Solid phase synthesis of glycopeptide dendrimers with Tn antigenic structure and their biological activities. Part I

Multiple antigenic peptides containing dimeric Tn antigen [Ac‐(Tn)₂‐γ‐Abu]₄‐(Lys‐X)₂‐Lys‐β‐Ala ( V : X=0; VIII : X=γ‐Abu) and [Ac‐(Tn)₂‐γ‐Abu]₈‐(Lys‐X)₄‐(Lys‐X)₂‐Lys‐β‐Ala ( XI : X=0; XIV : X=γ‐Abu), immobilized on biocompatible Tenta Gel S NH₂ support were prepared by SPPS. Rosetting tests of V , VIII , XI and XIV showed positive reactions with anti‐Tn (DAKO) and Tn+ erythrocytes, with anti‐Tn/A (BRIC 66) and Tn+ and A erythrocytes, other combinations were negative. In all the animals immunized with XIV , we found a remarkable increase in the level of anti‐Tn (titre 2000–64 000, score 105–167) and no change of anti‐A levels (titre 8, score 13–17). Neither non‐immune nor immune sera showed any reactivity with T⁺, Cad⁺ and blood group O erythrocytes. Immunized mice did not exhibit any sign of adverse reaction to the administered conjugates. Biological activities were correlated with molecular modelling and molecular dynamic calculations. The biological activities of these synthetic Tn antigen conjugates (good availability for the immunological interactions, highly specific immunogenicity, good biological tolerance) together with their precise chemical characterization seem to be a promising approach to preparation of anti‐tumour vaccine and affinity purification of anti‐Tn antibodies. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Independent regulation of serologically distinct idiotypes in F1 hybrid mice

We have investigated the regulation of expression of two distinct intrastrain cross-reactive idiotypes, CRI_A and CRI_C, characteristic of anti-p-azophenylarsonate (anti-Ar) antibodies of the A/J and BALB/c strains, respectively, in (BALB/c x A/J)F₁ (CAF₁) mice. Such hybrid mice were found to synthesize antibodies with each idiotype when immunized against the Ar hapten group, although the expression of each was significantly reduced as compared with the parental strain. CAF₁ mice were pretreated with idiotypic-specific antibody reagents and subsequently hyperimmunized against the Ar hapten. Analysis of the idiotypes present in immune sera showed that suppression of either CRI did not concomitantly suppress the expression of the other. Alteration of the expression of one idiotype was not, however, without influence on the other; the expression of CRI_C was markedly enhanced in mice suppressed for CRI_A.Abbreviations used in this paper anti-Id(A/J) idiotypic-specific antibodies against A/J serum Ar-specific antibodies - anti-Id(BALB/c) idiotypic-specific antibodies against BALB/c serum Ar-specific antibodies - Ar p-azophenylarsonate - BGG bovine -globulin - BSA bovine serum albumin - CAF₁ F(BALB/c x A/J) - CFA complete Freund's adjuvant - CRIA the major cross-reactive idiotype of A/J Ar-specific antibodies - CRI_C the major cross-reactive idiotype of BALB/c Ar-speck antibodies - CRI_m the minor cross-reactive idiotype of A/J Ar-specific antibodies - DTH delayed-type hypersensitivity - HP hybridoma product(s) - KLH keyhole limpet hemocyanin     

Activation of Macrophages by Sulfated Glycopeptides in Ovomucin,Yolk Membrane,and Chalazae in Chicken Eggs

Sulfated glycopeptides in ovomucin, chalazae and yolk membrane were found to activate cultured macrophage-like cells, J774.1, and TGC-induced macrophages from the peritoneal cavity of male mice. The macrophage-stimulating activity was estimated by the growth and morphology of the cells, H₂O₂ generation, and interleukin-1 (IL-1) production from the cells. The in vitro culture assay with macrophages showed that the protease digests of ovomucin, yolk membrane, and chalazae induced morphologic alteration and increased H₂O₂ generation and IL-1 production in lower concentration (100 μg/ml). The isolation of the components having macrophage-stimulating activity was attempted to elucidate the molecular mechanism. The O-linked carbohydrate chains, consisting of N-acetylgalactosamine, galactose, N-acetylneuraminic acid and sulfate, in the sulfated glycopeptide were identified as a component having macrophage-stimulating activity.     

