Stimulation Of Whole Blood Research Articles

Predicting Cerdulatinib Therapeutic Concentrations in Patients with Relapsed/Refractory B Cell Malignancies Using Preclinical Models and Clinical PK/PD and Tumor Response Correlates

▪Background: Preclinical data and early clinical studies demonstrate a reliance on B cell antigen receptor (BCR) signaling and/or cytokine-induced JAK/STAT signaling for survival of subsets of B cell malignancies. Increased serum concentrations of several cytokines are observed in CLL and NHL and predict a more aggressive disease progression (Yan, X. J., et al, 2011; Mahadevan, D., et al, 2009; el Far. M., et al, 2004; Lai, R., et al, 2002; Fayed, L., et al, 2001). The source of these cytokines may be derived from the tumor itself in an autocrine stimulation fashion, or from non-tumor leukocytes which have mounted an ineffective immune response within the tumor microenvironment. Experimentally, IL4 has been shown to promote the survival of CLL B cells in culture and protect them from death by treatment with fludarabine and chlorambucil (Steele, A.J., et al, 2010). The mechanism underlying this survival support appears to be cytokine-induced up-regulation of BCL2 family members (Castejon, R., et al, 1999; Jewell, A.P., et al, 1994; Panayiotidis, P., et al, 1993; Dancescu, M., et al, 1992). Combined SYK/JAK inhibition may therefore represent a powerful strategy to treat B cell malignancies. Cerdulatinib is a reversible ATP competitive inhibitor of SYK, JAK1, JAK3, and TYK2 that is currently in clinical development for the treatment of relapsed/refractory B cell lymphoma and leukemia. We review here cerdulatinib preclinical and clinical pharmacodynamic/pharmacokinetic relationships to tumor response in treated patients.Methods: Inhibition of SYK- and JAK-dependent and independent signaling was determined by intracellular phospho-flow following stimulation of human whole blood with various agonists against BCR and cytokine receptors. Anti-tumor activities of cerdulatinib and other selective inhibitors of BCR and JAK/STAT pathways were evaluated in fourteen DLBCL cell lines by CellTiter Glo, caspase 3 induction, and cell cycle arrest. In vivo anti-inflammatory activity of cerdulatinib was determined using the rat collagen induced arthritis model. The effective concentrations pre-clinically were compared to cerdulatinib-induced SYK and JAK inhibition, anti-inflammatory activity, and anti-tumor activity following oral dosing in relapsed/refractory CLL and NHL patients.Results: Cerdulatinib demonstrated broad anti-tumor activity in DLBCL cell lines, relative to other targeted agents. In a panel of 14 cell lines representing both ABC and GCB subtypes, 9 underwent apoptosis and an additional 2 underwent cell cycle arrest. Cooperative effects of SYK and JAK inhibition were observed in 4 of the cell lines, whereas 3 cell lines were sensitive to SYK but not JAK inhibition, and 1 cell line was sensitive to JAK but not SYK inhibition. Three of the 14 cell lines were resistant to cerdulatinib. Apoptosis was induced in the majority of DLBCL cell lines at 2 µM (achieved at clinical doses ≥40 mg). In healthy whole blood ex vivo spiking experiments, cerdulatinib selectively inhibited BCR/SYK and cytokine (IL2, IL4, IL6) JAK/STAT signaling with IC50's ranging from 0.2-0.9 µM (achieved at clinical doses of 15 mg-40 mg). Inflammation and joint destruction in the rat collagen-induced arthritis model was arrested at 0.3 µM average plasma concentration (achieved at clinical doses ≥30 mg). In cancer patients receiving cerdulatinib once daily, plasma concentrations above 2 µM have been safely achieved, while maintaining steady-state Cmin of ~1 µM. Following oral dosing in patients, SYK and JAK pathways were inhibited with similar potency as was observed in healthy volunteer ex vivo spiking experiments with >90% inhibition of SYK and JAK safely achieved for multiple cycles of therapy. Tumor response as measured by CT scan strongly correlated with percent inhibition of SYK and JAK signaling in patient-derived whole blood, and percent inhibition of serum markers of inflammation (e.g., β2M, CRP, IL10, VCAM1, sTNFR, CCL3).Conclusion: Once daily oral dosing of cerdulatinib at 40-65 mg achieves steady-state plasma concentrations in the 1-2µM range (trough to peak), which is sufficient to block SYK/JAK signaling, inflammatory mechanisms, and affect tumor viability both in DLBCL cell lines and in patients. We conclude that these exposure levels are sufficient to begin testing the clinical potential of cerdulatinib. We will begin enrolling phase Ib/IIa expansion cohorts this year. DisclosuresCoffey:Portola Pharmaceuticals Inc: Employment, Equity Ownership, Research Funding. Feng:Portola Pharmaceuticals: Employment, Equity Ownership, Research Funding. Michelson:Portola Pharmaceuticals Inc: Employment, Equity Ownership, Research Funding. Leads:Portola Pharmaceuticals: Employment, Equity Ownership, Research Funding. Curnutte:Portola Pharmaceuticals, Inc.: Employment; 3-V Biosciences: Equity Ownership; Sea Lane Biotechnologies: Consultancy. Pandey:Portola Pharmaceuticals Inc: Employment. Conley:Portola Pharmaceuticals, Inc.: Employment.

