Chlamydia pneumoniae infection induced allergic airway sensitization is controlled by regulatory T-cells and plasmacytoid dendritic cells

Chlamydia pneumoniae (CP) is associated with induction and exacerbation of asthma. CP infection can induce allergic airway sensitization in mice in a dose- and time-dependent manner. Allergen exposure 5 days after a low dose (mild-moderate), but not a high dose (severe) CP infection induces antigen sensitization in mice. Innate immune signals play a critical role in controlling CP infection induced allergic airway sensitization, however these mechanisms have not been fully elucidated. Wild-type, TLR2-/-, and TLR4-/- mice were infected intranasally (i.n.) with a low dose of CP, followed by i.n. exposure to human serum albumin (HSA) and challenged with HSA 2 weeks later. Airway inflammation, immunoglobulins, eosinophils, and goblet cells were measured. Low dose CP infection induced allergic sensitization in TLR2-/- mice, but not in TLR4-/- mice, due to differential Treg responses in these genotypes. TLR2-/- mice had reduced numbers of Tregs in the lung during CP infection while TLR4-/- mice had increased numbers. High dose CP infection resulted in an increase in Tregs and pDCs in lungs, which prevented antigen sensitization in WT mice. Depletion of Tregs or pDCs resulted in allergic airway sensitization. We conclude that Tregs and pDCs are critical determinants regulating CP infection-induced allergic sensitization. Furthermore, TLR2 and TLR4 signaling during CP infection may play a regulatory role through the modulation of Tregs.     

Overexpression of nucleoside diphosphate kinases induces neurite outgrowth and their substitution to inactive forms leads to suppression of nerve growth factor- and dibutyryl cyclic AMP-induced effects in PC12D cells

Whether nucleoside diphosphate kinase (NDPK) is involved in neuronal differentiation was investigated with special reference to its enzyme activity. Neurite outgrowth of PC12D cells induced by nerve growth factor or a cyclic AMP analog was suppressed to some extent when inactive NDPKs (the active site histidine 118 was replaced with alanine), not active forms, were transiently overexpressed. This suppression was more definite in their stably expressed clones. NDPKbeta-transfected clones and, to a lesser extent, NDPKalpha-transfected clones, but not inactive NDPK-transfected clones, extended neurites without differentiation inducers. These results imply that NDPKs may play a role by exerting their enzyme activity during differentiation of PC12 cells.     

Utilization of Methyl Proton Resonances in Cross-Saturation Measurement for Determining the Interfaces of Large Protein–Protein Complexes

Cross-saturation experiments allow the identification of the contact residues of large protein complexes (MW>50 K) more rigorously than conventional NMR approaches which involve chemical shift perturbations and hydrogen-deuterium exchange experiments [Takahashi et al. (2000) Nat. Struct. Biol., 7, 220–223]. In the amide proton-based cross-saturation experiment, the combined use of high deuteration levels for non-exchangeable protons of the ligand protein and a solvent with a low concentration of ¹H₂O greatly enhanced the selectivity of the intermolecular cross-saturation phenomenon. Unfortunately, experimental limitations caused losses in sensitivity. Furthermore, since main chain amide protons are not generally exposed to solvent, the efficiency of the saturation transfer directed to the main chain amide protons is not very high. Here we propose an alternative cross-saturation experiment which utilizes the methyl protons of the side chains of the ligand protein. Owing to the fast internal rotation along the methyl axis, we theoretically and experimentally demonstrated the enhanced efficiency of this approach. The methyl-utilizing cross-saturation experiment has clear advantages in sensitivity and saturation transfer efficiency over the amide proton-based approach. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

L-selectin or ICAM-1 deficiency reduces an immediate-type hypersensitivity response by preventing mast cell recruitment in repeated elicitation of contact hypersensitivity

Repeated Ag exposure results in a shift in the time course of contact hypersensitivity (CH) from a typical delayed-type to an immediate-type response followed by a late phase reaction. Chronic CH responses are clinically relevant to human skin allergic diseases, such as atopic dermatitis, that are usually caused by repeated stimulation with environmental Ags. Chronic inflammatory responses result in part from infiltrating leukocytes. To determine the role of leukocyte adhesion molecules in chronic inflammation, chronic CH responses were assessed in mice lacking L-selectin, ICAM-1, or both adhesion molecules. Following repeated hapten sensitization for 24 days at 2-day intervals, wild-type littermates developed an immediate-type response at 30 min after elicitation, followed by a late phase reaction. By contrast, loss of ICAM-1, L-selectin, or both, eliminated the immediate-type response and inhibited the late phase reaction. Similar results were obtained when wild-type littermates repeatedly exposed to hapten for 22 days were treated with mAbs to L-selectin and/or ICAM-1 before the elicitation on day 24. The lack of an immediate-type response on day 24 paralleled a lack of mast cell accumulation after 30 min of elicitation and decreased serum IgE production. Repeated Ag exposure in wild-type littermates resulted in increased levels of serum L-selectin, a finding also observed in atopic dermatitis patients. The current study demonstrates that L-selectin and ICAM-1 cooperatively regulate the induction of the immediate-type response by mediating mast cell accumulation into inflammatory sites and suggests that L-selectin and ICAM-1 are potential therapeutic targets for regulating human allergic reactions.     

