A novel monoclonal antibody recognizing β(1–3) glucans in intact cells of Candida and Cryptococcus

The cell walls of all medically important fungi contain a unique polyglucose compound, β(1–3) glucan. In the present study, murine monoclonal antibodies were produced against linear and β(1–6) branched β(1–3) glucans, and their specificities were characterized for reactivity to other β glucans, fungal cell wall fragments, and fungal cells. Their reactivity was also compared with that of rabbit polyclonal antibodies raised against the same immunogens. Two mouse monoclonal antibodies (AG and BG) recognized immunoreactive epitopes in β(1–3)(1–6) glucan by ELISA. In an inhibition assay of the anti‐β(1–3)(1–6) activity of the monoclonals, the homologous antigen effectively inhibited the activity as expected, while β(1–3) also inhibited the assay but to a much lesser extent. No inhibition was obtained by β(1–3)(1–4) or β(1–6), while a cell wall extract of Candida albicans (PPM) effectively inhibited both monoclonals. Cell wall fragments of C. albicans (CaCW) and Cryptococcus neoformans (CnCW) inhibited the anti‐β(1–3)(1–6) activity of AG, while BG was much less or not inhibited at all. Immunofluorescence confirmed the unique antibody specificity of AG by its recognition of a β(1–3)(1–6)‐associated epitope on the cell surfaces of C. albicans,C. krusei, C. glabrata, and nonencapsulated C. neoformans. The epitope for the AG antibody is suggested to be present in the branching point of β(1–3)(1–6), or in the randomly coiled β(1–3) polyglucan due to the presence of branches. Thus, monoclonal antibodies to β(1–3)(1–6) glucans may have potential as tools in the laboratory diagnosis of invasive yeast infections.     

Anti‐Asthma Simplified Herbal Medicine Intervention‐induced long‐lasting tolerance to allergen exposure in an asthma model is interferon‐γ, but not transforming growth factor‐β dependent

Background Chronic allergic asthma is the result of a T‐helper type 2 (Th2)‐biased immune status. Current asthma therapies control symptoms in some patients, but a long‐lasting therapy has not been established. Anti‐Asthma Simplified Herbal Medicine Intervention (ASHMI^?), a Chinese herbal formula improved symptoms and lung function, and reduced Th2 responses in a controlled trial of patients with persistent moderate to severe asthma. Objective We evaluated the persistence of ASHMI^? beneficial effects following therapy in a murine model of chronic asthma and the immunological mechanisms underlying such effects. Methods BALB/c mice sensitized intraperitoneally with ovalbumin (OVA) received 3 weekly intratracheal OVA challenges to induce airway hyper‐reactivity (AHR) and inflammation (OVA mice). Additionally, OVA mice were treated with ASHMI^? (OVA/ASHMI^?) or water (OVA/sham) for 4 weeks, and then challenged immediately and 8 weeks post‐therapy. In other experiments, OVA mice received ASHMI^? treatment with concomitant neutralization of IFN‐γ or TGF‐β. Effects on airway responses, cytokine‐ and OVA‐specific IgE levels were determined 8 weeks post‐therapy. Results Before treatment, OVA mice exhibited AHR and pulmonary eosinophilic inflammation following OVA challenge, which was almost completely resolved immediately after completing treatment with ASHMI^? and did not re‐occur following OVA re‐challenge up to 8 weeks post‐therapy. Decreased allergen‐specific IgE and Th2 cytokine levels, and increased IFN‐γ levels also persisted at least 8 weeks post‐therapy. ASHMI^? effects were eliminated by the neutralization of IFN‐γ, but not TGF‐β, during therapy. Conclusion ASHMI^? induced long‐lasting post‐therapy tolerance to antigen‐induced inflammation and AHR. IFN‐γ is a critical factor in ASHMI^? effects. Cite this as: K. Srivastava, T. Zhang, N. Yang, H. Sampson and X. M. Li, Clinical & Experimental Allergy, 2010 (40) 1678–1688.     

