Autoantibodies in lung cancer: possibilities for early detection and subsequent cure.

BACKGROUND: People with lung cancer usually present at a late stage in the course of their disease when their chances of long-term survival are low. At present there is little to offer for early diagnosis, even in those at high risk of developing the disease. Autoantibodies have been shown to be present in the circulation of people with various forms of solid tumour before cancer-associated antigens can be detected, and these molecules can be measured up to 5 years before symptomatic disease. OBJECTIVE: To assess the potential of a panel of tumour-associated autoantibody profiles as an aid to other lung cancer screening modalities. METHODS: Plasma from normal controls (n = 50), patients with non-small cell lung cancer (n = 82) and patients with small cell lung cancer (n = 22) were investigated for the presence of autoantibodies to p53, c-myc, HER2, NY-ESO-1, CAGE, MUC1 and GBU4-5 by enzyme-linked immunosorbent assay. RESULTS: Raised levels of autoantibodies were seen to at least 1/7 antigens in 76% of all the patients with lung cancer plasma tested, and 89% of node-negative patients, with a specificity of 92%. There was no significant difference between the detection rates in the lung cancer subgroups, although more patients with squamous cell carcinomas (92%) could be identified. CONCLUSION: Measurement of an autoantibody response to one or more tumour-associated antigens in an optimised panel assay may provide a sensitive and specific blood test to aid the early detection of lung cancer.

Expression of human beta-defensins in intraocular tissues.

PURPOSE: Defensins are naturally occurring antimicrobial peptides. Recently the authors published evidence of defensin production by the human ocular surface. A study was undertaken to look for intraocular defensins that may account for unexplained antimicrobial activity of intraocular fluids. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was performed on human postmortem ciliary body samples for beta defensins-1 (HBD-1) and beta defensin-2 (HBD-2), and alpha defensins 5 and 6. Induction of defensins by cytokines was analyzed in cultured human ciliary body epithelial (CBE) and retinal pigment epithelial (RPE) cells. Polyclonal antibodies were used to immunoblot aqueous and vitreous to detect HBD-1 and HBD-2 and to estimate their concentration. RESULTS: RT-PCR revealed constitutive HBD-1 message in ciliary body. HBD-2 and alpha defensin 5 and 6 messages were absent. HBD-2 message was induced by cytokine stimulation of both CBE and RPE cells. Immunoblots of vitreous and aqueous stained positively for HBD-1 but not HBD-2. The estimated aqueous concentration of HBD-1 was less than 16 ng/ml. CONCLUSIONS: This study demonstrates that HBD-1 is constitutively present in the aqueous and vitreous, probably at sub-bacteriocidal concentrations. HBD-2 was absent from aqueous, but cytokine stimulation studies suggest that it may be generated in response to inflammatory cytokines during infections. HBD-2 has a wider antibacterial spectrum, is 10-fold more potent, and may play a more significant role in antimicrobial defense than HBD-1. The use of defensins therapeutically may be indicated; however, caution is required because defensins also promote cell proliferation and fibrin formation, which are 2 key elements in ocular scarring processes such as proliferative vitreoretinopathy.

Ovine trophoblast interferon enhances MHC class I expression by sheep endometrial cells.

Type I interferons have a variety of important immunological functions, including effects on expression of class I molecules of the major histocompatibility complex (MHC). Interferon-tau (IFN-tau) is an important anti-luteolytic factor produced by the trophoblast of ruminants. Ovine endometrial cells were cultured with recombinant ovine IFN-tau and stained with monoclonal antibodies specific for ovine MHC class I molecules. The expression of MHC class I was then determined by immunocytochemical or immunofluorescent staining using microscopy or flow cytometry. IFN-tau was found to enhance the expression of MHC class I molecules on the endometrial cells, as did recombinant IFN-alpha. No significant effects on MHC class II expression were observed. These findings may have important implications for cellular interactions associated with immunological aspects of pregnancy.

Primary sequence and molecular model of the variable region of a mouse monoclonal anti-Der p 1 antibody showing a similar epitope specificity as human IgE.

