A class C CpG toll-like receptor 9 agonist successfully induces robust interferon-alpha production by plasmacytoid dendritic cells from patients chronically infected with hepatitis C.

Immunomodulators that induce local endogenous interferon-alpha (IFN-alpha) production by plasmacytoid dendritic cells (pDCs) may offer new strategies for the treatment of patients chronically infected with the hepatitis C virus (HCV). However, such an approach may be compromised if reports are true that IFN-alpha production by pDCs from patients with chronic HCV (cHCV) is profoundly impaired. To address the question of pDC dysfunction in cHCV more definitively, in the present study a panel of four prototypic synthetic agonists of toll-like receptor 7 (TLR7) or TLR9 were administered in vitro to pDCs purified from cHCV patients and from normal uninfected donors and their responses compared in terms of not only IFN-alpha production but also the global expression of other cytokines and phenotypic maturation. Plasmacytoid DCs from uninfected donors produced substantial levels of IFN-alpha in response to three of the four agonists and yet only one TLR9 agonist, a class C CpG oligodeoxynucleotide (ODN), induced robust IFN-alpha production by pDCs from cHCV patients. Proinflammatory cytokine production and phenotypic maturation in response to all four agonists was equivalent in infected and uninfected pDCs. These data point to a profound but selective defect in IFN-alpha production by pDCs from cHCV donors. Nonetheless, a class C CpG ODN successfully induced robust IFN-alpha production, suggesting that this class of TLR9 agonist may have utility as a future immunotherapeutic for the treatment of chronic HCV infection.

A novel, helminth-derived immunostimulant enhances human recall responses to hepatitis C virus and tetanus toxoid and is dependent on CD56+ cells for its action.

We have described previously an immunostimulant derived from Onchocerca volvulus, the helminth parasite that causes onchocerciasis. Recombinant O. volvulus activation-associated secreted protein-1 (rOv-ASP-1) was a potent adjuvant for antibody and cellular responses to protein, polypeptide and small peptide antigens. Our aims were to determine whether rOv-ASP-1 is immunostimulatory for human peripheral blood mononuclear cells (PBMC) and, if so, whether it could augment cellular responses against human pathogen antigens in vitro. Cytokines from rOv-ASP-1-stimulated human PBMC were measured by a fluorescence activated cell sorter-based multiplex assay. Recall responses of normal healthy donor (NHD) and chronic hepatitis C virus (c-HCV)-infected patient PBMC to tetanus toxoid (TT) or HCV core (HCVco) antigen, respectively, were measured by interferon-gamma enzyme-linked immunospot assays. Interferon-gamma was the predominant cytokine induced by rOv-ASP-1. 77.3% of NHD anti-TT and 88.9% of c-HCV anti-HCVco responses were enhanced by rOv-ASP-1. The immunostimulant effect was dependent upon contact between CD56+ and CD56- fractions of PBMC. We have described a helminth-derived protein that can act as an immunostimulant for human recall responses in vitro to TT and, perhaps more importantly, HCV antigens in patients with chronic HCV infection. Our longer-term goal would be to boost anti-viral responses in chronic infections such as HCV.

Monocyte-derived dendritic cell function in chronic hepatitis C is impaired at physiological numbers of dendritic cells.

Monocyte-derived dendritic cells (MoDCs) are a promising cellular adjuvant for effector immune responses against tumours and chronic viral infections, including hepatitis C virus (HCV). If autologous DC therapeutic approaches are to be applied in persistent HCV infections in patients, it is important to have an unambiguous understanding of the functional status of the cell type used, namely MoDCs from patients with chronic hepatitis C (CHC) infection. Because of conflicting published reports of either impaired or normal MoDC function in CHC infection, we re-examined the ability of MoDCs from CHC and normal healthy donors (NHD) to mature to an inflammatory stimulus [tumour necrosis factor (TNF)-alpha] and their subsequent functional capabilities. Expression of maturation-associated phenotypic markers [human leucocyte antigen (HLA)-DR, CD83, CD86, CD40], allostimulatory capacity in mixed lymphocyte reactions (MLRs) and CD40-ligand-induced cytokine and chemokine generation were compared in CHC- versus NHD-MoDCs. TNF-alpha-stimulated CHC-MoDCs up-regulated phenotypic markers, but to significantly lower levels than NHD-MoDCs. At physiological ratios of DCs to T cells, CHC-MoDCs were less allostimulatory than NHD-MoDCs, but not when DC numbers were substantially increased. CHC- and NHD-MoDCs generated equivalent amounts of cytokines [TNF-alpha, interleukin (IL)-1beta, IL-6, IL-12p70, IL-15, IL-10] and chemokines [interferon-inducible protein (IP)-10, macrophage inflammatory protein (MIP)-1alpha, regulated upon activation, normal T expressed and secreted (RANTES)] after CD40 ligation. Because the functional defect was not apparent at high MoDC : T cell ratios, autologous MoDC therapy with sufficiently high numbers of DCs could, in theory, overcome any impairment of MoDC function in CHC.

Expression of the high-affinity IgE receptor on peripheral blood dendritic cells: differential binding of IgE in atopic asthma.

