Innate Immune Responses to Bacterial Ligands in the Peripheral Human Lung – Role of Alveolar Epithelial TLR Expression and Signalling

It is widely believed that the alveolar epithelium is unresponsive to LPS, in the absence of serum, due to low expression of TLR4 and CD14. Furthermore, the responsiveness of the epithelium to TLR-2 ligands is also poorly understood. We hypothesised that human alveolar type I (ATI) and type II (ATII) epithelial cells were responsive to TLR2 and TLR4 ligands (MALP-2 and LPS respectively), expressed the necessary TLRs and co-receptors (CD14 and MD2) and released distinct profiles of cytokines via differential activation of MAP kinases. Primary ATII cells and alveolar macrophages and an immortalised ATI cell line (TT1) elicited CD14 and MD2-dependent responses to LPS which did not require the addition of exogenous soluble CD14. TT1 and primary ATII cells expressed CD14 whereas A549 cells did not, as confirmed by flow cytometry. Following LPS and MALP-2 exposure, macrophages and ATII cells released significant amounts of TNFα, IL-8 and MCP-1 whereas TT1 cells only released IL-8 and MCP-1. P38, ERK and JNK were involved in MALP-2 and LPS-induced cytokine release from all three cell types. However, ERK and JNK were significantly more important than p38 in cytokine release from macrophages whereas all three were similarly involved in LPS-induced mediator release from TT1 cells. In ATII cells, JNK was significantly more important than p38 and ERK in LPS-induced MCP-1 release. MALP-2 and LPS exposure stimulated TLR4 protein expression in all three cell types; significantly more so in ATII cells than macrophages and TT1 cells. In conclusion, this is the first study describing the expression of CD14 on, and TLR2 and 4 signalling in, primary human ATII cells and ATI cells; suggesting that differential activation of MAP kinases, cytokine secretion and TLR4 expression by the alveolar epithelium and macrophages is important in orchestrating a co-ordinated response to inhaled pathogens

Properties, production, and applications of camelid single-domain antibody fragments

Camelids produce functional antibodies devoid of light chains of which the single N-terminal domain is fully capable of antigen binding. These single-domain antibody fragments (VHHs or Nanobodies®) have several advantages for biotechnological applications. They are well expressed in microorganisms and have a high stability and solubility. Furthermore, they are well suited for construction of larger molecules and selection systems such as phage, yeast, or ribosome display. This minireview offers an overview of (1) their properties as compared to conventional antibodies, (2) their production in microorganisms, with a focus on yeasts, and (3) their therapeutic applications

Comparative evaluation of NASBA HIV-1 RNA QT, AMPLICOR-HIV monitor, and QUANTIPLEX HIV RNA assay, three methods for quantification of human immunodeficiency virus type 1 RNA in plasma.

Three commercial assays for quantifying plasma human immunodeficiency virus type 1 (HIV-1) RNA were evaluated. The assays differed in their sample volumes, the means of preparing samples, and methods of amplification and detection. Plasma samples were obtained from 36 HIV-1-infected patients representing all stages of HIV-1 infection and were analyzed as coded specimens. Measurement of HIV-1 RNA baseline levels revealed no significant difference in sensitivity between the three assays. The assays were also applied to the quantitation of HIV-1 RNA levels in the plasma of patients who were changing their antiretroviral therapy. The changes measured in HIV-1 RNA levels in plasma in response to therapy were comparable by the three assays. No close correlation was found between the amount of HIV-1 RNA and the CD4 T-cell count; HIV-1 RNA assays were more sensitive than p24 antigen assays as an indicator of plasma viremia. Overall, the study demonstrates that all three quantitative assays for HIV-1 RNA can be used to measure the HIV-1 RNA copy number representing the HIV-1 viremia status in patients with HIV-1 infection. Since this copy number is likely to be useful in monitoring the effectiveness of antiviral therapy, these quantitative assays for HIV-1 RNA are ready to be built into clinical trials

Detection of Differentially Modified Pathogen Proteins by Western Blot after 2D Gel Electrophoresis and Identification by MALDI-TOF/TOF

The detection of proteomic changes after viral infection, especially those which are due to post-translational modifications of host and pathogen proteins is of particular importance for the understanding of the fast interplay between pathogen and host components in viral infections. The characterization of modified isoforms of such proteins can benefit considerably from the combination of fluorescence labelled monospecific antibodies and 2D-DIGE differential proteomic studies. The potential of this approach is illustrated with a study of essential proteins in a measles virus-host cell system using small 2D gels and low sample amounts

Comprehensive mapping of immune tolerance yields a regulatory TNF receptor 2 signature in a murine model of successful Fel d 1-specific immunotherapy using high-dose CpG adjuvant.

Background: The prevalence of allergy to cat is expanding worldwide. Allergen-specific immunotherapy (AIT) has advantages over symptomatic pharmacotherapy and promises long-lasting disease control in allergic patients. However, there is still a need to improve cat AIT regarding efficacy, safety, and adherence to the treatment. Here, we aim to boost immune tolerance to the major cat allergen Fel d 1 by increasing the anti-inflammatory activity of AIT with the established immunomodulatory adjuvant CpG, but at a higher dose than previously used in AIT. Methods: Together with CpG, we used endotoxin-free Fel d 1 as therapeutic allergen throughout the study in a BALB/c model of allergy to Fel d 1, thus mimicking the conditions of human AIT trials. Multidimensional immune phenotyping including mass cytometry (CyTOF) was applied to analyze AIT-specific immune signatures. Results: We show that AIT with high-dose CpG in combination with endotoxin-free Fel d 1 reverts all major hallmarks of allergy. High-dimensional CyTOF analysis of the immune cell signatures initiating and sustaining the AIT effect indicates the simultaneous engagement of both, the pDC-Treg and B-cell axis, with the emergence of a systemic GATA3+ FoxP3hi biTreg population. The regulatory immune signature also suggests the involvement of the anti-inflammatory TNF/TNFR2 signaling cascade in NK and B cells at an early stage and in Tregs later during AIT. Conclusion: Our results highlight the potential of CpG adjuvant in a novel formulation to be further exploited for inducing allergen-specific tolerance in patients with cat allergy or other allergic diseases