foreign human leukocyte antigen (HLA) Class I antigens after exposure to allogeneic cells. These anti-HLA class I antibodies can bind transfused platelets (PLTs) and mediate their destruction, thus leading to PLT refractoriness. Patients with PLT refractoriness need HLA-matched PLTs, which require expensive HLA typing of donors, antibody analyses of patient sera and/or crossmatching. An alternative approach is to reduce PLT HLA Class I expression using a brief incubation in citric acid on ice at low pH. METHODS AND MATERIALS: Apheresis PLT concentrates were depleted of HLA Class I complexes by 5 minutes incubation in ice-cold citric acid, at pH 3.0. Surface expression of HLA Class I complexes, CD62P, CD63, phosphatidylserine, and complement factor C3c was analyzed by flow cytometry. PLT functionality was tested by thromboelastography (TEG). RESULTS: Acid treatment reduced the expression of HLA Class I complexes by 71% and potential for C3c binding by 11.5-fold compared to untreated PLTs. Acid-treated PLTs were significantly more activated than untreated PLTs, but irrespective of this increase in steady-state activation, CD62P and CD63 were strongly upregulated on both acid-treated and untreated PLTs after stimulation with thrombin receptor agonist peptide. Acid treatment did not induce apoptosis over time. X-ray irradiation did not significantly influence the expression of HLA Class I complexes, CD62P, CD63, and TEG variables on acid treated PLTs. CONCLUSION: The relatively simple acid stripping method can be used with irradiated apheresis PLTs and may prevent transfusion-associated HLA sensitization and overcome PLT refractoriness.

Antitumor, Anti-Inflammatory and Antiallergic Effects of Agaricus blazei Mushroom Extract and the Related Medicinal Basidiomycetes Mushrooms, Hericium erinaceus and Grifola frondosa: A Review of Preclinical and Clinical Studies.

Since the 1980s, medicinal effects have been documented in scientific studies with the related Basidiomycota mushrooms Agaricus blazei Murill (AbM), Hericium erinaceus (HE) and Grifola frondosa (GF) from Brazilian and Eastern traditional medicine. Special focus has been on their antitumor effects, but the mushrooms' anti-inflammatory and antiallergic properties have also been investigated. The antitumor mechanisms were either direct tumor attack, e.g., apoptosis and metastatic suppression, or indirect defense, e.g., inhibited tumor neovascularization and T helper cell (Th) 1 immune response. The anti-inflammatory mechanisms were a reduction in proinflammatory cytokines, oxidative stress and changed gut microbiota, and the antiallergic mechanism was amelioration of a skewed Th1/Th2 balance. Since a predominant Th2 milieu is also found in cancer, which quite often is caused by a local chronic inflammation, the three conditions-tumor, inflammation and allergy-seem to be linked. Further mechanisms for HE were increased nerve and beneficial gut microbiota growth, and oxidative stress regulation. The medicinal mushrooms AbM, HE and GF appear to be safe, and can, in fact, increase longevity in animal models, possibly due to reduced tumorigenesis and oxidation. This article reviews preclinical and clinical findings with these mushrooms and the mechanisms behind them.

NETs analysed by novel calprotectin-based assays in blood donors and patients with multiple myeloma or rheumatoid arthritis: A pilot study.

Two novel enzyme-linked immunosorbent assays (ELISAs), designed to detect complexes containing DNA, leucocyte calprotectin and S100A12 proteins, were generated for improved specificity and rapid measurement of neutrophil extracellular traps (NETs). The assays were applied on plasma and serum samples from blood donors for establishment of reference values, and from patients with multiple myeloma (MM) or rheumatoid arthritis (RA) in order to examine putatively increased values in the two different inflammatory conditions. Although NETs were hardly detectable in healthy individuals, NET levels were as expected highly and statistically significantly increased in RA patients. The detection of statistically significantly increased NET levels in MM is a novel finding.

Agaricus blazei-Based Mushroom Extract Supplementation to Birch Allergic Blood Donors: A Randomized Clinical Trial.

