Serum Apolipoprotein E and Other Inflammatory Markers Can Identify Non-Responding Patients to a Dendritic Cell Vaccine.

BACKGROUND: Despite the majority of patients do not gain any benefit from dendritic cells (DC) vaccines, this approach has occasionally given rise to dramatic responses in melanoma. Biomarkers are crucial to identify which patients are more likely to respond. We looked for correlations between pre- or post- vaccination biomarkers and clinical outcomes to DC therapy in a cohort of patients with stage IV melanoma receiving a vaccine with autologous ex-vivo expanded DCs pulsed with allogeneic tumor cell lysate. METHODS: Serial serum samples were collected at baseline, week 4 and 12 and they were analyzed for a panel of different inflammatory markers using cytometric bead array technology and ELISA. RESULTS: Twenty-one patients were evaluable for response. Patients were separated into responders and non-responders based on clinical benefit. Responders were defined as patients who achieved a complete response, partial response or stable disease the latter lasting for at least 6 months. Responders (N = 9) showed a significantly longer Progression-free Survival (PFS; HR 0.23; 95% CI 0.08-062; P < .001) and Overall Survival (OS; HR 0.22; 95% CI 0.08-0.59; P < .001). The clinical non-responder phenotype correlated with an elevated pre-vaccination level of cytokines associated with inflammation compared to clinical responders (Apolipoprotein C111; IL-12 p40; MiP1α; Stem Cell Factor and TNFα). Apolipoprotein E (ApoE) was also significantly elevated in the pre-vaccine sera of the clinically non-responding group and in addition it was found to correlate with outcomes. Patients with increased levels of ApoE had a significantly shorter PFS (HR 3.02; 95% CI 1.09-8.35; P = .015) and OS (HR 2.40; 95% CI 0.9-6.3; P = .034). CONCLUSION: Our findings support the notion that treating the inflammatory background may have an impact on clinical outcome for patients receiving immunotherapy. A larger study is needed to confirm the significance of ApoE as a predictive biomarker for response to DC vaccines.

Infection of monocytes with European porcine reproductive and respiratory syndrome virus (PRRSV-1) strain Lena is significantly enhanced by dexamethasone and IL-10.

Monocytes are considered refractory to porcine reproductive and respiratory syndrome virus type 1 (PRRSV-1) infection. However, monocytes are only short-lived in blood, being able to differentiate into macrophages and dendritic cells (DC). It was therefore merited to revisit PRRSV-1 interaction with monocytes, particularly those treated with cytokines influencing monocyte biology. Thus, several factors were screened, particularly those modulating monocyte differentiation and expression of putative PRRSV-1 receptors (CD169 and CD163). M-CSF, known to stimulate macrophage differentiation, did not increase their susceptibility to PRRSV-1. Nor did GM-CSF or IL-4, known drivers for monocyte-derived DC (MoDC) differentiation. In contrast, monocyte treatment with IL-10 or the corticosteroid, dexamethasone, known to be potent suppressors of monocyte differentiation, was correlated with increased susceptibility to PRRSV-1 infection. While this effect was strongly correlated to CD163 and CD169 expression, our data suggest that receptor expression is not the only factor driving successful infection of PPRSV-1 in monocytes.

Establishing Porcine Monocyte-Derived Macrophage and Dendritic Cell Systems for Studying the Interaction with PRRSV-1.

Monocyte-derived macrophages (MoMØ) and monocyte-derived dendritic cells (MoDC) are two model systems well established in human and rodent systems that can be used to study the interaction of pathogens with host cells. Porcine reproductive and respiratory syndrome virus (PRRSV) is known to infect myeloid cells, such as macrophages (MØ) and dendritic cells (DC). Therefore, this study aimed to establish systems for the differentiation and characterization of MoMØ and MoDC for subsequent infection with PRRSV-1. M-CSF differentiated MoMØ were stimulated with activators for classical (M1) or alternative (M2) activation. GM-CSF and IL-4 generated MoDC were activated with the well established maturation cocktail containing PAMPs and cytokines. In addition, MoMØ and MoDC were treated with dexamethasone and IL-10, which are known immuno-suppressive reagents. Cells were characterized by morphology, phenotype, and function and porcine MØ subsets highlighted some divergence from described human counterparts, while MoDC, appeared more similar to mouse and human DCs. The infection with PRRSV-1 strain Lena demonstrated different replication kinetics between MoMØ and MoDC and within subsets of each cell type. While MoMØ susceptibility was significantly increased by dexamethasone and IL-10 with an accompanying increase in CD163/CD169 expression, MoDC supported only a minimal replication of PRRSV These findings underline the high variability in the susceptibility of porcine myeloid cells toward PRRSV-1 infection.