Antigen-Specific Mammary Inflammation Depends on the Production of IL-17A and IFN-γ by Bovine CD4+ T Lymphocytes.

Intramammary infusion of the antigen used to sensitize cows by the systemic route induces a local inflammation associated with neutrophil recruitment. We hypothesize that this form of delayed type hypersensitivity, which may occur naturally during infections or could be induced intentionally by vaccination, can impact the outcome of mammary gland infections. We immunized cows with ovalbumin to identify immunological correlates of antigen-specific mammary inflammation. Intraluminal injection of ovalbumin induced a mastitis characterized by a prompt tissue reaction (increase in teat wall thickness) and an intense influx of leukocytes into milk of 10 responder cows out of 14 immunized animals. The magnitude of the local inflammatory reaction, assessed through milk leukocytosis, correlated with antibody titers, skin thickness test, and production of IL-17A and IFN-γ in a whole-blood antigen stimulation assay (WBA). The production of these two cytokines significantly correlated with the magnitude of the milk leukocytosis following the ovalbumin intramammary challenge. The IL-17A and IFN-γ production in the WBA was dependent on the presence of CD4+ cells in blood samples. In vitro stimulation of peripheral blood lymphocytes with ovalbumin followed by stimulation with PMA/ionomycin allowed the identification by flow cytometry of CD4+ T cells producing either IL-17A, IFN-γ, or both cytokines. The results indicate that the antigen-specific WBA, and specifically IL-17A and IFN-γ production by circulating CD4+ cells, can be used as a predictor of mammary hypersensitivity to protein antigens. This prompts further studies aiming at determining how Th17 and/or Th1 lymphocytes modulate the immune response of the mammary gland to infection.

Reduced Responsiveness of Blood Leukocytes to Lipopolysaccharide Does not Predict Nosocomial Infections in Critically Ill Patients.

Critically ill patients show signs of immune suppression, which is considered to increase vulnerability to nosocomial infections. Whole-blood stimulation is frequently used to test the function of the innate immune system. We here assessed the association between whole-blood leukocyte responsiveness to lipopolysaccharide (LPS) and subsequent occurrence of nosocomial infections in critically ill patients admitted to the intensive care unit (ICU). All consecutive critically ill patients admitted to the ICU between April 2012 and June 2013 with two or more systemic inflammatory response syndrome criteria and an expected length of ICU stay of more than 24 h were enrolled. Age- and sex-matched healthy individuals were included as controls. Blood was drawn the first morning after ICU admission and stimulated ex vivo with 100 ng/mL ultrapure LPS for 3 h. Tumor necrosis factor-α, interleukin-1β (IL-1β), and IL-6 were measured in supernatants. Seventy-three critically ill patients were included, of whom 10 developed an ICU-acquired infection. Compared with healthy subjects, whole-blood leukocytes of patients were less responsive to ex vivo stimulation with LPS, as reflected by strongly reduced tumor necrosis factor-α, IL-1β, and IL-6 levels in culture supernatants. Results were not different between patients who did and those who did not develop an ICU-acquired infection. The extent of reduced LPS responsiveness of blood leukocytes in critically ill patients on the first day after ICU admission does not relate to the subsequent development of ICU-acquired infections. These results argue against the use of whole-blood stimulation as a functional test applied early after ICU admission to predict nosocomial infection.

Lack of LCAT reduces the LPS-neutralizing capacity of HDL and enhances LPS-induced inflammation in mice