Glycosylations versus conformational preferences of cancer associated mucin core

Synthetic oligosaccharide vaccines based on core STn (sialyl 2-6 GalNAc) carbohydrate epitopes are being evaluated by a number of biopharmaceutical firms as potential immunotherapeutics in the treatment of mucin-expressing adenocarcinomas. The STn carbohydrate epitopes exist as discontinuous clusters, O-linked to proximal serine and threonine residues within the mucin sequence. In an effort to probe the structure and dynamics of STn carbohydrate clusters as they may exist on the cancer-associated mucin, we have used NMR spectroscopy and MD simulations to study the effect of O-glycosylation of adjacent serine residues in a repeating (Ser)n sequence. Three model peptides/glyco-peptides were studied: a serine trimer containing no carbohydrate groups ((Ser)₃ trimer); a serine trimer containing three Tn (GalNAc) carbohydrates -linked to the hydroxyls of adjacent serine sidechains ((Ser.Tn)₃ trimer); and a serine trimer containing three STn carbohydrates -linked to the hydroxyls of adjacent serine sidechains ((Ser.STn)₃ trimer). Our results demonstrate that clustering of carbohydrates shifts the conformational equilibrium of the underlying peptide backbone into a more extended and rigid state, an arrangement that could function to optimally present the clustered carbohydrate antigen to the immune system. Steric effects appear to drive these changes since an increase in the size of the attached carbohydrate (STn versus Tn) is accompanied by a stronger shift in the equilibrium toward the extended state. In addition, NMR evidence points to the formation of hydrogen bonds between the peptide backbone NH protons and the proximal GalNAc groups in the (Ser.Tn)₃ and (Ser.STn)₃ trimers. The putative peptide-sugar hydrogen bonds may also play a role in influencing the conformation of the underlying peptide backbone, as well as the orientation of the O-linked carbohydrate. The significance of these results will be discussed within the framework of developing clustered STn-based vaccines, capable of targeting the clustered STn epitopes on the cancer-associated mucin.     

Linker Length Matters,Fynomer-Fc Fusion with an Optimized Linker Displaying Picomolar IL-17A Inhibition Potency

Fynomers are small binding proteins derived from the human Fyn SH3 domain. Using phage display technology, Fynomers were generated inhibiting the activity of the proinflammatory cytokine interleukin-17A (IL-17A). One specific Fynomer called 2C1 inhibited human IL-17A in vitro with an IC₅₀ value of 2.2 nm. Interestingly, when 2C1 was genetically fused to the Fc part of a human antibody via four different amino acid linkers to yield bivalent IL-17A binding proteins (each linker differed in length), the 2C1-Fc fusion protein with the longest linker displayed the most potent inhibitory activity. It blocked homodimeric IL-17A with an IC₅₀ value of 21 pm, which corresponds to a hundredfold improved IC₅₀ value as compared to the value obtained with monovalent Fynomer 2C1. In contrast, the 2C1-Fc fusion with the shortest linker showed only an ∼8-fold improved IC₅₀ value of 260 pm. Furthermore, in a mouse model of acute inflammation, we have shown that the most potent 2C1-Fc fusion protein is able to efficiently inhibit IL-17A in vivo. With their suitable biophysical properties, Fynomer-Fc fusion proteins represent new drug candidates for the treatment of IL-17A mediated inflammatory conditions such as psoriasis, psoriatic arthritis, or rheumatoid arthritis.     