Mycolactone-Dependent Depletion of Endothelial Cell Thrombomodulin Is Strongly Associated with Fibrin Deposition in Buruli Ulcer Lesions.

A well-known histopathological feature of diseased skin in Buruli ulcer (BU) is coagulative necrosis caused by the Mycobacterium ulcerans macrolide exotoxin mycolactone. Since the underlying mechanism is not known, we have investigated the effect of mycolactone on endothelial cells, focussing on the expression of surface anticoagulant molecules involved in the protein C anticoagulant pathway. Congenital deficiencies in this natural anticoagulant pathway are known to induce thrombotic complications such as purpura fulimans and spontaneous necrosis. Mycolactone profoundly decreased thrombomodulin (TM) expression on the surface of human dermal microvascular endothelial cells (HDMVEC) at doses as low as 2 ng/ml and as early as 8 hrs after exposure. TM activates protein C by altering thrombin's substrate specificity, and exposure of HDMVEC to mycolactone for 24 hours resulted in an almost complete loss of the cells' ability to produce activated protein C. Loss of TM was shown to be due to a previously described mechanism involving mycolactone-dependent blockade of Sec61 translocation that results in proteasome-dependent degradation of newly synthesised ER-transiting proteins. Indeed, depletion from cells determined by live-cell imaging of cells stably expressing a recombinant TM-GFP fusion protein occurred at the known turnover rate. In order to determine the relevance of these findings to BU disease, immunohistochemistry of punch biopsies from 40 BU lesions (31 ulcers, nine plaques) was performed. TM abundance was profoundly reduced in the subcutis of 78% of biopsies. Furthermore, it was confirmed that fibrin deposition is a common feature of BU lesions, particularly in the necrotic areas. These findings indicate that there is decreased ability to control thrombin generation in BU skin. Mycolactone's effects on normal endothelial cell function, including its ability to activate the protein C anticoagulant pathway are strongly associated with this. Fibrin-driven tissue ischemia could contribute to the development of the tissue necrosis seen in BU lesions.

Effect of selenium on markers of risk of pre-eclampsia in UK pregnant women: a randomised, controlled pilot trial.

Pre-eclampsia is a serious hypertensive condition of pregnancy associated with high maternal and fetal morbidity and mortality. Se intake or status has been linked to the occurrence of pre-eclampsia by our own work and that of others. We hypothesised that a small increase in the Se intake of UK pregnant women of inadequate Se status would protect against the risk of pre-eclampsia, as assessed by biomarkers of pre-eclampsia. In a double-blind, placebo-controlled, pilot trial, we randomised 230 primiparous pregnant women to Se (60 µg/d, as Se-enriched yeast) or placebo treatment from 12 to 14 weeks of gestation until delivery. Whole-blood Se concentration was measured at baseline and 35 weeks, and plasma selenoprotein P (SEPP1) concentration at 35 weeks. The primary outcome measure of the present study was serum soluble vascular endothelial growth factor receptor-1 (sFlt-1), an anti-angiogenic factor linked with the risk of pre-eclampsia. Other serum/plasma components related to the risk of pre-eclampsia were also measured. Between 12 and 35 weeks, whole-blood Se concentration increased significantly in the Se-treated group but decreased significantly in the placebo group. At 35 weeks, significantly higher concentrations of whole-blood Se and plasma SEPP1 were observed in the Se-treated group than in the placebo group. In line with our hypothesis, the concentration of sFlt-1 was significantly lower at 35 weeks in the Se-treated group than in the placebo group in participants in the lowest quartile of Se status at baseline (P= 0·039). None of the secondary outcome measures was significantly affected by treatment. The present finding that Se supplementation has the potential to reduce the risk of pre-eclampsia in pregnant women of low Se status needs to be validated in an adequately powered trial.

The acute-phase reactant C-reactive protein binds to phosphorylcholine-expressing Neisseria meningitidis and increases uptake by human phagocytes.