HDL has important immunomodulatory properties, including the attenuation of lipopolysaccharide (LPS)-induced inflammatory response. As lecithin–cholesterol acyltransferase (LCAT) is a critical enzyme in the maturation of HDL we investigated whether LCAT-deficient (Lcat−/−) mice present an increased LPS-induced inflammatory response. LPS (100μg/kg body weight)-induced cytokine response in Lcat−/− mice was markedly enhanced and prolonged compared to wild-type mice. Importantly, reintroducing LCAT expression using adenovirus-mediated gene transfer reverted their phenotype to that of wild-type mice. Ex vivo stimulation of whole blood with LPS (1–100ng/mL) showed a similar enhanced pro-inflammatory phenotype. Further characterization in RAW 264.7 macrophages in vitro showed that serum and HDL, but not chylomicrons, VLDL or the lipid-free protein fraction of Lcat−/− mice, had a reduced capacity to attenuate the LPS-induced TNFα response. Analysis of apolipoprotein composition revealed that LCAT-deficient HDL lacks significant amounts of ApoA-I and ApoA-II and is primarily composed of ApoE, while HDL from Apoa1−/− mice is highly enriched in ApoE and ApoA-II. ApoA-I-deficiency did not affect the capacity of HDL to neutralize LPS, though Apoa1−/− mice showed a pronounced LPS-induced cytokine response. Additional immunophenotyping showed that Lcat−/− , but not Apoa1−/− mice, have markedly increased circulating monocyte numbers as a result of increased Cd11b+Ly6Cmed monocytes, whereas ‘pro-inflammatory’ Cd11b+Ly6Chi monocytes were reduced. In line with this observation, peritoneal macrophages of Lcat−/− mice showed a markedly dampened LPS-induced TNFα response. We conclude that LCAT-deficiency increases LPS-induced inflammation in mice due to reduced LPS-neutralizing capacity of immature discoidal HDL and increased monocyte number.

PrimeFlow™ RNA assay as a method to elucidate the effect of protein transport inhibitors on cytokine gene expression in whole blood (TECH3P.941)

Abstract Intracellular antibody staining followed by flow cytometric analysis is commonly used to study cytokine expression in leukocytes. Detection of cytokines typically requires that cells be treated with protein transport inhibitors such as Brefeldin A and Monensin in order to accumulate sufficient levels of intracellular protein. However, it is not known whether the use of these inhibitors affects gene expression. PrimeFlow™ RNA is a new flow cytometric assay that allows the simultaneous interrogation of RNA and protein levels within single cells. We have used this assay to investigate the effect of protein transport inhibitors on the expression of human IL-2, TNF alpha and IFN gamma during the course of a 5 hour stimulation of whole blood with PMA and ionomycin. This represents first time that the effect of protein transport inhibitors on gene expression in whole blood has been examined in single cells.

Abstract 447: β-cyclodextrin Reduces Cholesterol Crystal-induced Inflammation Through Modulating Complement Activation

Atherosclerosis is an inflammatory condition and the underlying cause for cardiovascular disease. Cholesterol crystals (CC) are found to be abundant in atherosclerotic plaques and we have previously shown that CC initiate an inflammatory response via the complement system and inflammasome activation. Cyclic oligosaccharide 2-hydroxypropyl-β-cyclodextrin (BCD) is a compound that solubilizes lipophilic substances and is commonly used in pharmaceuticals or drug delivery. BCD is reported to increase cholesterol solubility and to promote the removal of cholesterol from foam cells. However, it remains unknown whether BCD has any effect on crystalline cholesterol. We here show that BCD attenuates the CC -induced inflammatory cytokine response as well as regulates a range of CC-related genes in human peripheral blood mononuclear cells. BCD binds to CC and prevents deposition of complement factors on CC in human plasma. Furthermore, BCD also decreases the formation of soluble terminal complement complex (TCC) and the expression of complement receptor 3 in response to CC stimulation in human whole blood. Induction of TCC by mono sodium urate crystals or zymosan was not affected by BCD. Of interest, after 1 hr of incubation, BCD is starting to dissolve the CC. These data demonstrate that BCD is a strong inhibitor of CC-induced inflammation, which might be explained by BCD-mediated attenuation of complement activation. These data suggest that BCD is a potential candidate for treatment of atherosclerosis.

Endogenous microparticles drive the proinflammatory host immune response in severely injured trauma patients.

Severe trauma affects the immune system, which in its turn is associated with poor outcome. The mediators driving the immune responses in trauma are largely unknown. The aim of this study was to investigate the role of endogenous microparticles (MPs) in mediating the immune response following severe trauma. A prospective, observational substudy of the ACIT II (Activation of Coagulation and Inflammation in Trauma II) study was performed at our academic level I trauma center. Adult multiple-trauma patients with an injury severity score of 15 or higher were included between May 2012 and June 2013. Ex vivo whole-blood stimulation with lipopolysaccharide was performed on aseptically collected patient plasma containing MPs and in plasma depleted of MPs. Flow cytometry and transmission electronic microscopy were performed on plasma samples to investigate the numbers and cellular origin of MPs. Healthy individuals served as a control group. Ten trauma patients and 10 control subjects were included. Trauma patients were significantly injured with a median injury severity score of 19 (range, 17-45). Patients were neither in shock nor bleeding. On admission to the hospital, the host response to bacterial stimulation was blunted in trauma patients compared with control subjects, as reflected by decreased production of interleukin 6 (IL-6), IL-10, and tumor necrosis factor α (P < 0.001). In trauma patients, MP-positive plasma was associated with a significantly higher synthesis of IL-6 and tumor necrosis factor α compared with plasma depleted from MPs (P = 0.047 and 0.002, respectively). Compared with control subjects, the number of circulating MPs was significantly decreased in trauma patients (P = 0.009). Most MPs originated from platelets. Multiple cellular protrusions, which result in MP formation, were observed in plasma from trauma patients, but not in control subjects. On admission, trauma patients have a reduced immune response toward endotoxin challenge, which is, at least in part, mediated by MPs, which circulate in low numbers and in early stages. Most MPs originate from platelets, which indicates that these cells may be the most important source of MPs involved in initiating an inflammatory host response after injury.