Exacerbated intestinal inflammation in P2Y6 deficient mice is associated with Th17 activation

Extracellular nucleotides are released as constitutive danger signals by various cell types and activate nucleotide (P2) receptors such as P2Y₆ receptor. P2Y₆ activation on monocytes induces the secretion of the chemokine CXCL8 which may propagate intestinal inflammation. Also, P2Y₆ expression is increased in infiltrating T cells of Crohn's disease patients. As inflammatory bowel disease (IBD) is associated with immune cell recruitment, we hypothesised that P2Y₆ would participate to the establishment of inflammation in this disease. To address this, we used P2Y₆ deficient (P2ry6^‐−/−) mice in the dextran sodium sulfate (DSS) murine model of IBD. In disagreement with our hypothesis, P2Y₆ deficient mice were more susceptible to inflammation induced by DSS than WT mice. DSS treated-P2ry6^−/− mice showed increased histological damage and increased neutrophil and macrophage infiltration that correlated with increased mRNA levels of the chemokines KC and MCP-1. DSS treated-P2ry6^−/− mice exhibited also higher levels of Th17/Th1 lymphocytes in their colon which correlated with increased levels of IFN-γ and IL-17A in the sera as well as increased mRNA levels of IFN-γ, IL-17A, IL-6, IL-23 and IL-1β in P2ry6^−/− colons. This inflammation was also accompanied by a decreased cell proliferation and goblet cell number. Importantly, injection of anti-IL-17 intraperitoneally partially protected P2ry6^−/− mice from DSS-induced colitis. Taken together, in the absence of P2Y₆, an exacerbated intestinal inflammation to DSS was observed which correlated with increased recruitment of Th17/Th1 lymphocytes. These data suggest a protective role of P2Y₆ expressed on leukocytes in intestinal inflammation.     

Gene therapy using IL-27 ameliorates Sj?gren's syndrome-like autoimmune exocrinopathy

Introduction

Sjögren''s syndrome (SjS) is a systemic autoimmune disease characterized by decreased salivary and lacrimal gland secretions, resulting in severe dry mouth and dry eyes. Recent studies have suggested that T_H17 cells and its signature cytokine IL-17 are involved in the underlying pathogenic mechanisms leading to destructive inflammation and autoimmunity. In the present study, we examined whether IL-27, a natural inhibitor of T_H17 activity, could down-regulate or reverse SjS in C57BL/6.NOD-Aec1Aec2 mice, a model of primary-SjS.

Methods

Recombinant serotype 2 adeno-associated viral (AAV2) vectors expressing either IL-27 (rAAV2-IL27) or LacZ (rAAV2-LacZ) were injected into 6 or 14 week-old C57BL/6.NOD-Aec1Aec2 mice. Changes in IL-27, IL-17, and IL-10 cytokine levels in peripheral blood were determined by ELISAs, while flow cytometry analyses were used to quantify cytokine-positive splenocytes. Histological assessment of salivary glands, anti-nuclear autoantibody (ANA) staining, and stimulated saliva flow rates were used to profile SjS disease severity.

Results

Mice systemically treated with intravenous rAAV2-IL27 injections at either 6 or 14 weeks of age exhibited long-term elevated levels of serum IL-27 with concomitantly reduced levels of IL-17 compared with sera from mice injected with rAAV2-LacZ or saline out to 20 weeks post-inoculation. Most importantly, disease profiles revealed that rAAV2-IL27 treatment had little effect on lymphocytic focus (LF) scores, but resulted in structural changes in LF, lower titers of ANAs with changes in staining patterns, and a less severe clinical disease as determined by saliva flow rates.

Conclusions

These data support the concept that IL-27, when provided exogenously, can induce a suppressive effect on SjS development and thus may be an effective therapeutic agent for regulating T_H17 pro-inflammatory activity in autoimmune diseases where the T_H17 system has been shown to play an important role in their pathogenesis.     

Microarray analysis of the human antibody response to synthetic Cryptosporidium glycopeptides