Impaired phagocytosis directs human monocyte activation in response to fungal derived β‐glucan particles

Recognition of the fungal cell wall carbohydrate β‐glucan by the host receptor Dectin‐1 elicits broad immunomodulatory responses, such as phagocytosis and activation of oxidative burst. These responses are essential for engulfing and killing fungal pathogens. Phagocytic monocytes are key mediators of these early host inflammatory responses to infection. Remarkably, whether phagocytosis of fungal β‐glucan leads to an inflammatory response in human monocytes remains to be established. Here, we show that phagocytosis of heat‐killed Candida albicans is essential to trigger inflammation and cytokine release. By contrast, inhibition of actin‐dependent phagocytosis of particulate (1‐3,1‐6)‐β‐glucan induces a strong inflammatory signature. Sustained monocyte activation, induced by fungal β‐glucan particles upon actin cytoskeleton disruption, relies on Dectin‐1 and results in the classical caspase‐1 inflammasome formation through NLRP3, generation of an oxidative burst, NF‐κB activation, and increased inflammatory cytokine release. PI3K and NADPH oxidase were crucial for both cytokine secretion and ROS generation, whereas Syk signaling mediated only cytokine production. Our results highlight the mechanism by which phagocytosis tightly controls the activation of phagocytes by fungal pathogens and strongly suggest that actin cytoskeleton dynamics are an essential determinant of the host's susceptibility or resistance to invasive fungal infections.     

Involvement of α‐ and β‐catenins and E‐cadherin in the development of mammary phyllodes tumours

Tsang J Y S, Mendoza P, Lam C C F, Yu A M C, Putti T C, Karim R Z, Scolyer R A, Lee C S, Tan P H & Tse G M
(2012) Histopathology  61, 667–674 Involvement of α‐ and β‐catenins and E‐cadherin in the development of mammary phyllodes tumours Aims: Phyllodes tumours (PT) are rare but clinically important fibroepithelial tumours of the breast. β‐Catenin, a key component in Wnt signalling, has been shown to be important in the development of PT. It also functions as a component of the cadherin complex, which may therefore be implicated in PT pathogenesis. By assessing stromal α‐catenin, β‐catenin and E‐cadherin expression in 158 PT cases using immunohistochemistry and examining associations with clinicopathological features, we aimed to determine the role of these proteins in PT pathogenesis. Methods and results: Cytoplasmic β‐catenin correlated with α‐catenin expression. A significantly higher expression of both markers was observed in borderline than in benign PT (P = 0.003 and <0.001, respectively), but a lower level was found in malignant PT. Cytoplasmic E‐cadherin expression was significantly higher in borderline and malignant than in benign PT (P = 0.001 and 0.012, respectively), but was not correlated with other markers. Both E‐cadherin and α‐catenin showed stronger correlations with histological parameters than β‐catenin. α‐Catenin showed a significant correlation with recurrence (P = 0.005 and 0.016, respectively). Conclusions: α‐ and β‐catenins may be important in the early stages of PT development, while E‐cadherin may be required for malignant development. The correlation of α‐catenin expression with tumour recurrence may be relevant in predicting PT behaviour.     

Inhibition of murine γδ lymphocyte expansion and effector function by regulatory αβ T cells is cell‐contact‐dependent and sensitive to GITR modulation

γδ T cells are highly cytolytic lymphocytes that produce large amounts of pro‐inflammatory cytokines during immune responses to multiple pathogens. Furthermore, their ability to kill tumor cells has fueled the development of γδ‐T‐cell‐based cancer therapies. Thus, the regulation of γδ‐T‐cell activity is of great biological and clinical relevance. Here, we show that murine CD4⁺CD25⁺ αβ T cells, the vast majority of which express the Treg marker, Foxp3, abolish key effector functions of γδ T cells, namely the production of the pro‐inflammatory cytokines, IFN‐γ and IL‐17, cytotoxicity, and lymphocyte proliferation in vitro and in vivo. We further show that suppression is dependent on cellular contact between Treg and γδ T cells, results in the induction of an anergic state in γδ lymphocytes, and can be partially reversed by manipulating glucocorticoid‐induced TNF receptor‐related protein (GITR) signals. Our data collectively dissect a novel mechanism by which the expansion and pro‐inflammatory functions of γδ T cells are regulated.     

Synthesis and Characterization of Polyrotaxanes Comprising γ‐CDs and Distal Azide‐Terminated PHEMA Using Propargylamine Monosubstituted β‐CDs as End Stoppers

To highlight whether γ‐cyclodextrins (CDs) facilitate propargylamine monosubstituted β‐CDs (PA‐β‐CDs) as end stoppers to get threaded onto a distal azide terminated poly(2‐hydroxyethylmethacrylate) (PHEMA) homopolymer (PH‐46‐2N₃) to create linear and hyperbranched polyrotaxanes (PRs) via the in situ copper‐catalyzed azide/alkyne cycloaddition (CuAAC), PH‐46‐2N₃ is self‐assembled with a varying amount of γ‐CDs in water and then subjected the CuAAC with PA‐β‐CDs to end‐cap the resulting γ‐CD‐PHEMA polypseudorotaxanes (PPRs) into the γ‐CD‐PHEMA PRs. It demonstrates that γ‐CDs cannot promote PA‐β‐CDs to be entrapped on the PHEMA chain most likely due to their different cavity size and molecular framework and linear PRs are always formed with up to 29% γ‐CD coverage ratio along the PHEMA axis thereof.     