Genetic Variation in Pattern Recognition Receptors and Adaptor Proteins Associated With Development of Chronic Q Fever.

Q fever is an infection caused by Coxiella burnetii. Persistent infection (chronic Q fever) develops in 1%-5% of patients. We hypothesize that inefficient recognition of C. burnetii and/or activation of host-defense in individuals carrying genetic variants in pattern recognition receptors or adaptors would result in an increased likelihood to develop chronic Q fever. Twenty-four single-nucleotide polymorphisms in genes encoding Toll-like receptors, nucleotide-binding oligomerization domain-like receptor-2, αvβ3 integrin, CR3, and adaptors myeloid differentiation primary response protein 88 (MyD88), and Toll interleukin 1 receptor domain-containing adaptor protein (TIRAP) were genotyped in 139 patients with chronic Q fever and in 220 controls with cardiovascular risk-factors and previous exposure to C. burnetii. Associations between these single-nucleotide polymorphisms and chronic Q fever were assessed by means of univariate logistic regression models. Cytokine production in whole-blood stimulation assays was correlated with relevant genotypes. Polymorphisms in TLR1 (R80T), NOD2 (1007fsX1), and MYD88 (-938C>A) were associated with chronic Q fever. No association was observed for polymorphisms in TLR2, TLR4, TLR6, TLR8, ITGAV, ITGB3, ITGAM, and TIRAP. No correction for multiple testing was performed because only genes with a known role in initial recognition of C. burnetii were included. In the whole-blood assays, individuals carrying the TLR1 80R-allele showed increased interleukin 10 production with C. burnetii exposure. Polymorphisms in TLR1 (R80T), NOD2 (L1007fsX1), and MYD88 (-938C>A) are associated with predisposition to development of chronic Q fever. For TLR1, increased interleukin 10 responses to C. burnetii in individuals carrying the risk allele may contribute to the increased risk of chronic Q fever.

Diminished ROS Production in Neonatal Polymorphonuclear Leukocytes Stimulated with Escherichia coli

Bacterial infection is an important cause of death and long-term morbidity in neonates, especially in immature preterm neonates. The present study clarified the defense mechanism in neonates by measuring superoxide anion (one of reactive oxygen species: ROS) production and phagocytic activity of polymorphonuclear leukocytes (PMNs) caused by live bacterial stimulation in whole blood. In neonatal PMNs, superoxide anion production was significantly diminished when stimulated with an appropriate amount of Escherichia coli (E. coli). There was no significant difference in ROS production with stimulation of Staphylococcus aureus (S. aureus) between adult and neonatal PMNs. Both adult and neonatal PMNs showed equal activity in E. coli phagocytosis. The expression of Toll-Like Receptor 2 (TLR2), Toll-Like Receptor 4 (TLR4) and CD14 on the PMNs showed no difference between neonate and adult blood. The p38 mitogen-activated protein kinase (p38 MAPK) was also activated equally with the stimulation of live bacteria in both PMNs. Since p38 MAPK is a key molecule in lipopolysaccharide binding protein (LBP)-TLR4 mediating intracellular pathway, the receptor mediated signal transduction pathways have no difference between adult and neonatal PMNs. Diminished superoxide production of neonatal PMNs with E. coli stimulation was reconstituted, when neonatal PMNs were stimulated in adult plasma. This phenomenon was abolished by the heat inactivation of adult plasma but not by the addition of anti-interferon γ or anti-tumor necrosis factor α blocking antibody. These data suggest that some heat-labile factors in whole blood might have a role in augmentation of the ROS production of neonatal PMNs, responding to E. coli.

Development and pharmacological validation of novel methods of B cell activation in rat whole blood.