Glycoproteins expressed by Cryptosporidium parvum are immunogenic in infected individuals but the nature of the epitopes recognised in C. parvum glycoproteins is poorly understood. Since a known immunodominant antigen of Cryptosporidium, the 17 kDa glycoprotein, has previously been shown to bind to lectins that recognise the Tn antigen (GalNAcα1-Ser/Thr-R), a large number of glycopeptides with different Tn valency and presentation were prepared. In addition, glycopeptides were synthesised based on a 40 kDa cryptosporidial antigen, a polymorphic surface glycoprotein with varying numbers of serine residues, to determine the reactivity with sera from C. parvum-infected humans. These glycopeptides and non-glycosylated peptides were used to generate a glycopeptide microarray to allow screening of sera from C. parvum-infected individuals for the presence of IgM and IgG antibodies. IgG but not IgM in sera from C. parvum-infected individuals bound to multivalent Tn antigen epitopes presented on glycopeptides, suggesting that glycoproteins from C. parvum that contain the Tn antigen induce immune responses upon infection. In addition, molecular differences in glycosylated peptides (e.g. substituting Ser for Thr) as well as the site of glycosylation had a pronounced effect on reactivity. Lastly, pooled sera from individuals infected with either Toxoplasma or Plasmodium were also tested against the modified Cryptosporidium peptides and some sera showed specific binding to glycopeptide epitopes. These studies reveal that specific anti-glycopeptide antibodies that recognise the Tn antigen may be useful diagnostically and in defining the roles of parasite glycoconjugates in infections.     

Strong immunogenicity of a multicomponent peptide vaccine developed with the branched lysine oligopeptide method for human immunodeficiency virus infection

We synthesized one V3 peptide each from HTLV-III_B, Thai A and Thai B, conjugating then to the T cell epitope of the env region, and we also synthesized a p17 protein peptide of the gag region (HGP-30). These peptide were then coupled to 8-lysine copolymers using N-succinimidyl maleimido carboxylate (M_r = ca 60 000). We designated this the branched lysine oligopeptide method. The large peptide complexes constructed from these four macromolecular peptide were used with aluminium hydroxide or complete Freund's adjuvant to immunize mice and rabbits four times. ELISA assay with aluminium hydroxide or complete Freund's adjuvant to immunize mice and rabbits four times. ELISA assay showed high titres of anti-peptide antibodies to each V3loop peptide and the HGP-30 peptide. Strong inhibition of CD4⁺ dependent cell fusion was obtained with these antisera when III_b, Thai A and Thai B strains of human immunodeficiency virus (HIV) were used. Strong anti-fusion inhibition was also observed with two other HIV strains. In addition, an increase of the anti-HIV effect was observed when we used sera obtained by multicomponent vaccine immunization. The same kind of inhibition was also observed in p24 assay systems using these immunized antisera. Activation of IL-2 production in lymphocytes was observed in p24 assay systems using these immunized antisera. Activation of IL-2 production in lymphocytes was observed in mice immunized with this vaccine. These results suggest that immunization with macromolecular peptide complexes can result in strong immunogenicity towards HIV-1.     

The liquid Kangfuxin (KFX) has efficient antifungal activity and can be used in the treatment of vulvovaginal candidiasis in mice

Vulvovaginal candidiasis (VVC) is an infectious disease caused mainly by Candida albicans. Kangfuxin (KFX) is a traditional Chinese medicine preparation made from Periplaneta americana extracts, which promotes wound healing and enhances body immunity and also acts as an antifungal agent. Here, we evaluated the effect of KFX in the treatment of VVC in vitro and in vivo. The minimum inhibitory concentration (MIC₅₀) of KFX against C. albicans ranged from 7·65 to 20·57%. In addition, KFX was more efficient than fluconazole (FLC) in inhibiting the drug-resistant C. albicans, and the effect was more intense after 8 h. The KFX treatment also exhibited good activity in vivo. It restored the body weight and reduced the vulvovaginal symptoms in mice induced with VVC. It downregulated the expression of the hyphae-related gene, HWP1, thus inhibiting the growth and development of C. albicans hyphae. It also increased the number of neutrophils and promoted the secretion of interleukin-17A (IL-17A); however, the levels of interleukin-8 (IL-8) and interleukin-1β (IL-1β) decreased in mice with VVC. We deduce that KFX effectively treats vaginal candidiasis in two ways: by inhibiting the growth and development of mycelia to reduce colonization of C. albicans and by promoting the secretion and release of IL-17A and neutrophils in high numbers to fight C. albicans infection. This study provides a theoretical basis for the use of KFX for the clinical treatment of VVC.     