Submandibular gland morphogenesis: Stage‐specific expression of TGF‐α/EGF,IGF, TGF‐β, TNF,and IL‐6 signal transduction in normal embryonic mice and the phenotypic effects of TGF‐β2, TGF‐β3, and EGF‐r null mutations

Branching morphogenesis of the mouse submandibular gland (SMG) is dependent on cell‐cell conversations between and within epithelium and mesenchyme. Such conversations are typically mediated in other branching organs (lung, mammary glands, etc.) by hormones, growth factors, cytokines, and the like in such a way as to translate endocrine, autocrine, and paracrine signals into specific gene responses regulating cell division, apoptosis, and histodifferentiation. We report here the protein expression in embryonic SMGs of four signal transduction pathways: TGF‐α/EGF/EGF‐R; IGF‐II/IGF‐IR/IGF‐IIR; TGF‐βs and cognate receptors; TNF, IL‐6, and cognate receptors. Their in vivo spatiotemporal expression is correlated with specific stages of progressive SMG development and particular patterns of cell proliferation, apoptosis, and mucin expression. Functional necessity regarding several of these pathways was assessed in mice with relevant null mutations (TGF‐β2, TGF‐β₃, EGF‐R). Among many observations, the following seem of particular importance: (1) TGF‐α and EGF‐R, but not EGF, are found in the Initial and Pseudoglandular Stages of SMG development; (2) ductal and presumptive acini lumena formation was associated with apoptosis and TNF/TNF‐R1 signalling; (3) TGF‐β2 and TGF‐β3 null mice have normal SMG phenotypes, suggesting the presence of other pathways of mitostasis; (4) EGF‐R null mice displayed an abnormal SMG phenotype consisting of decreased branching. These and other findings provide insight into the design of future functional studies. Anat Rec 256:252–268, 1999. © 1999 Wiley‐Liss, Inc.     

αβ Lineage-committed thymocytes can be rescued by the γδ T cell receptor (TCR) in the absence of TCR β chain

Commitment of the αβ and γδ T cell lineages within the thymus has been studied in T cell receptor (TCR)-transgenic and TCR mutant murine strains. TCRγδ-transgenic or TCRβ knockout mice, both of which are unable to generate TCRαβ-positive T cells, develop phenotypically αβ-like thymocytes in significant proportions. We provide evidence that in the absence of functional TCRβ protein, the γδTCR can promote the development of αβ-like thymocytes, which, however, do not expand significantly and do not mature into γδ T cells. These results show that commitment to the αβ lineage can be determined independently of the isotype of the TCR, and suggest that αβ versus γδ T cell lineage commitment is principally regulated by mechanisms distinct from TCR-mediated selection. To accommodate our data and those reported previously on the effect of TCRγ and δ gene rearrangements on αβ T cell development, we propose a model in which lineage commitment occurs independently of TCR gene rearrangement.     

Interleukin‐17‐producing γδ+ T cells protect NOD mice from type 1 diabetes through a mechanism involving transforming growth factor‐β

Whether interleukin (IL)‐17 promotes a diabetogenic response remains unclear. Here we examined the effects of neutralization of IL‐17 on the progress of adoptively transferred diabetes. IL‐17‐producing cells in non‐obese diabetic (NOD) mice were identified and their role in the pathogenesis of diabetes examined using transfer and co‐transfer assays. Unexpectedly, we found that in vivo neutralization of IL‐17 did not protect NOD–severe combined immunodeficiency (SCID) mice against diabetes transferred by diabetic splenocytes. In NOD mice, γδ⁺ T cells were dominated by IL‐17‐producing cells and were found to be the major source of IL‐17. Interestingly, these IL‐17‐producing γδ T cells did not exacerbate diabetes in an adoptive transfer model, but had a regulatory effect, protecting NOD mice from diabetes by up‐regulating transforming growth factor (TGF)‐β production. Our data suggest that the presence of IL‐17 did not increase the chance of the development of diabetes; γδ T cells protected NOD mice from diabetes in a TGF‐β‐dependent manner, irrespective of their role as major IL‐17 producers.