Whole blood functional assays are pharmacologically relevant in the drug discovery process to evaluate potency in a relevant biological matrix, to support establishment of PK/PD relationships and to aid in human dose predictions. However development of B cell activation assays by BCR ligation in rat whole blood has not been previously described. The aim of the present study was to develop novel methods of B cell activation in rat whole blood. B cell activation in rat whole blood was evaluated by measuring CD86 up-regulation via flow cytometry. Rat B cells in whole blood were stimulated with dextran-coupled anti-IgD or a combination of anti-IgD and TLR9 agonist. BTK, SYK, and PI3Kδ inhibitors were added to rat whole blood prior to activation with dextran-coupled anti-IgD or anti-IgD and TLR9 agonist combination for pharmacological validation of the assay. Both methods of stimulation in rat whole blood evoked robust B cell activation in a uni-modal fashion. Highly selective inhibitors of BTK, SYK, and PI3Kδ dose-dependently attenuated B cell activity evoked by both dextran-coupled anti-IgD and combined anti-IgD and TLR9 agonist. Compound potencies and rank order determined by the two assays were comparable. Two novel methods were developed to stimulate B cells in rat whole blood, that have the potential to be used to support drug discovery efforts in the therapeutic targeting of B cells. Furthermore, we pharmacologically validated these whole blood assays using highly selective inhibitors of BTK, SYK, and PI3Kδ, signaling kinases which are downstream of the B cell receptor.

Diagnostic Performance of a Cytokine and IFN-γ–Induced Chemokine mRNA Assay after Mycobacterium tuberculosis–Specific Antigen Stimulation in Whole Blood from Infected Individuals

Interferon (IFN)-γ release assays have limited sensitivity and cannot differentiate between active tuberculosis (TB) disease and latent TB infection (LTBI). Numerous cytokines and regulator factors have been implicated in the pathogenesis and control of Mycobacterium tuberculosis infection. Additional cytokines and chemokines associated with M. tuberculosis infection may improve the performance of IFN-γ release assays. We developed a real-time RT-PCR TaqMan assay for targeting levels of eight human targets [IFN-γ, tumor necrosis factor (TNF)-α, IL-2R, IL-4, IL-10, CXCL9, CXCL10, and CXCL11] and evaluated the assay with three different study groups. Results showed that the sensitivity of TNF-α, IL-2R, and CXCL10 in the active pulmonary tuberculosis (PTB) group was 96.43%, 96.43%, and 100%, respectively. The sensitivity of IL-2R and CXCL10 in the latent tuberculosis infection group was 86.36% and 81.82%, respectively. Statistical results showed that TNF-α and CXCL9 were the best individual markers for differentiating between the PTB, LTBI, and non-TB groups. For optimal sensitivity and differentiation of M. tuberculosis infection status, the simultaneous detection of multiple targets was attempted. The combination of IFN-γ, TNF-α, and IL-2R, and the combination of TNF-α, IL-2R, CXCL9, and CXCL10 showed the best performance for detecting active PTB (both 100% positivity) and LTBI (86.36% and 81.82% positivity, respectively). These results imply that the combination of suitable markers is useful in efficiently diagnosing TB and differentiating M. tuberculosis infection status.

Qualification of a whole blood intracellular cytokine staining assay to measure mycobacteria-specific CD4 and CD8 T cell immunity by flow cytometry

BackgroundQualified or validated assays are essential in clinical trials. Short-term stimulation of whole blood and intracellular cytokine staining assay is commonly used to measure immunogenicity in tuberculosis vaccine clinical trials. Previously, the short-term stimulation process of whole blood with BCG was optimized. We aimed to qualify the intracellular cytokine staining process and assess the effects of long-term cryopreservation. Our hypotheses were that the assay is robust in the measurement of the mycobacteria-specific T cells, and long-term cryopreservation of fixed cells from stimulated whole blood would not compromise reliable measurement of mycobacteria induced CD4 T cell immunity. MethodsWhole blood from healthy adults was collected in sodium heparinized tubes. The blood was left unstimulated or stimulated with mycobacterial antigens or mitogens for 12h. Cells were harvested, fixed and multiple aliquots from each participant cryopreserved. Later, mycobacteria-specific CD4 and CD8 T cells expressing IFN-γ, TNF-α, IL-2 and IL-17 were quantitated by flow cytometry. Assay performance characteristics evaluated included limit of quantification and detection, reproducibility, precision, robustness, specificity and sensitivity. To assess the effects of long-term cryopreservation, fixed cells from the stimulated bloods were analysed one week post-cryopreservation and at 3-month intervals over a 3-year period. ResultsThe limit of quantification for the different cytokines was variable: 0.04% for frequencies of IFN-γ- and IL-2-expressing T cells and less than 0.01% for TNF-α- and IL-17-expressing T cells. When measurement of the mycobacteria-specific T cells was assessed at levels above the detection limit, the whole blood intracellular cytokine assay showed high precision that was operator-independent. The assay was also robust: variation in staining conditions including temperature (4°C or 20–23°C) and time (45, 60 or 90min) did not markedly affect quantification of specific T cells. Finally, prolonged periods of cryopreservation also did not significantly influence quantification of mycobacteria-specific CD4 T cells. ConclusionsThe whole blood intracellular cytokine assay is robust and reliable in quantification of the mycobacteria-specific T cells and is not significantly affected by cryopreservation of fixed cells.