Hepatitis C Virus NS3/4A Protease Inhibits Complement Activation by Cleaving Complement Component 4

Background

It has been hypothesized that persistent hepatitis C virus (HCV) infection is mediated in part by viral proteins that abrogate the host immune response, including the complement system, but the precise mechanisms are not well understood. We investigated whether HCV proteins are involved in the fragmentation of complement component 4 (C4), composed of subunits C4α, C4β, and C4γ, and the role of HCV proteins in complement activation.

Methods

Human C4 was incubated with HCV nonstructural (NS) 3/4A protease, core, or NS5. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then subjected to peptide sequencing. The activity of the classical complement pathway was examined using an erythrocyte hemolysis assay. The cleavage pattern of C4 in NS3/4A-expressing and HCV-infected cells, respectively, was also examined.

Results

HCV NS3/4A protease cleaved C4γ in a concentration-dependent manner, but viral core and NS5 did not. A specific inhibitor of NS3/4A protease reduced C4γ cleavage. NS3/4A protease–mediated cleavage of C4 inhibited classical pathway activation, which was abrogated by a NS3/4A protease inhibitor. In addition, co-transfection of cells with C4 and wild-type NS3/4A, but not a catalytic-site mutant of NS3/4A, produced cleaved C4γ fragments. Such C4 processing, with a concomitant reduction in levels of full-length C4γ, was also observed in HCV-infected cells expressing C4.

Conclusions

C4 is a novel cellular substrate of the HCV NS3/4A protease. Understanding disturbances in the complement system mediated by NS3/4A protease may provide new insights into the mechanisms underlying persistent HCV infection.     

Micronucleus test with potassium chromate(VI) administered intraperitoneally and orally to mice

The effect of route of administration, intraperitoneal (i.p.) or oral gavage (p.o.), in the mouse micronucleus test was studied with K2CrO4 in 2 mouse strains (MS/Ae and CD-1). A simplified acute toxicity test to estimate the toxic dose levels of K2CrO4 showed that the LD50S were 50 mg/kg i.p. and 300 mg/kg p.o. for MS/Ae and 32 mg/kg i.p. and 180 mg/kg p.o. for CD-1. Based on results of a pilot micronucleus test to determine appropriate dose levels and the optimal sampling time, it was decided to sample bone marrow cells of both strains of mice 24 h after i.p. doses of 10-80 mg/kg and p.o. doses ranging from 20 to 320 mg/kg. K2CrO4 administered i.p. induced micronucleated polychromatic erythrocytes (MNPCEs) dose-dependently in both strains. In contrast, when administered p.o. the chemical failed to induce MNPCEs. These results suggest that this difference between i.p. and p.o. routes is related to a difference of absorption or metabolic fate of chromate in vivo.     

Enhanced antigen-presenting capacity of cultured Langerhans' cells is associated with markedly increased expression of Ia antigen

Recent studies indicate that when epidermal Langerhans' cells (LC) are cultured for 2 to 3 days they, in comparison to freshly prepared LC, exhibit markedly enhanced ability to stimulate T cell proliferative responses in oxidative mitogenesis and in the mixed epidermal-leukocyte reaction. In this study, we determined whether cultured LC enhance antigen-specific T cell responses, and whether such enhanced stimulatory capacity correlates with the level of Ia antigen expressed on LC. We used C3H/He (Iak) epidermal cells as stimulators and, as responder cells, both the trinitrophenyl-specific clones D8 and SE4, which were assayed for [3H]dThd incorporation, and the pigeon cytochrome c specific hybridoma 2C2, which was assayed for interleukin 2 production. Cultured LC induced 10 to 100 times greater proliferation or interleukin 2 production by responder cells than did freshly prepared LC. The intensity of I-Ak and I-Ek, expressed on cultured LC as assessed by immunofluorescence and flow cytometry, was found to be 10 to 36 times greater on a per cell basis than that on freshly prepared LC. Depletion of LC from fresh epidermal cell suspensions by anti-Iak and complement or treatment with 50 mJ/cm2 medium range ultraviolet light or cycloheximide before culture abrogated both the increase in Ia expression and antigen-specific clonal proliferation. The results suggest that when LC are removed from their usual epidermal milieu, they express increased amounts of Ia and become more potent stimulators of T cell responses.