Since Agaricus blazei Murill (AbM) extract reduced specific IgE and ameliorated a skewed Th1/Th2 balance in a mouse allergy model, it was tested in blood donors with self-reported, IgE-positive, birch pollen allergy and/or asthma. Sixty recruited donors were randomized in a placebo-controlled, double-blinded study with pre-seasonal, 7-week, oral supplementation with the AbM-based extract AndosanTM. Before and after the pollen season, questionnaires were answered for allergic rhino-conjunctivitis, asthma, and medication; serum IgE was measured, and Bet v 1-induced basophil activation was determined by CD63 expression. The reported general allergy and asthma symptoms and medication were significantly reduced in the AbM compared to the placebo group during pollen season. During the season, there was significant reduction in specific IgE anti-Bet v 1 and anti-t3 (birch pollen extract) levels in the AbM compared with the placebo group. While the maximal allergen concentrations needed for eliciting basophil activation before the season, changed significantly in the placebo group to lower concentrations (i.e., enhanced sensitization) after the season, these concentrations remained similar in the AndosanTM AbM extract group. Hence, the prophylactic effect of oral supplementation before the season with the AbM-based AndosanTM extract on aeroallergen-induced allergy was associated with reduced specific IgE levels during the season and basophils becoming less sensitive to allergen activation.

Cytotoxic Effect on Human Myeloma Cells and Leukemic Cells by the Agaricus blazei Murill Based Mushroom Extract, Andosan™.

Agaricus blazei Murill is an edible mushroom of the Basidiomycetes family, which has been found to contain a number of compounds with antitumor properties, such as proteoglycans and ergosterol. In the present investigation, we show that the commercial mushroom product Andosan, which contains 82.4% Agaricus blazei Murill, together with medicinal mushrooms Hericium erinaceus (14.7%) and Grifola frondosa (2.9%), has a cytotoxic effect on primary myeloma cells, other myeloma cell lines, and leukemia cell lines in vitro. Although the exact content and hence the mechanisms of action of the Andosan extract are unknown, we have found in this investigation indications of cell cycle arrest when myeloma cell lines are cultivated with Andosan. This may be one of the possible explanations for the cytotoxic effects of Andosan.

Human Adipose-Derived Mesenchymal Stem Cells Respond to Short-Term Hypoxia by Secreting Factors Beneficial for Human Islets In Vitro and Potentiate Antidiabetic Effect In Vivo.

Adipose-derived mesenchymal stem cells (ASCs) release factors beneficial for islets in vitro and protect against hyperglycemia in rodent models of diabetes. Oxygen tension has been shown to induce metabolic changes and alter ASCs' release of soluble factors. The effects of hypoxia on the antidiabetic properties of ASCs have not been explored. To investigate this, we incubated human ASCs for 48 h in 21% (normoxia) or 1% O2 (hypoxia) and compared viability, cell growth, surface markers, differentiation capability, and soluble factors in the conditioned media (CM). Human islets were exposed to CM from ASCs incubated in either normoxia or hypoxia, and islet function and apoptosis after culture with or without proinflammatory cytokines were measured. To test hypoxic preconditioned ASCs' islet protective effects in vivo, ASCs were incubated for 48 h in normoxia or hypoxia before being injected into Balb/c Rag 1-/- immunodeficient mice with streptozotocin-induced insulitis. Progression of diabetes and insulin content of pancreas were measured. We found that incubation in hypoxia was well tolerated by ASCs and that levels of VEGF-A, FGF-2, and bNGF were elevated in CM from ASCs incubated in hypoxia compared to normoxia, while levels of HGF, IL-8, and CXCL1 were reduced. CM from ASCs incubated in hypoxia significantly improved human islet function and reduced apoptosis after culture, and reduced cytokine-induced apoptosis. In our mouse model, pancreas insulin content was higher in both groups receiving ASCs compared to control, but the mice receiving preconditioned ASCs had lower random and fasting blood glucose, as well as improved oral glucose tolerance compared to untreated mice. In conclusion, our in vitro results indicate that the islet protective potential of ASCs improves in hypoxia, and we give insight into factors involved in this. Finally we show that hypoxic preconditioning potentiates ASCs' antidiabetic effect in vivo.

3',4'-Dimethoxyflavone and valproic acid promotes the proliferation of human hematopoietic stem cells.