Separation and concentration of Δ-6-keto-PGF1α using monoclonal antibody to ω3-olefin structure of trienoic prostanoids

A monoclonal antibody against cis-3-hexen-1-ol was prepared and used to separate and/or concentrate Δ¹⁷-6-keto-prostaglandin F_1α (PGF_1α) in the human sera. cis-3-Hexen-1-ol was conjugated with the human serum albumin (HSA) according to the N-succinimidylester method and hyperimmunized to BALB/c mouse. The monoclonal afntibodies were obtained from hybridoma clones established by a fusion between SP2/0-Ag14-k13 mouse myeloma cells and splenocytes of a mouse. A monoclonal antibody, named 4G9-12B, recognized the epitope characteristic for ω3-olefin structure. The 4G9-12B antibody became more specific for Δ¹⁷-6-keto-PGF_1α than 6-keto-PGF_1α by applying inhibition ELISA using amino-residue coating plates. Using the prepared immunoaffinity columns of this antibody, Δ¹⁷-6-keto-PGF_1α was clearly detected in 6 pg/ml of the human blood sera by GC/MS analysis. These results suggest that the monoclonal antibody to the partial structure of trienoic prostanoid, ω3-olefin unit, and that its immunoaffinity columns are useful in separating and concentrating Δ¹⁷-6-keto-PGF_1α in the human blood or urine.     

Attachment Sites of Carbohydrate Moieties to Peptide Chain of Bovine κ-Casein from Normal Milk

The attachment site of the carbohydrate moiety to the peptide chain of normal κ-casein was investigated with κ-casein component P-6 containing the most carbohydrates. Three short glycopeptides 6-IB₁, 6-IB_{2 ? 1} and 6-IB_{2 ? 2} were prepared by cyanogen bromide cleavage and digestion of proteases (pronase P and thermolysin). Glycopeptides 6-IB₁, 6-IB_{2? 1} and 6-IB_{2 ? 2} corresponded to residues 128–139 (Gly-Glu-Pro-Thr-Ser-Thr-Pro-Thr-Thr-Glu-Ala-Val), residues 128–132 (or 127–131) and residues 135–139 of κ-casein A, respectively, and contained threonine and/or serine, but not asparagine. Glycopeptide 6-IB₁ was considered to have three carbohydrate chains because it contained three galactosamine residues. The results of alkali treatment of 6-IB, under reduction condition excluded serine residue as the binding site, and confirmed the existence of three binding sites in the carbohydrate moieties. The carbohydrate moiety was shown to attach to threonine residue No. 131 from analysis of 6-IB_{2? 1} and to threonine residue No. 135 (or 136) from analysis of 6-IB_{2 ? 2}. It was concluded that the carbohydrate chains attached to threonine residues No. 131, 133 and 135 (or 136).     

IL-17A inhibits the expansion of IL-17A-producing T cells in mice through "short-loop" inhibition via IL-17 receptor

IL-23 and IL-17A regulate granulopoiesis through G-CSF, the main granulopoietic cytokine. IL-23 is secreted by activated macrophages and dendritic cells and promotes the expansion of three subsets of IL-17A-expressing neutrophil-regulatory T (Tn) cells; CD4(-)CD8(-)alphabeta(low), CD4(+)CD8(-)alphabeta(+) (Th17), and gammadelta(+) T cells. In this study, we investigate the effects of IL-17A on circulating neutrophil levels using IL-17R-deficient (Il17ra(-/-)) mice and Il17ra(-/-)Itgb2(-/-) mice that lack both IL-17R and all four beta(2) integrins. IL-17R deficiency conferred a reduction in neutrophil numbers and G-CSF levels, as did Ab blockade against IL-17A in wild-type mice. Bone marrow transplantation revealed that IL-17R expression on nonhemopoietic cells had the greatest effects on regulating blood neutrophil counts. Although circulating neutrophil numbers were reduced, IL-17A expression, secretion, and the number of IL-17A-producing Tn cells were elevated in Il17ra(-/-) and Il17ra(-/-)Itgb2(-/-) mice, suggesting a negative feedback effect through IL-17R. The negative regulation of IL-17A-producing T cells and IL-17A and IL-17F gene expression through the interactions of IL-17A or IL-17F with IL-17R was confirmed in splenocyte cultures in vitro. We conclude that IL-17A regulates blood neutrophil counts by inducing G-CSF production mainly in nonhemopoietic cells. IL-17A controls the expansion of IL-17A-producing Tn cell populations through IL-17R.     