Comparative Analysis of Infused “Static”, Ex Vivo-Generated Platelets Vs. Infused Megakaryocytes-Generated Platelets: A Cautionary Tale

Ex vivo-generated (EV) platelets beginning with embryonic stem cells or induced pluripotent stem cells (iPSCs) or hematopoietic progenitors cells (HPCs) may have clinical utility over donor-derived platelets, and efforts to produce such EV-platelets have been pursued in several laboratories under static megakaryocyte (Meg) culture conditions. Success in generating these has been reported, even demonstrating EV-platelet incorporation into growing thrombi in murine models. We have pursued an alternative strategy for thrombopoiesis using EV-Megs, grown from either human adult HPCs or from iPSCs or fetal livers, and directly infusing them into NOD-SCID gamma-interferon-deficient (NSG) mice. These studies were based on our prior observation that infused murine EV-Megs into wildtype mice are entrapped in the pulmonary bed and over the subsequent 1-4 hours release a wave of functional platelets at a significant level. We now show that infusion of human EV-Megs do the same in NSG mice, but resulting in two different pools of derived platelets: (1) A pool of young (as determined by thiazole orange staining) platelets having the same bell-shaped size distribution was seen as after infusion of human donor-derived platelets in these mice. These platelets take several hours to appear, but then have the same half-life as donor-derived platelets. These platelets are derived from the infused EV-Megs and were termed in vivo-generated (IV)-platelets. (2) A second pool of mostly older platelets was present that originated during the static growth of the EV-Megs, and these EV-platelets varied widely in size and age. Initially, these platelets accounted for a third of all the human platelets seen. Unlike IV-platelets, EV-platelets are immediately present and circulate with a markedly short half-life of 2-3 hours unless the recipient NSG mice were pre-treated with clodronate-ladened liposomes to delete their macrophage pools. Rapid removal of EV-platelets by macrophages is due to their being preactivated as determined by surface P-selectin expression in whole mice blood. These EV-platelets also had very limited further responsiveness to convulxin activation. On the other hand, human IV-platelets were quiescent prior to agonist stimulation in whole mice blood and responded strongly to agonist, similar to human donor-derived platelets infused into NSG mice. The IV-platelets were also selectively incorporated into cremaster arteriole laser injury thrombi over EV-platelets. Finally, directly harvested “platelets” from EV static-grown Megs were isolated and analyzed both in vitro and in vivo. Only a third of these particles are CD41+/CD42+ platelets and approximately half are actually CD41-/CD42-. Both pools showed the same wide size distribution in vitro and in vivo after infusion into mice. The CD41+/CD42+ fraction behaved just as the EV-platelets, but the CD41-/CD42- fraction half-life was unaffected by pre-treatment with clodronate-ladened liposomes. In summary, infused human Megs grown under static growth conditions released platelets in a recipient mouse’s lung with features characteristic of donor-derived platelets. On the other hand, “platelets” harvested from the same Megs were predominantly not even platelets as measured using CD41/CD42 markers. The portion that were CD41+/CD42+ platelets were preactivated, poorly responsive to agonists, and cleared rapidly. These findings set a standard on how to judge the potential clinical value of platelets derived from EV-Megs and also raise concerns whether direct visual imaging of “platelet release” in static culture is biologically meaningful given that most particles released were not CD41+/CD42+ platelets, and the ones that were CD41+/CD42+ were mis-sized and functionally limited. DisclosuresNo relevant conflicts of interest to declare.

Endogenous circadian regulation of pro-inflammatory cytokines and chemokines in the presence of bacterial lipopolysaccharide in humans.

Various aspects of immune response exhibit 24-hour variations suggesting that infection susceptibility and treatment efficacy may vary by time of day. Whether these 24-hour variations are endogenous or evoked by changes in environmental or behavioral conditions is not known. We assessed the endogenous circadian control and environmental and behavioral influences on ex-vivo lipopolysaccharide stimulation of whole blood in thirteen healthy participants under 48 hours of baseline conditions with standard sleep-wake schedules and 40–50 hours of constant environmental and behavioral (constant routine; CR) conditions. Significant 24-hour rhythms were observed under baseline conditions in Monocyte Chemotactic Protein, Granulocyte-Macrophage Colony-Stimulating Factor and Interleukin 8 but not Tumor Necrosis Factor alpha whereas significant 24-hour rhythms were observed in all four immune factors under CR conditions. The rhythm amplitudes, expressed as a percentage of mean, were comparable between immune factors and across conditions. In contrast, the acrophase time (time of the fitted peak) was different between immune factors, and included daytime and nighttime peaks and changes across behavioral conditions. These results suggest that the endogenous circadian system underpins the temporal organization of immune responses in humans with additional effects of external environmental and behavioral cycles. These findings have implications for understanding the adverse effects of recurrent circadian disruption and sleep curtailment on immune function.

