Performance of monocyte activation test supplemented with human serum compared to fetal bovine serum.

The monocyte activation test (MAT) is used to detect pyrogens in pharmaceutical products and serves as replacement of the rabbit pyrogen test. The peripheral blood mononuclear cell-based MAT assay requires the addition of serum to the medium and is performed with either fetal bovine serum (FBS) or human serum (HS). Since the capacity to detect non-endotoxin pyrogens (NEPs) in a sensitive manner is an important strength of MAT compared to the bacterial endo­toxin test, the performance of the MAT using FBS and HS was compared using endotoxin and several NEPs. The MAT was more sensitive for endotoxin when FBS was used, however for most NEPs the MAT was more sensitive when per­formed in HS. Furthermore, heat-inactivation of FBS affected the performance of the MAT for endotoxin to some extent but not for the NEPs. Interestingly, heat-inactivation of HS led to an almost complete loss of reactivity towards endotoxin, reduced the response towards heat-killed Staphylococcus aureus and peptidoglycan, but had minor or no effects on the responses towards R848, flagellin, and Pam3CSK4. Product testing of a human blood-derived product in MAT using HS was beneficial since endotoxin spike recoveries were improved. This product is therefore currently batch released with the HS-based MAT assay. Overall, to guarantee optimal performance of MAT, heat-inactivated serum should be avoided. The HS-based MAT appears to be the first choice to replace the rabbit pyrogen test, while in some cases the FBS-based MAT may be favored.

TLR4 and C5aR crosstalk in dendritic cells induces a core regulatory network of RSK2, PI3Kß, SGK1, and FOXO transcription factors.

Crosstalk between complement component 5a receptors (C5aRs) and TLRs in dendritic cells (DCs) occurs upon pathogen invasion; however, studies on C5aR and TLR crosstalk mainly focused on the modulating effect of C5a on TLR-induced cytokine production. To elucidate the breadth of C5aR and TLR4 crosstalk, the effect of simultaneous treatment with C5a and LPS was investigated in human monocyte-derived DCs (moDCs) 2 h after stimulation using whole transcriptome sequencing analysis. Although the effect of C5a on hallmark genes defining TLR4-induced DC maturation was limited at this time point, RNA sequencing analysis revealed a great variety of novel C5a targets, of which many interfere with TLR4-mediated immune activation. Analysis of functional relationships among these genes uncovered induction of a central immune regulatory network upon C5aR and TLR4 crosstalk, involving the transcription factors forkhead box (FOX)O1 and FOXO3 and the signaling molecules serum- and glucocorticoid-inducible kinase (SGK1), ribosomal S6 kinase 2 (RSK2), and PI3Kß. C5aR and TLR crosstalk, furthermore, yielded down-regulation of mainly proinflammatory network branches, including IL-12B, IL-2Rα (IL-2RA), and jagged 1 (JAG1) and cooperative induction of predominantly anti-inflammatory network branches, including sphingosine kinase 1 (SPHK1), ß2 adrenergic receptor (ADRB2), gastric inhibitory polypeptide receptor (GIPR), and four-and-a-half Lin11, Isl-1, and Mec-3 domains protein 2 (FHL2). Together, these data point toward induction of generalized immune regulation of DC function. Motif enrichment analysis indicate a prominent role for basic leucine zipper (bZIP) and IFN regulatory factor 4 (IRF4) transcription factors upon C5aR and TLR4 crosstalk. Additionally, differences were observed in the modulating capacity of C5a on DCs in the absence or presence of a pathogen (TLR stimulus). Our findings shed new light on the depth and complexity of C5aR and TLR4 crosstalk and provide new foci of research for future studies.

Anaphylatoxin C5a Regulates 6-Sulfo-LacNAc Dendritic Cell Function in Human through Crosstalk with Toll-Like Receptor-Induced CREB Signaling.

Activation of antigen-presenting dendritic cells (DCs) and the complement system are essential early events in the immune defense against invading pathogens. Recently, we and others demonstrated immunological crosstalk between signaling from receptors recognizing complement activation products and PAMPs on DCs. This affects DC effector function, as demonstrated by the finding that C5a prevents induction of pro-inflammatory cytokines by toll-like receptor (TLR) ligands in human monocyte-derived DCs (moDCs). Here, we demonstrate that this regulatory crosstalk is specifically important in 6-sulfo LacNAc dendritic cells (slanDCs), the most pro-inflammatory DC subset found in human. C5aR and TLR signaling show profound interference in the ERK/p38/CREB1 signaling pathways. C5aR signaling accelerates TLR-induced CREB1 phosphorylation both in moDC and slanDC. This is key in the regulatory effect of C5a on pro-inflammatory DC maturation by mediating induction of IL-10, which subsequently inhibits pro-inflammatory cytokine production via negative feedback signaling. Importantly, the regulatory effect of C5a affects T-cell immunity by decreasing Th1 and cytotoxic CD8 T-cell responses. The finding that the pro-inflammatory effector function of slanDC can be down modulated by activation products of the complement system highlights the existence of intricate regulatory interactions between various arms of the immune system. Intensive immune monitoring of patients suffering from complement-mediated diseases or patients receiving complement modulating compounds can give more inside in the contribution of complement receptor and TLR crosstalk in APCs in disease.

