A novel canine model of immune thrombocytopenia: has immune thrombocytopenia (ITP) gone to the dogs?

Canine immune thrombocytopenia (ITP) is analogous to human ITP, with similar platelet counts and heterogeneity in bleeding phenotype among affected individuals. With a goal of ultimately investigating this bleeding heterogeneity, a canine model of antibody-mediated ITP was developed. Infusion of healthy dogs with 2F9, a murine IgG2a monoclonal antibody to the canine platelet glycoprotein GPIIb (a common target of autoantibodies in ITP) resulted in profound, dose-dependent thrombocytopenia. Model dogs developed variable bleeding phenotypes, e.g. petechiae and haematuria, despite similar degrees of thrombocytopenia. 2F9 infusion was not associated with systemic inflammation, consumptive coagulopathy, or impairment of platelet function. Unexpectedly however, evaluation of cytokine profiles led to the identification of platelets as a potential source of serum interleukin-8 (IL8) in dogs. This finding was confirmed in humans with ITP, suggesting that platelet IL8 may be a previously unrecognized modulator of platelet-neutrophil crosstalk. The utility of this model will allow future study of bleeding phenotypic heterogeneity including the role of neutrophils and endothelial cells in ITP.

Clonal evolution of lymphoblastoid cell lines.

Lymphoblastoid cell lines represent Epstein-Barr virus (EBV)-immortalized B lymphocytes, which are typically prepared by in vitro culture of normal blood cells. In this study, we evaluated the kinetics of clonal evolution in lymphoblastoid cell lines (LCLs) established from five different donors. Immunoglobulin heavy chain (IGH) and T-cell receptor gamma (TRG) gene rearrangements were tracked over time using PCR, while EBV clonality was tracked using Southern blot analysis of the viral terminal repeat fragment. All five cultures evolved towards monoclonal B cells within 8 weeks, while T lymphocytes disappeared over the same period. No significant association was found between the rapidity of clonal emergence and either the proliferation rate or the size of the EBV terminal repeat fragment, suggesting the random nature of clonal selection. Our results suggest that EBV-driven B lymphocytes rapidly progress from polyclonal to virtually monoclonal, which has implications for the pace at which lymphoma might evolve in vivo. In addition, our findings indicate that established lymphobastoid cell lines are not an ideal 'normal control' for human B lymphocytes because they do not represent the spectrum of polyclonal B cells found in healthy humans; instead, they primarily represent the progeny of a single B lymphocyte.

Radiation clastogenesis and cell cycle checkpoint function as functional markers of breast cancer risk.

BACKGROUND: Familial breast cancer is associated with mutations in several genes (BRCA1, BRCA2, p53, ATM) whose protein products protect against radiation-induced genotoxicity. This study tested whether sporadic breast cancer was associated with constitutive radiation hypersensitivity. METHODS: Blood lymphocytes and EBV-transformed lymphoblasts from patients with newly diagnosed breast cancer and controls without cancer were evaluated for ionizing radiation (IR)-induced chromosomal aberrations and cell cycle delays. Lymphoblasts from patients with ataxia telangiectasia (AT) and heterozygous AT carriers were tested as positive controls for radiation hypersensitivity. RESULTS: Lymphoblasts from AT patients and AT carriers displayed G2-irradiation, chromosomal hypersensitivity (GICH). Irradiated G2 phase lymphocytes from breast cancer cases and controls displayed 3-fold inter-individual variation in frequencies of chromatid damage. However, the percentage of breast cancer cases with damage frequencies in excess of 2 SD of the control mean (8/102 or 8%) was not significantly elevated compared to controls (2/48 or 4%, P=0.5). Lymphoblasts sampled 24 h after 3 Gy of IR also varied in the ratios of cells with 4N and 2N DNA content (4N/2N ratio), as a measure of cell cycle checkpoint function. 4N/2N ratios in irradiated lymphoblasts were strongly correlated with the fractions of S phase cells in un-irradiated control cultures (Pearson's correlation coefficient, r=0.87). After normalization to S fraction, the radiation-induced increment in the 4N/2N ratio was significantly elevated in AT lymphoblasts but not in lymphoblasts from AT carriers. The fraction of breast cancer cases with reduced checkpoint function (2/45 or 4%) was equal to the control fraction (2/45 or 4%). For breast cancer cases and controls, GICH in primary lymphocytes was not associated with reduced cell cycle checkpoint function in lymphoblasts. CONCLUSION: Constitutive radiation hypersensitivity in blood lymphocytes and lymphoblasts was not a useful biomarker for identifying women at increased risk of breast cancer.