Immunogenicity of 60 novel latency-related antigens of Mycobacterium tuberculosis.

The aim of our work here was to evaluate the immunogenicity of 60 mycobacterial antigens, some of which have not been previously assessed, notably a novel series of in vivo-expressed Mycobacterium tuberculosis (IVE-TB) antigens. We enrolled 505 subjects and separated them in individuals with and without latent tuberculosis infection (LTBI) vs. patients with active tuberculosis (TB). Following an overnight and 7 days stimulation of whole blood with purified recombinant M. tuberculosis antigens, interferon-γ (IFN-γ) levels were determined by ELISA. Several antigens could statistically significantly differentiate the groups of individuals. We obtained promising antigens from all studied antigen groups [dormancy survival regulon (DosR regulon) encoded antigens; resuscitation-promoting factors (Rpf) antigens; IVE-TB antigens; reactivation associated antigens]. Rv1733, which is a probable conserved transmembrane protein encoded in DosR regulon, turned out to be very immunogenic and able to discriminate between the three defined TB status, thus considered a candidate biomarker. Rv2389 and Rv2435n, belonging to Rpf family and IVE-TB group of antigens, respectively, also stood out as LTBI biomarkers. Although more studies are needed to support our findings, the combined use of these antigens would be an interesting approach to TB immunodiagnosis candidates.

Stimulation of duodenal biopsies and whole blood from dogs with food-responsive chronic enteropathy and healthy dogs with Toll-like receptor ligands and probiotic Enterococcus faecium.

The composition of the microbiome plays a significant role in the pathogenesis of inflammatory bowel disease (IBD) in humans and chronic enteropathies (CE) in dogs. The administration of probiotic micro-organisms is one way of modulating the microbiome, but experiments elucidating mechanisms of action of probiotics in the intestine of healthy and CE dogs are lacking. The aim of our study was to investigate the effects of different Toll-like receptor (TLR) ligands and Enterococcus faecium (EF) on ex vivo cultured duodenal samples and whole blood (WB) from dogs with food-responsive chronic enteropathy (FRE) when compared to healthy dogs. Biopsy stimulation was performed in 17 FRE and 11 healthy dogs; WB stimulation was performed in 16 FRE and 16 healthy dogs. Expression of TLR2, 4, 5 and 9, IL-17A, IL-22, IFNy, TNFα, IL-4, IL-10, TGFβ and PPARy was determined in biopsies by quantitative polymerase chain reaction (PCR). In addition, production of TNFα, IL-10, IFNy and IL-17A protein in WB and biopsy supernatants was assessed by ELISA. Treatment with individual TLR ligands or EF induced a variety of changes in the expression of different TLRs and cytokines, but not necessarily a consistent change with a single stimulating agent. Even though cytokine protein could not be detected in supernatants from ex vivo stimulated biopsies, we found TNFα protein responses in blood to be opposite of the transcriptional responses seen in the biopsies. Stimulation of canine duodenal biopsies with TLR ligands can potentially induce anti-inflammatory gene expression, especially in healthy tissue, whereas the effects of EF were limited.

Altered innate immune development in HIV-exposed uninfected infants.

Early in life, HIV-exposed uninfected (HEU) infants are at an increased risk of morbidity and mortality from infectious disease compared with HIV-unexposed (UE) infants. To improve our understanding of the mechanisms underlying their increased risk, we contrasted innate immune development between HEU and UE infants in a developing world setting, where early life infectious disease risk is exceptionally high. A prospective longitudinal cohort of HEU and UE newborns was established, and the most detailed characterization to date of HEU infant immune development was performed. Single-cell cytokine production was analyzed by flow cytometry after stimulation of whole blood with pathogen-associated molecular patterns (PAMPs). Monocyte, classical dendritic cell, and plasmacytoid dendritic cell composition was similar between HEU and UE infants throughout the first year of life. However, HEU mononuclear cells mounted an enhanced pro-inflammatory response to PAMP stimulation, both in quantity of cytokine produced per cell and in proportion of responder cells. Significant differences in cytokine production were detected on the single-cell level in a PAMP-specific pattern, but only at 2 and 6 weeks of age; all differences normalized by 12 months of age. This time course of innate immune deviation early in life corresponds to the clinical window of vulnerability to infections in HEU infants and may be at least partially responsible for their increased morbidity and mortality from infectious disease.