Role of IL-1 beta in the development of human T(H)17 cells: lesson from NLPR3 mutated patients

Background

T helper 17 cells (T_H-17) represent a lineage of effector T cells critical in host defence and autoimmunity. In both mouse and human IL-1β has been indicated as a key cytokine for the commitment to T_H-17 cells. Cryopyrin-associated periodic syndromes (CAPS) are a group of inflammatory diseases associated with mutations of the NLRP3 gene encoding the inflammasome component cryopyrin. In this work we asked whether the deregulated secretion of IL-1β secondary to mutations characterizing these patients could affect the IL-23/IL-17 axis.

Methodology/Principal Findings

A total of 11 CAPS, 26 systemic onset juvenile idiopathic arthritis (SoJIA) patients and 20 healthy controls were analyzed. Serum levels of IL-17 and IL-6 serum were assessed by ELISA assay. Frequency of T_H17 cells was quantified upon staphylococcus enterotoxin B (SEB) stimulation. Secretion of IL-1β, IL-23 and IL-6 by monocyte derived dendritic cells (MoDCs), were quantified by ELISA assay. A total of 8 CAPS and 11 SoJIA patients were also analysed before and after treatment with IL-1β blockade. Untreated CAPS patients showed significantly increased IL-17 serum levels as well as a higher frequency of T_H17 compared to control subjects. On the contrary, SoJIA patients displayed a frequency of T_H17 similar to normal donors, but were found to have significantly increased serum level of IL-6 when compared to CAPS patients or healthy donors. Remarkably, decreased IL-17 serum levels and T_H17 frequency were observed in CAPS patients following in vivo IL-1β blockade. On the same line, MoDCs from CAPS patients exhibited enhanced secretion of IL-1β and IL-23 upon TLRs stimulation, with a reduction after anti-IL-1 treatment.

Conclusion/Significance

These findings further support the central role of IL-1β in the differentiation of T_H17 in human inflammatory conditions.     

Immunochemical characterization of polylysine conjugates containing reductively aminated cellulose oligosaccharides

Neoglycoproteins prepared by direct reductive amination of cellobiose, cellotetraose and cellopentaose to polylysine, were found to be effective antigens in rabbits, and the antisera were found by quantitative inhibition techniques to be predominantly hapten specific. Several analogs incorporating various structural features of the carbohydrate hapten were synthesized and examined as inhibitors of the precipition reaction between the neoglycoproteins and homologous antisera in order to identify those structural features of the hapten important in antibody recognition. Antibodies to the reductively aminated cellobiose-polylysine conjugate were found to recognize the terminal -glucopyranosyl residue, theacyclic reduced glucose residue, and the secondary ammonium linkage and methylene arm of the lysyl residue of the hapten. Antibodies to the cellotetraose-polylysine conjugate, in contrast, displayed no recognition for the secondary ammonium linkage region; they were found to recognize the non-reducing terminal -glucopyranosyl residue, the two internal 1,4-linked glucopyranosyl residues, and the reducing end glucose residue in anacyclic orcyclic form. Inhibition studies with antisera to the cellopentaose-polylysine conjugate again established that there was no recognition of the secondary ammonium linkage region, and demonstrated that the upper limit to the size, of the antibody combining site was four 1,4-linked glucopyranosyl residues.Abbreviations BSA bovine serum albumin - Glc glucose - (Glc)₂ (Glc)₄, (Glc)₅ compounds derived by reductive amination of cellobiose, cellotetraose and cellopentaose     

Prolonged, granulocyte-macrophage colony-stimulating factor-dependent, neutrophil survival following rheumatoid synovial fibroblast activation by IL-17 and TNFalpha

Introduction

A surprising feature of the inflammatory infiltrate in rheumatoid arthritis is the accumulation of neutrophils within synovial fluid and at the pannus cartilage boundary. Recent findings suggest that a distinct subset of IL-17-secreting T-helper cells (T_H17 cells) plays a key role in connecting the adaptive and innate arms of the immune response and in regulating neutrophil homeostasis. We therefore tested the hypothesis that synovial fibroblasts bridge the biological responses that connect T_H17 cells to neutrophils by producing neutrophil survival factors following their activation with IL-17.