INTRODUCTION: Human hematopoietic stem cells (HSCs) have been clinically used for transplantation and gene and cellular therapy for more than 4 decades. However, this use is limited because of the challenges in the ex vivo culturing of HSCs. The major hurdle is to amplify these cells without losing their self-renewing property. METHODS: In our study, we tested 3',4'-dimethoxyflavone (3'4'-DMF) and valproic acid (VPA) on the ex vivo expansion of HSCs under both normoxic (20% O2) and hypoxic (1% O2) conditions. 3'4'-DMF is a widely used anticancer drug that acts as a competitive antagonist of the aryl hydrocarbon receptor. VPA is a potent inhibitor of histone deacetylase and is used in the treatment of neurologic disorders. RESULTS: Culturing HSCs (from mobilized peripheral blood) under normoxia, with 3'4'-DMF and VPA, highly preserved the CD34 positivity (3'4'-DMF, 22.1%, VPA, 20.3%) after 1 week and strongly enhanced the CD34(+) cells (3'4'-DMF, 27.8 fold; VPA, 34.1 fold) compared with the control cultures (11.6% and 14.4 fold). Addition of 3'4'-DMF and VPA also resulted in more primary colonies and replating efficiency compared with control cultures. Although no significant effect was observed on the enhancement of CD34(+) cells under hypoxia, the number of primary colonies was significantly higher than the control cultures. CONCLUSIONS: Based on these findings, this study presents, for the first time, in vitro evidence for a new and relevant effect of 3'4'-DMF on human HSCs. In addition, the results suggest a potential clinical use of 3'4'-DMF and VPA in HSC therapy.

Biological response modifiers in photochemically pathogen-reduced versus untreated apheresis platelet concentrates.

BACKGROUND: Lipids and other biologically active substances accumulate in platelet concentrates (PCs) during storage. Some of these substances have been suggested to modulate immune responses and to play a pathogenic role in the development of transfusion-related acute lung injury. This study compared the content and impact of some biological response modifiers in PCs treated with pathogen reduction (PR) technology and nontreated PCs. STUDY DESIGN AND METHODS: Apheresis PCs (n = 12) were split in two: one split was subjected to PR treatment (INTERCEPT, Cerus Corp.) and the other split was left untreated. Basic characterization and content of vascular endothelial growth factor (VEGF) and sCD154 were measured. Lipopolysaccharide (LPS)-induced secretion of interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) was measured after incubation of heparinized whole blood with platelet (PLT) supernatants. The supernatants' neutrophil (PMN)-priming capacity, and thereby activation of the NADPH oxidase, was measured as the rate of superoxide anion production after formyl-Met-Leu-Phe activation. Lipids were extracted from the supernatants on Day 6 and tested for PMN-priming activity. RESULTS: Supernatants from PR-treated PCs demonstrated significantly higher mean PLT volume (MPV) and O(2) , lower pH, CO(2) , and HCO(3-) , and significantly less LPS-induced TNF-α secretion compared to untreated PCs. No differences in swirling, PLT count, potassium levels, glucose consumption, lactate production, IL-10, VEGF, sCD154, or PMN-priming activity were found between the groups over time. CONCLUSION: INTERCEPT PR treatment caused no substantial differences in PCs, except for minor changes in MPV and metabolic variables. Further studies are needed to explain the differences in the LPS-induced TNF-α secretion.

Glycogen synthase kinase-3 (GSK-3) inhibition induces apoptosis in leukemic cells through mitochondria-dependent pathway.

The roles of glycogen synthase kinase-3 (GSK-3) in cell survival and apoptosis are controversial. We examined the effect of a specific GSK-3 inhibitor (SB-415286) on the regulation of leukemic cells proliferation and apoptosis. SB-415286 (40 µM) induced cell growth inhibition, ß-catenin stabilization, cell cycle arrest in G(2)/M phase, cyclin B1 downregulation, and apoptosis in leukemic cell lines KG1a, K562, and CMK. Blocking the death receptor pathway by using a specific inhibitor of caspase-8, did not inhibit SB-415286-induced apoptosis. This indicates that activation of caspase-8 is part of the intrinsic apoptotic pathway and occurs downstream of mitochondria membrane potential depolarization mediated by other caspases. Furthermore, we found that depolarization of mitochondria membrane caused by GSK-3 inhibition is regulated by dephosphorylation of anti-apoptotic protein Bcl-2 and downregulation of Bcl-xL. Thus, inhibition of GSK-3-induced apoptosis of leukemic cells could be an attractive target for treatment of leukemia.

Evaluation of nonleukoreduced red blood cell transfusion units collected at delivery from the placenta.