BACKGROUND: Der p 1, a major mite allergen, elicits IgE antibody responses in 80% of patients suffering from dust mite allergy. Given the potent IgE eliciting properties of Der p 1, there is considerable interest in studying the molecular architecture of the variable (Fv) region of IgE antibodies specific for this allergen. OBJECTIVES: IgE is present in human serum at extremely low concentrations, and as such it is practically impossible to purify sufficient quantities for structural studies. We have therefore sought to sequence and model a representative murine monoclonal (MoAb) anti-Der p 1 antibody, as a surrogate human IgE. METHODS: The cDNA coding for the Fv region of an anti-Der p 1 MoAb (2C7), that mimics the binding of human IgE to Der p 1, was amplified by PCR, cloned and sequenced. The predicted amino acid sequences were then compared with a directory of human germline V-gene segments. Modelling of the Fv region of MoAb 2C7 was carried out using the extensive database of existing immunoglobulin structures in the Brookhaven PDB. RESULTS: The MoAb 2C7 heavy chain showed greater than 70% homology with three members of the VH3 family, DP-35, DP-53 and DP-54. Similarly, the light chain showed greater than 70% homology with 11 VK sequences, including the VKII sequences DPK18, DPK19 and DPK28. A molecular model of the Fv region of MoAb 2C7 was generated and can be accessed from the EMBL databank. CONCLUSIONS: Antibodies similar to MoAb 2C7 could be generated as part of the human repertoire. The availability of 3-dimensional model of MoAb 2C7, as a surrogate human IgE antibody, combined with further data on its epitope specificity, will facilitate studies into IgE antibody responses to Der p 1.

The production and characterisation of a chimaeric human IgE antibody, recognising the major mite allergen Der p 1, and its chimaeric human IgG1 anti-idiotype.

BACKGROUND: Two mouse monoclonal antibodies have been described, namely: mAb 2C7 (IgG2bkappa), which is directed against the major house dust mite allergen Der p 1, and mAb 2G10 (IgG1kappa), which is an anti-idiotypic antibody raised against mAb 2C7. Given its broad IgE specificity, anti-idiotype mAb 2G10 could potentially have immunomodulatory applications. For example, a chimaeric human IgG version of mAb 2G10 could prove to be a useful molecule for binding to mast cell and basophil FcepsilonRI bound IgE, and in doing so co-ligating FcepsilonRI with FcgammaRIIB, which has been reported to have downregulatory effects. AIMS: To produce a chimaeric human IgE version of mAb 2C7 (mAb 2C7huE) and a chimaeric human IgG1 version of its anti-idiotype mAb 2G10 (mAb 2G10huG1). METHODS: The Vkappa and VH regions of mAb 2C7 and its anti-idiotype mAb 2G10 were engineered into human constant regions of the IgE and IgG1 isotypes, respectively. RESULTS: The production of chimaeric mAb 2C7huE and its anti-idiotype mAb 2G10huG1 confirmed that the respective mouse antibody V regions were successfully engineered into human constant regions and still retained the specificity of the original murine V regions. CONCLUSION: The newly constructed chimaeric antibodies will be useful to investigate the downregulation of IgE mediated hypersensitivity by the crosslinking of FcepsilonRI with FcgammaRIIB.

Characterisation of a mouse monoclonal anti-idiotype reactive with a V region sequence commonly used by human immunoglobulins.

BACKGROUND: A mouse monoclonal antibody (2C7/IgG2b kappa) has been described recently, which is directed against the major house dust mite allergen Der p 1, and whose epitope specificity is representative of a major component of the human IgE anti-Der p 1 response. AIMS: To characterise an anti-idiotypic antibody (2G10/IgG1 kappa) raised against monoclonal antibody 2C7 as surrogate human IgE anti-Der p 1. METHODS: The specificity of the anti-idiotype antibody 2G10 was determined by competitive inhibition experiments using human and mouse immunoglobulins of known VH gene families. The epitope recognised by monoclonal antibody 2G10 was located on the molecular model of the Fv (fragment variable) region of monoclonal antibody 2C7. RESULTS: The data suggest that monoclonal antibody 2G10 is directed against a crossreactive idiotype on human IgE that is shared by polyclonal IgG. Competitive inhibition studies against human immunoglobulins, representative of VH2, VH3, and VH4 gene families, showed that monoclonal antibody 2G10 is mostly likely to be directed against sequences encoded by either VH3 or VH4 genes. The fact that monoclonal antibody 2G10 binds to the humanized (complementarity determining region (CDR) grafted) CAMPATH-1H antibody, but not to the original rat CAMPATH-1 YTH34.5.6 antibody, indicates that it is directed against a framework region rather than the CDRs. Analysis of amino acids in the VH region for charge, hydrophobicity, and accessibility suggests that reactivity with monoclonal antibody 2G10 is defined by a hexapeptide spanning residues 74-79 within framework region 3. CONCLUSION: The anti-idiotype monoclonal antibody 2G10 could potentially be used as a probe for determining the contribution of the VH3 and VH4 gene segments to antigenic specificity.