Neisseria meningitidis is a global cause of meningitis and septicemia. Immunity to N. meningitidis involves both innate and specific mechanisms with killing by serum bactericidal activity and phagocytic cells. C-reactive protein (CRP) is an acute-phase serum protein that has been shown to help protect the host from several bacterial pathogens, which it recognizes by binding to phosphorylcholine (PC) on their surfaces. Pathogenic Neisseria species can exhibit phase-variable PC modification on type 1 and 2 pili. We have shown that CRP can bind to piliated meningococci in a classical calcium-dependent manner. The binding of CRP to the meningococcus was concentration dependent, of low affinity, and specific for PC. CRP appears to act as an opsonin for N. meningitidis, as CRP-opsonized bacteria showed increased uptake by human macrophages and neutrophils. Further investigation into the downstream effects of CRP-bound N. meningitidis may lead us to a better understanding of meningococcal infection and help direct more effective therapeutic interventions.

FcgammaRIIa expression with FcgammaRI results in C-reactive protein- and IgG-mediated phagocytosis.

C-reactive protein (CRP) is a pattern-recognition molecule, which can bind to phosphorylcholine and certain phosphorylated carbohydrates found on the surface of a number of microorganisms. CRP has been shown recently to bind human Fc receptor for immunoglobulin G (IgG; FcgammaR)I and mediate phagocytosis and signaling through the gamma-chain. To date, binding of monomeric CRP to FcgammaRII has been contentious. We demonstrate that erythrocytes opsonized with CRP bind FcgammaRIIa-transfected COS-7 cells. In addition, we demonstrate that FcgammaRI can use FcgammaRIIa R131 and H131 to phagocytose erythrocytes coated with IgG or purified or recombinant CRP in the absence of the gamma-chain. COS-7 cells expressing FcgammaRIIa or FcgammaRI alone did not phagocytose opsonized erythrocytes. Such phagocytosis required the cytoplasmic domain of FcgammaRIIa, as mutation of tyrosine at position 205 and truncation of the cytoplasmic domain from the end of the transmembrane region (position 206), resulting in the loss of the immunoreceptor tyrosine activatory motif, abrogated phagocytosis. FcgammaRIIa R131 was more efficient than FcgammaRIIa H131 at mediating CRP-dependent phagocytosis.

C-reactive protein-mediated phagocytosis and phospholipase D signalling through the high-affinity receptor for immunoglobulin G (FcgammaRI).

C-reactive protein (CRP) is the prototypic acute-phase protein in man which performs innate immune functions. CRP-mediated phagocytosis may be indirect, through activation of complement and complement receptors, or direct, through receptors for the Fc portion of immunoglobulin G (IgG; FcgammaRs) or even a putative CRP-specific receptor. No strong evidence has been shown to indicate which receptors may be responsible for phagocytosis or signalling responses. Using BIAcore technology, we confirm that CRP binds directly to the extracellular portion of FcgammaRI with a threefold higher affinity than IgG (KD = 0.81 x 10-9 m). Binding is Ca2+ dependent and is inhibited by IgG1 but not by phosphorylcholine (PC). CRP opsonization (using CRP concentrations within the normal human serum range) of PC-conjugated sheep erythrocytes increased phagocytosis of these particles by COS-7 cells transfected with FcgammaRI-II chimaera or FcgammaRI/gamma-chain. Interferon-gamma-treated U937 cells, which signal through FcgammaRI to activate phospholipase D (PLD) in response to cross-linked IgG, were also activated by CRP without any requirement for further cross-linking. These studies indicate that CRP is capable of binding to and cross-linking FcgammaRI thereby resulting in PLD activation and increased phagocytosis. Uptake by FcgammaRI has been reported to promote various acquired immune responses suggesting that CRP could act in a similar way.

C-reactive protein-mediated phagocytosis of Leishmania donovani promastigotes does not alter parasite survival or macrophage responses.

C-reactive protein (CRP) is an acute phase protein that binds to surface structures of a number of different organisms. Leishmania donovani express CRP ligand when first entering the mammalian host and CRP has been shown to alter macrophage function. The aim of this study was to investigate the functional significance of CRP-mediated uptake of L. donovani on survival of the parasite within human macrophages and macrophage cell responses to the infection. CRP opsonized L. donovani uptake was inhibitable by including excess CRP in the fluid phase, suggesting Fc receptor usage rather than indirect complement-mediated uptake. Comparing equivalent initial infection loads, parasite survival over 72 h within peripheral blood derived macrophages (PBMs) and differentiated U937 cells was unaltered by CRP. Whereas CRP increased macrophage responses to phosphorylcholine coated erythrocytes, no significant alteration in tumour necrosis factor-alpha, interleukin (IL)-10 or IL-12 production from PBMs was observed between CRP opsonized or unopsonized L. donovani promastigotes. Thus, in contrast to other systems, where CRP opsonization results in macrophage activation, Leishmania can use CRP to improve infection without inducing detrimental macrophage activation.