BACKGROUND: The objective of this study was to evaluate the suitability of cord blood (CB) as a source of red blood cells (RBCs) for autologous transfusion. STUDY DESIGN AND METHODS: CB was collected in 150-mL storage containers with citrate phosphate dextrose (CPD) as anticoagulant and stored in either saline, adenine, glucose, and mannitol (SAG-M; n = 18) or phosphate, adenine, glucose, guanosine, saline, and mannitol (PAGGS-M; n = 18) for 35 days at 4 degrees C. Hematologic status and hemolysis were studied. The lipopolysaccharide (LPS)-induced production of tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta1 from CB monocytes was analyzed after incubation with addition of weekly sampled supernatants from the CB RBC units. Five additional units (PAGGS-M) were leukoreduced and thereafter analyzed as indicated above. RESULTS: Hemolysis increased significantly over time, in SAG-M more than in PAGGS-M. During storage in both media, the number of white blood cells (WBCs) decreased, and the LPS-induced production of TNF-alpha and TGF-beta1 decreased and increased, respectively. There were no significant changes in the LPS-induced production of TNF-alpha and TGF-beta1 in the leukoreduced CB RBC units. CONCLUSION: Hemolysis in CB RBC units increased significantly over time, and PAGGS-M appears to be superior to SAG-M as a preservation solution for CB RBC. The changes in LPS-induced TNF-alpha and TGF-beta1 production over time were probably caused by substances released from apoptotic and/or necrotic WBCs. Further studies are needed to identify both which substances are responsible for the changes in LPS-induced cytokine release and the clinical significance hereof.

Evaluation of platelet activation and cytokine release during storage of platelet concentrates processed from buffy coats either manually or by the automated OrbiSac system.

BACKGROUND: This study aimed to compare platelet (PLT) quality during storage of buffy coat (BC) PLT concentrates (PCs), prepared either manually or by the automated OrbiSac system (Gambro BCT). STUDY DESIGN AND METHODS: Following overnight storage at 20 to 22 degrees C, five BCs were pooled with 300 mL of PLT additive solution. Twenty-one PCs were produced manually (M-PCs) and 21 by the automated OrbiSac system (A-PCs). Swirling, PLT count, mean PLT volume, blood gas analyses, potassium, glucose, and lactate were assessed. Expression of the activation markers CD42a, CD62P, and PAC-1 was analyzed by flow cytometry on resting PLTs and PLTs stimulated with thrombin receptor agonist peptide (TRAP). Levels of CCL5 and transforming growth factor-beta1 (TGF-beta1) were measured by enzyme-linked immunosorbent assay. RESULTS: The A-PCs had significantly larger volume and higher PLT yield, PLT recovery, and white blood cell concentration than the M-PCs, whereas the red blood cell content was significantly highest in the M-PCs. pH levels were between 6.9 and 7.2 in all PCs. Neither glucose consumption nor lactate production differed significantly over time. A-PCs had, compared to M-PCs, significantly higher expression of CD62P on resting PLTs, lower capacity for up regulating CD62P on TRAP-stimulated PLTs, and higher levels of CCL5 during storage. TRAP-stimulated A-PCs had a significantly higher potential for down regulation of CD42a than M-PCs. No difference was found in TGF-beta1 levels or TRAP-induced up regulation of PAC-1. CONCLUSION: The levels of CCL5 and the expression of CD62P in resting and stimulated PLTs indicate that PLTs in A-PCs are slightly more activated than in M-PCs, but the clinical importance of this finding is yet unknown.

Platelet activation and residual activation potential during storage of hyperconcentrated platelet products in two different platelet additive solutions.