Methods

IL-17-expressing cells in the rheumatoid synovium, and IL-17-expressing cells in the peripheral blood, and synovial fluid were examined by confocal microscopy and flow cytometry, respectively. Peripheral blood neutrophils were cocultured either with rheumatoid arthritis synovial fibroblasts (RASF) or with conditioned medium from RASF that had been pre-exposed to recombinant human IL-17, TNFα or a combination of the two cytokines. Neutrophils were harvested and stained with the vital mitochondrial dye 3,3''-dihexyloxacarbocyanine iodide before being enumerated by flow cytometry.

Results

T_H17-expressing CD4⁺ cells were found to accumulate within rheumatoid synovial tissue and in rheumatoid arthritis synovial fluid. RASF treated with IL-17 and TNFα (RASF_IL-17/TNF) effectively doubled the functional lifespan of neutrophils in coculture. This was entirely due to soluble factors secreted from the fibroblasts. Specific depletion of granulocyte–macrophage colony-stimulating factor from RASF_IL-17/TNF-conditioned medium demonstrated that this cytokine accounted for approximately one-half of the neutrophil survival activity. Inhibition of phosphatidylinositol-3-kinase and NF-κB pathways showed a requirement for both signalling pathways in RASF_IL-17/TNF-mediated neutrophil rescue.

Conclusion

The increased number of neutrophils with an extended lifespan found in the rheumatoid synovial microenvironment is partly accounted for by IL-17 and TNFα activation of synovial fibroblasts. T_H17-expressing T cells within the rheumatoid synovium are likely to contribute significantly to this effect.     

Antiproliferative effect of T/Tn specific <Emphasis Type="Italic">Artocarpus lakoocha</Emphasis> agglutinin (ALA) on human leukemic cells (Jurkat,U937, K562) and their imaging by QD-ALA nanoconjugate

T/Tn specificity of Artocarpus lakoocha agglutinin (ALA), isolated from the seeds of A. lakoocha (Moraceae) fruit and a heterodimer (16 kD and 12 kD) of molecular mass 28 kD, was further confirmed by SPR analysis using T/Tn glycan containing mammalian glycoproteins. N-terminal amino acid sequence analysis of ALA showed homology at 15, 19–21, 24–27, and 29 residues with other lectin members of Moraceae family viz., Artocarpus integrifolia (jacalin) lectin, Artocarpus hirsuta lectin, and Maclura pomifera agglutinin. It is mitogenic to human PBMC and the maximum proliferation was observed at 1 ng/ml. It showed an antiproliferative effect on leukemic cells, with the highest effect toward Jurkat cells (IC₅₀ 13.15 ng/ml). Synthesized CdS quantum dot-ALA nanoconjugate was employed to detect the expression of T/Tn glycans on Jurkat, U937, and K562 leukemic cells surfaces as well as normal lymphocytes by fluorescence microscopy. No green fluorescence was observed with normal lymphocytes indicating that T/Tn determinants, which are recognized as human tumor associated structures were cryptic on normal lymphocyte surfaces, whereas intense green fluorescent dots appeared during imaging of leukemic cells, where such determinants were present in unmasked form. The above results indicated that QD-ALA nanoconjugate is an efficient fluorescent marker for identification of leukemic cell lines that gives rise to high quality images.     