A Novel Immune Function Biomarker Predicts Early Clinical Outcomes Following Liver Transplantation.

Immune function monitoring post liver transplantation (OLTx) remains substandard. QuantiFERON-Monitor (QFM, Qiagen, USA) is a novel immune function biomarker based on the same laboratory platform as the readily used QFN-gold assay (Qiagen, USA) and measures IFN-γ production after overnight stimulation of 1mL whole blood with both innate (R848) and adaptive (CD3) immune ligands with potential for next-day results. We present a blinded, prospective, observational study, representing the first clinical use of QFM in predicting clinical outcomes post-OLTx. QFM was examined in 69 adult OLTx recipients at the following time-points: pre-OLTX and post-OLTx days 1,3,5, 7, 14 and 30. Patients were monitored for treated and biopsy proven acute rejection (tBPAR) (n=17 (24.6%) median 9 days) and infections (n=21 (30.4%) median 12 days). QFM fell post-OLTx to a nadir at D3, then gradually increased to D30 (p<0.0001). Elevated D7 QFM in clinically stable patients (n=57, median 1.35IU/mL) was significantly associated with future tBPAR (ROC 0.84, p<0.0001), conversely low QFM was associated with infection (ROC 0.74, p=0.008). In those with the most impaired immune response (bottom quartile, <0.62IU/mL) 0/15 (0%) developed tBPAR, whilst 6/15 (40%) developed an infection. Conversely, in those with a robust immune response (upper quartile, >4.0IU/mL) 8/14 (57%) developed tBPAR whilst only 1/14 (7%) developed an infection.Figure: No Caption available.Notably, 4 of 5 patients with the lowest QFM developed opportunistic infections including cryptococcal meningitis, HSV and fungal peritonitis. Importantly, therapeutic drug levels at each time point were not associated with QFM or clinical events. D7 QFM predicts early post-OLTx clinical events, particularly identifying patients (QFM <1.35IU/mL) who are at risk of infection with a low probability of rejection, who may benefit from immunosuppression reduction. This may facilitate individualisation of immunosuppression and fundamentally alter the management of OLTx, and possibly other solid organ recipients. DISCLOSURE:Sood, S.: Grant/Research Support, Cellestis. Yu, J.: Employee, Cellestis Ltd, (Past). Visvanathan, K.: Grant/Research Support, Cellestis Ltd. Testro, A.: Grant/Research Support, Cellestis Ltd.

Difference in immune response in vaccinated and unvaccinated Swedish individuals after the 2009 influenza pandemic.

BackgroundPrevious exposures to flu and subsequent immune responses may impact on 2009/2010 pandemic flu vaccine responses and clinical symptoms upon infection with the 2009 pandemic H1N1 influenza strain. Qualitative and quantitative differences in humoral and cellular immune responses associated with the flu vaccination in 2009/2010 (pandemic H1N1 vaccine) and natural infection have not yet been described in detail. We designed a longitudinal study to examine influenza- (flu-) specific immune responses and the association between pre-existing flu responses, symptoms of influenza-like illness (ILI), impact of pandemic flu infection, and pandemic flu vaccination in a cohort of 2,040 individuals in Sweden in 2009–2010.MethodsCellular flu-specific immune responses were assessed by whole-blood antigen stimulation assay, and humoral responses by a single radial hemolysis test.ResultsPrevious seasonal flu vaccination was associated with significantly lower flu-specific IFN-γ responses (using a whole-blood assay) at study entry. Pandemic flu vaccination induced long-lived T-cell responses (measured by IFN-γ production) to influenza A strains, influenza B strains, and the matrix (M1) antigen. In contrast, individuals with pandemic flu infection (PCR positive) exhibited increased flu-specific T-cell responses shortly after onset of ILI symptoms but the immune response decreased after the flu season (spring 2010). We identified non-pandemic-flu vaccinated participants without ILI symptoms who showed an IFN-γ production profile similar to pandemic-flu infected participants, suggesting exposure without experiencing clinical symptoms.ConclusionsStrong and long-lived flu-M1 specific immune responses, defined by IFN-γ production, in individuals after vaccination suggest that M1-responses may contribute to protective cellular immune responses. Silent flu infections appeared to be frequent in 2009/2010. The pandemic flu vaccine induced qualitatively and quantitatively different humoral and cellular immune responses as compared to infection with the 2009 H1N1 pandemic H1N1 influenza strain.

SAT0371 Ustekinumab Treatment in Ankylosing Spondylits (AS) Patients Does not Modulate Effector CD4+ T Cell Frequencies Including TH17 Cells

BackgroundUstekinumab blocks the action of IL-12 and IL-23 by interfering with binding of the p40 subunit to the IL-12Rβ1 subunit used by IL-12 and IL-23 receptors. IL-12 and IL-23 are...