Generation of dendritic cells for immunotherapy is minimally impaired by granulocytes in the monocyte preparation.

The growing number of clinical studies, using monocyte-derived DC therapy, requires protocols where a sufficient number of dendritic cell (DCs) are produced according to current Good Manufacturing Practice guidelines. Therefore, a closed culture system for the generation of DCs is inevitable. One cost-effective way to isolate monocytes directly from leukapheresis material in a closed system is by elutriation with the Elutra cell separation system. In the Elutra, granulocytes co-purify with the monocytes. Therefore, we studied if and to what extent the presence of granulocytes in a monocyte product affects the generation of mature DCs. The presence of up to 16% granulocytes in the monocyte product had no significant effects on the quality of the DCs formed. The presence of higher granulocyte percentages, however, gradually altered DC quality. In this respect, the presence of higher number of granulocytes induced significant lower migratory capacity of the DCs and lower expression levels of CD80, CD40 and CD86. No effects were observed on the DC yield, cytokine production or the stimulatory capacity of the DCs in MLR. In conclusion, the presence of 20-30% granulocytes in a monocyte product has no major influence on the quality of the DCs generated from monocytes. Therefore, the Elutra is a suitable closed system apparatus to separate monocytes from other blood components for the generation of DCs, even from leukapheresis material which contains a high number of granulocytes.

TLR4-mediated pro-inflammatory dendritic cell differentiation in humans requires the combined action of MyD88 and TRIF.

TLR4 ligation can activate both the MyD88 and the Toll-IL-1 receptor domain-containing adaptor inducing IFN-ß (TRIF) signaling route. Whereas MyD88 is generally recognized as a universal adaptor for pro-inflammatory responses, TRIF is mainly thought to contribute to specific type I IFN responses. Here, we investigated the contribution of both MyD88 and TRIF to TLR4-mediated pro-inflammatory dendritic cell (DC) differentiation in human. Pro-inflammatory cytokine induction was strongly decreased in monophosphoryl lipid A- and LPS-matured monocyte-derived DCs when either MyD88 or TRIF were down-regulated by small interfering RNA electroporation. Induction of co-stimulatory molecule expression was entirely dependent on the TRIF pathway. Our results demonstrate that in human DCs the TRIF pathway is important for overall pro-inflammatory DC differentiation via TLR4 by mediating co-stimulation and playing a non-redundant role in pro-inflammatory cytokine induction.

The clinical grade maturation cocktail monophosphoryl lipid A plus IFNgamma generates monocyte-derived dendritic cells with the capacity to migrate and induce Th1 polarization.

Ex vivo generated monocyte-derived dendritic cells (DCs) are used as a cellular vaccine against cancer in clinical trials. In order to be able to induce an efficient tumour-specific CTL response during immunotherapy, DCs have to be able to migrate to the lymph node and produce the Th1 polarizing cytokine, IL-12p70, upon encounter of T cells in the lymph node. However, most clinically used DCs do not produce IL-12p70 upon T cell contact. In this study, we compared a newly developed clinical grade DC maturation cocktail consisting of MPLA and IFNgamma with two clinically available maturation cocktails, the 'gold standard' (TNFalpha, IL-1beta, IL-6 and PGE(2)) and the 'alpha type 1 polarizing' (TNFalpha, IL-1beta, IFNalpha, IFNgamma and pI:C) cocktail. All three cocktails induced phenotypically mature DCs. However, in contrast to 'gold standard' DCs, which produce no IL-12p70 and as a result induce mainly Th2 cells, DCs matured with MPLA and IFNgamma produce high levels of IL-12p70 upon CD40 triggering. Subsequently, these DCs induce mainly Th1 cells in vitro, even slightly more than by the alpha type 1 polarized DCs. In addition, MPLA plus IFNgamma matured DCs have an intermediate migratory capacity towards CCL21. In conclusion, we here present MPLA plus IFNgamma as a simple clinical grade maturation cocktail to generate immunostimulatory DCs with superior capacity to induce type 1 immunity.

Production, purification and characterisation of recombinant Fahsin, a novel antistasin-type proteinase inhibitor.