Estradiol Enhances CD4+ T-Cell Anti-Viral Immunity by Priming Vaginal DCs to Induce Th17 Responses via an IL-1-Dependent Pathway

Clinical and experimental studies have shown that estradiol (E2) confers protection against HIV and other sexually transmitted infections. Here, we investigated the underlying mechanism. Better protection in E2-treated mice, immunized against genital HSV-2, coincided with earlier recruitment and higher proportions of T_h1 and T_h17 effector cells in the vagina post-challenge, compared to placebo-treated controls. Vaginal APCs isolated from E2-treated mice induced 10-fold higher T_h17 and T_h1 responses, compared to APCs from progesterone-treated, placebo-treated, and estradiol-receptor knockout mice in APC-T cell co-cultures. CD11c⁺ DCs in the vagina were the predominant APC population responsible for priming these T_h17 responses, and a potent source of IL-6 and IL-1β, important factors for T_h17 differentiation. T_h17 responses were abrogated in APC-T cell co-cultures containing IL-1β KO, but not IL-6 KO vaginal DCs, showing that IL-1β is a critical factor for T_h17 induction in the genital tract. E2 treatment in vivo directly induced high expression of IL-1β in vaginal DCs, and addition of IL-1β restored T_h17 induction by IL-1β KO APCs in co-cultures. Finally, we examined the role of IL-17 in anti-HSV-2 memory T cell responses. IL-17 KO mice were more susceptible to intravaginal HSV-2 challenge, compared to WT controls, and vaginal DCs from these mice were defective at priming efficient T_h1 responses in vitro, indicating that IL-17 is important for the generation of efficient anti-viral memory responses. We conclude that the genital mucosa has a unique microenvironment whereby E2 enhances CD4⁺ T cell anti-viral immunity by priming vaginal DCs to induce T_h17 responses through an IL-1-dependent pathway.     

IL-27 affects helper T cell responses via regulation of PGE2 production by macrophages

IL-27 is a heterodimeric cytokine that regulates both innate and adaptive immunity. The immunosuppressive effect of IL-27 largely depends on induction of IL-10-producing Tr1 cells. To date, however, effects of IL-27 on regulation of immune responses via mediators other than cytokines remain poorly understood. To address this issue, we examined immunoregulatory effects of conditional medium of bone marrow-derived macrophages (BMDMs) from WSX-1 (IL-27Rα)-deficient mice and found enhanced IFN-γ and IL-17A secretion by CD4⁺ T cells as compared with that of control BMDMs. We then found that PGE₂ production and COX-2 expression by BMDMs from WSX-1-deficient mice was increased compared to control macrophages in response to LPS. The enhanced production of IFN-γ and IL-17A was abolished by EP2 and EP4 antagonists, demonstrating PGE₂ was responsible for enhanced cytokine production. Murine WSX-1-expressing Raw264.7 cells (mWSX-1-Raw264.7) showed phosphorylation of both STAT1 and STAT3 in response to IL-27 and produced less amounts of PGE₂ and COX-2 compared to parental RAW264.7 cells. STAT1 knockdown in parental RAW264.7 cells and STAT1-deficiency in BMDMs showed higher COX-2 expression than their respective control cells. Collectively, our result indicated that IL-27/WSX-1 regulated PGE₂ secretion via STAT1–COX-2 pathway in macrophages and affected helper T cell response in a PGE₂-mediated fashion.     

IL-17F deficiency inhibits small intestinal tumorigenesis in Apc mice

IL-17 plays an important role in gut homeostasis. However, the role of IL-17F in intestinal tumorigenesis has not been addressed. Here we demonstrate that ablation of IL-17F significantly inhibits spontaneous intestinal tumorigenesis in the small intestine of Apc^Min/+ mice. IL-17F ablation decreased IL-1β and Cox-2 expression as well as IL-17 receptor C (IL-17RC) expression, which were increased in tumors from Apc^Min/+ mice. Lack of IL-17F did not reverse the splenomegaly but partially restored thymic atrophy, suggesting a local effect of IL-17F in the intestine. IL-17F deficient Apc^Min/+ mice showed a significant decrease in immune cell infiltration in the lamina propria. Interestingly, the expression of IL-17A from CD4 T cells in the lamina propria remains unchanged in the absence of IL-17F. Collectively, our results suggest the proinflammatory and essential role of IL-17F to develop spontaneous intestinal tumorigenesis in Apc^Min/+ mice in the presence of IL-17A.