BACKGROUND: To improve platelet (PLT) quality, hyperconcentrated PLT concentrates (hcPCs) were compared to standard PLT concentrates (stdPCs) in two different PLT additive solutions, T-Sol and PAS-27a. PAS-27a differs from T-Sol by containing glucose, phosphate, potassium, magnesium, and bicarbonate. STUDY DESIGN AND METHODS: PLTs were harvested by apheresis twice from 14 donors; each unit was divided into two. Four units from each donor were produced: hcPCs, 2000 x 10(9) per L in T-Sol or PAS-27a; and stdPCs, 1400 x 10(9) per L in 65 percent T-Sol or PAS-27a and 35 percent acid citrate dextrose-plasma. On Days 1 through 4, swirling was scored and PLT count, mean PLT volume, pH, blood gas, glucose, and lactate were measured. Expression of CD42a, CD62P, CD63, and PAC-1 was analyzed by flow cytometry on resting PLTs and PLTs stimulated with thrombin receptor agonist peptide (TRAP). RESULTS: Glucose consumption and lactate production were significantly higher in hcPCs stored in PAS-27a than in T-Sol. Both stdPC and hcPC PLTs in T-Sol expressed CD62P and PAC-1 significantly higher than in PAS-27a. Over time the T-Sol hcPCs revealed highest expression of CD62P and CD63. A significantly higher capacity for up regulation of CD62P, CD63, and PAC-1 upon TRAP stimulation was found for stdPCs and hcPCs in PAS-27a compared to PLTs in T-Sol. TRAP-stimulated PLTs in stdPCs and hcPCs suspended in PAS-27a showed significantly higher potential for down regulation of CD42a than the T-Sol concentrates. CONCLUSIONS: PLTs appear better preserved in vitro in PAS-27a than in T-Sol, and this suggests that storage of hcPCs in PAS-27a could be extended beyond 24 hours.

Expression and involvement of Toll-like receptors (TLR)2, TLR4, and CD14 in monocyte TNF-alpha production induced by lipopolysaccharides from Neisseria meningitidis.

BACKGROUND: The present study was undertaken to examine the ability of lipopolysaccharide-containing outer membrane vesicles (OMV-LPS) and purified LPS (P-LPS) from the same meningococcal strain to induce the expression of Toll-like receptors (TLR2 and TLR4) and TNF-alpha production in leukocytes, and further to study the involvement of TLRs, and CD14 in monocyte TNF- alpha production in an ex vivo human whole blood system. MATERIAL/METHODS: OMV-LPS or P-LPS were added to human whole blood and expression of TLR2/4 and production of TNF- alpha in leukocytes were measured by flow cytometry. To study involvement of TLRs and CD14 in monocyte cytokine production we used monoclonal antibodies against TLR2/4 and CD14. RESULTS: OMV-LPS and P-LPS induced surface expression (maximal at 120 min) of TLR2 and TLR4 on granulocytes and monocytes. LPS incorporated in OMV was less potent (weight basis) than P-LPS in inducing monocyte TNF- alpha production. When inducing monocyte TNF-alpha by OMV-LPS, antibodies directed against TLR2 and TLR4 caused 45 and 78% inhibition, respectively. When inducing TNF- alpha by P-LPS, antibodies against TLR2 had no effect, whereas anti-TLR4 antibodies caused 63% inhibition. Antibodies against CD14 inhibited nearly completely the monocyte TNF- alpha response induced by meningococcal LPS irrespective of whether LPS was presented in purified form or incorporated in membrane vesicles. CONCLUSIONS: OMV-LPS and P-LPS from the same meningococcal strain induced expression of TLR2/4 on monocytes and granulocytes. Surface receptors TLR2/4 and CD14 are essential for in vitro cellular activation induced by OMV-LPS and P-LPS, but the functional significance of these receptors during meningococcal infections remains elusive.

Outer membrane vesicles from Neisseria meningitidis.

Flow cytometry was used to study the expression of leukocyte adhesion molecules CD11a, CD11b, CD11c, CD14, and CD62L (L-selectin) and production of reactive oxygen species (ROS) in an ex vivo human whole-blood system stimulated with lipopolysaccharide-containing outer membrane vesicles (LPS-OMV) from N. meningitidis. Results demonstrated a dose-dependent increase in surface expression of CD11a, CD11b, CD11c and CD14 in granulocytes and monocytes (maximal at 30-120 min) upon OMV-LPS challenge, whereas CD62L expression was heavily downregulated (maximal at 30-120 min). The OMV-associated LPS was almost as potent (on a weight basis) as purified LPS from E. coli in inducing adhesion molecule modulation but the response was delayed. Upon stimulation with OMV-LPS or E. coli-LPS, the production of intracellular ROS increased in both granulocytes and monocytes when dihydroethidium (DHE, mainly reflecting superoxide anion) was used as a probe, whereas peroxynitrite production monitored with dihydrorhodamine 123 (DHR) was not significantly changed. The OMV-mediated modulation of leukocyte adhesion molecule expression and increased ROS production may certainly lead to increased entrapment of leukocytes in the microcirculation and contribute to untoward inflammatory reactions as seen in systemic meningococcal disease.