Serine proteinases from inflammatory cells, including polymorphonuclear neutrophils, are involved in various inflammatory disorders, like pulmonary emphysema and rheumatoid arthritis. Inhibitors of these serine proteinases are potential drug candidates for the treatment of these disorders, since they prevent the unrestricted proteolysis. This study describes a novel specific antistasin-type inhibitor of neutrophil serine proteinases, we called Fahsin. This inhibitor was purified from the Nile leech Limnatis nilotica, sequenced and heterologously expressed using a synthetic gene in the methylotrophic yeast Pichia pastoris, yielding 0.5 g(-l) of the protein in the culture medium. Recombinant Fahsin was purified to homogeneity and characterised by N-terminal amino acid sequencing and mass spectrometry. Inhibition-kinetic analysis showed that recombinant Fahsin is a fast, tight-binding inhibitor of human neutrophil elastase with inhibition constant in the nanomolar range. Furthermore, recombinant Fahsin was, in contrast to various other neutrophil elastase inhibitors, insensitive to chemical oxidation and biological oxidation via myeloperoxidase-generated free oxygen radicals. Thus, Fahsin constitutes a novel member of a still expanding family of naturally occurring inhibitors of serine proteinases with potential therapeutic use for treatment of human diseases.

The major grass pollen group 5 allergen from Dactylis glomerata and its C-terminal split product both behave as dimers: implications for allergen standardization.

BACKGROUND: On SDS-PAGE grass pollen group-5 allergens migrate as a doublet with an apparent molecular mass (M(r)) of 25 kDa. Immunoblot analysis revealed additional group 5 reactivity at double and half this M(r). The aim of this study was to investigate these group 5 molecular entities and to compare their allergenicity and behavior in quantitative immunoassays. METHODS: Group-5-specific monoclonal antibodies were produced and used for the development of a group-5-specific sandwich ELISA. Affinity-purified Dac g 5 was separated by SDS-PAGE/Western blotting; individual bands were analyzed by N-terminal sequencing. Size exclusion chromatography (SEC) in conjunction with group-5-specific ELISA, competitive RIA and RAST inhibition were used to analyze the size distribution of Dac g 5. Basophil histamine release assays were used to assess biological activity. RESULTS: The lower band of the typical group 5 doublet was identified as a truncated form lacking the typical group 5 N-terminus AD(L)/(A)GY, observed in the upper band. The 12-kDa peptide was shown to be the C-terminal half of Dac g 5 (amino acid 127 onwards). SEC in conjunction with competitive RIA revealed that around 45% of Dac g 5 is represented by the 12-kDa peptide. Both the C-terminal half and the whole allergen dimerize under nondenaturing conditions. In competitive RIA and RAST inhibition both forms are equally well detected. In contrast, the half molecule is poorly recognized in sandwich ELISA and displays negligible biological activity in basophil histamine release tests with purified IgE. CONCLUSIONS: These observations stress the need to evaluate the performance of allergen standardization protocols in detail, with special attention to allergen size distribution.

Characterization of natural Dac g 1 variants: an alternative to recombinant group 1 allergens.

BACKGROUND: Production of soluble correctly folded recombinant group 1 allergens has proven to be difficult. Purified natural group 1 allergens could be an alternative for application in immunotherapy. OBJECTIVE: Cloning and expression of recombinant Dac g 1; purification of natural Dac g 1 variants and immunochemical characterization of these molecules. METHODS: Dac g 1 was cloned and expressed in the yeast Pichia pastoris. Hydrophobic interaction (HIC), size exclusion, and/or affinity chromatography were used to purify Dac g 1 from Dactylis glomerata pollen extract. Dac g 1 variants were analyzed by N-terminal sequencing. Immune reactivity was assessed by sandwich ELISA, competitive RIA, RAST (inhibition), and in vitro basophil histamine release tests. RESULTS: Dac g 1 was cloned, revealing up to 98% amino acid sequence homology to other group 1 allergens. Purification of natural Dac g 1 revealed at least 3 variants, with an apparent molecular mass (Mr) on SDS-PAGE of 33 kd (HMr), 30 kd (IMr) and 28 kd (LMr). Extraction of IMr Dac g 1 required 0.9% saline, whereas the other 2 variants were also extractable in water. The N-terminus of HMr and IMr Dac g 1 differs at 2 positions, and LMr Dac g 1 was shown to be N-terminally truncated, lacking the first 30 amino acids. The nonretarded fraction of HIC commonly used in group 1 purification protocols does not contain this LMr molecule. IMr Dac g 1 was poorly recognized in 2 of 3 sandwich ELISAs and competitive RIA but demonstrated similar biological activity compared with HMr Dac g 1. CONCLUSIONS: Natural Dac g 1 variants can be separated by extraction of pollen in the presence or absence of saline followed by HIC and size exclusion chromatography. Thus, purified Dac g 1 is an alternative to recombinant group 1 allergens.