BACKGROUND: Dendritic cells can express the high-affinity IgE receptor (FcepsilonRI), which, in the presence of specific IgE, facilitates the uptake of allergen, leading to increased activation of allergen-specific T cells. FcepsilonRI expression by dendritic cells is higher in the airways of atopic asthmatic subjects than in those of healthy, nonatopic control subjects. OBJECTIVE: The aims of this study were to determine whether a similar difference in FcepsilonRI expression occurs between dendritic cells in the peripheral blood of atopic asthmatic subjects and healthy individuals and also whether an altered ability of FcepsilonRI(+) peripheral blood dendritic cells to bind IgE accompanies the atopic asthmatic state. METHODS: Flow cytometry was used to analyze the surface expression of FcepsilonRI and exogenously bound IgE on dendritic cells identified as lineage negative (CD3, CD14, CD16, CD19, and CD56) and HLA-DR bright. RESULTS: The total expression of FcepsilonRI on the surface of dendritic cells from healthy and asthmatic subjects was not significantly different. However, in vivo, dendritic cells from atopic asthmatic subjects had higher levels of receptor occupancy by IgE and bound exogenous IgE in vitro more efficiently than dendritic cells from healthy subjects. CONCLUSION: The similar levels of expression of FcepsilonRI on peripheral blood dendritic cells from healthy and asthmatic subjects suggest that the local environment in the airway is responsible for the upregulation of surface FcepsilonRI on airway dendritic cells in asthma. The results also suggest that the functional ability of FcepsilonRI to bind IgE is differentially controlled in the atopic state.

Cytokine profiles of BAL T cells and T-cell clones obtained from human asthmatic airways after local allergen challenge.

BACKGROUND: This study assessed the heterogeneity of cytokine expression in asthma before and after local allergen challenge. METHODS: BAL T cells were obtained 10 min or 24 h after local endobronchial allergen challenge in atopic asthmatic subjects. T cells were cloned by direct limiting dilution. mRNA expression was assessed by RT-PCR, and cytokine protein production by ELISA. RESULTS: Unstimulated baseline BAL T cells expressed mRNA for IFN-gamma, IL-13, and TNF-alpha. A minority of samples expressed IL-4 and IL-5, but no IL-3 mRNA was detected. PHA stimulation increased expression of IL-3, IL-4, and IL-5 mRNA in 4/6 samples. IL-13 and GM-CSF mRNA were found in BAL cells after allergen challenge, but expression of IFN-gamma was reduced. Both IL-4 and IL-3 were strongly upregulated after PHA stimulation, while the expression of TNF-alpha and IFN-gamma was reduced, compared to equivalent baseline samples. Seventeen panels of BAL T-cell clones were derived (average cloning efficiency 1/40 T cells). Seven panels survived to 8 weeks for analysis. Clones derived 4 h after saline challenge showed strong mRNA signals for IL-13, IL-4, and IFN-gamma, whereas clones derived 24 h after allergen challenge expressed IL-13, GM-CSF, IL-3, IL-4, and often IL-5 (i.e., closer to the Th2 profile). There was considerable heterogeneity in the patterns of cytokine mRNA and protein production by different clones. CONCLUSIONS: T cells from asthmatic airways produce IL-13, IFN-gamma, and TNF-alpha, but after allergen challenge, type 2 cytokines are upregulated. mRNA and protein analysis provide complementary information on airways T-cell cytokine profiles.

Dendritic cells in normal and asthmatic airways: expression of the alpha subunit of the high affinity immunoglobulin E receptor (Fc epsilon RI -alpha).

BACKGROUND: Immunoglobulin E (IgE) plays an important role in asthma, with total serum IgE levels closely related to both clinical expression of the disease and airway hyperresponsiveness. IgE binds to a high affinity cell-surface receptor (Fc epsilon RI) which is present on mast cells and which has also recently been demonstrated on cutaneous dendritic cells. If pulmonary dendritic cells were also able to express this receptor, this would have important implications with regard to their potential role in asthma. OBJECTIVES: The aims of the study were to investigate the expression of the alpha subunit of the high affinity IgE receptor (Fc epsilon RI-alpha) in normal and asthmatic airways, and to analyse its cellular provenance with particular emphasis on the dendritic cell. METHODS: Bronchial biopsy specimens were obtained using fibreoptic bronchoscopy from 10 atopic asthmatics and nine non-atopic non-asthmatic control subjects. Specimens were processed into glycolmethacrylate resin and analysed by immunohistochemistry using specific monoclonal antibodies against Fc epsilon RI-alpha, and against tryptase and CD1a, markers for mast cells and dendritic cells, respectively. RESULTS: The numbers of dendritic cells were significantly higher in the airways of asthmatics compared with those of control subjects (P < 0.02). Analysis of sequential sections revealed that the alpha subunit of Fc epsilon RI was localized to both mast cells and dendritic cells. The proportion of dendritic cells expressing Fc epsilon RI-alpha was significantly increased in the asthmatic group (P < 0.003). CONCLUSION: These results support the hypothesis that dendritic cells play an important role in allergic asthma although the functional significance of Fc epsilon RI-alpha expression needs further investigation.

Ultrastructural identification of type 1 fibres in human skeletal muscle. Immunogold labelling of thin cryosections with a monoclonal antibody against slow myosin.

Existing methods for the ultrastructural identification of fibre types in human skeletal muscle are fallible. This has prompted us to develop a reliable immunoelectron microscopic approach for the identification of human skeletal muscle fibre types. Here we report the unambiguous electron microscopic identification of human type 1 muscle fibres, achieved by combining cryoultramicrotomy with colloidal gold immunocytochemical labelling, using a monoclonal antibody (N0Q7.5.4D) which is specific for the heavy chain of the slow myosin isoform of human skeletal muscle. This method for the identification of muscle fibre types and determination of myosin isoform distributions may have important applications in the ultrastructural study of pathological muscle and in the analysis of myofibrillar assembly during myogenesis.