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Neutralizing antibodies (inhibitors) to replacement factor VIII (FVIII, either plasma derived or recombinant) impair the effective management of hemophilia A. Individuals with hemophilia A due to major deletions of the FVIII gene (F8) lack antigenically cross-reactive material in their plasma ('CRM-negative'), and the prevalence of inhibitors in these individuals may be as high as 90%. Conversely, individuals with hemophilia A caused by F8 missense mutations are CRM-positive, and their overall prevalence of inhibitors is <10% (ref. 2). Individuals with the F8 intron 22 inversion (found in ∼50% of individuals with severe hemophilia A) have been grouped with the former on the basis of their genetic defect and CRM-negative status. However, only ∼20% of these individuals develop inhibitors. Here we demonstrate that the levels of F8 mRNA and intracellular FVIII protein in B lymphoblastoid cells and liver biopsies from individuals with the intron 22 inversion are comparable to those in healthy controls. These results support the hypothesis that most individuals with the intron 22 inversion are tolerized to FVIII and thus do not develop inhibitors. Furthermore, we developed a new pharmacogenetic algorithm that permits the stratification of inhibitor risk for individuals and subpopulations by predicting the immunogenicity of replacement FVIII using, as input, the number of putative T cell epitopes in the infused protein and the competence of major histocompatibility complex class II molecules to present such epitopes. This algorithm showed statistically significant accuracy in predicting the presence of inhibitors in 25 unrelated individuals with the intron 22 inversion. [ABSTRACT FROM AUTHOR]

Exosomes, microvesicles of endocytic origin released by normal and tumor cells, play an important role in cell-to-cell communication. Angiogenesis has been shown to regulate progression of chronic myeloid leukemia (CML). The mechanism through which this happens has not been elucidated. We isolated and characterized exosomes from K562 CML cells and evaluated their effects on human umbilical endothelial cells (HUVECs). Fluorescent-labeled exosomes were internalized by HUVECs during tubular differentiation on Matrigel. Exosome localization was perinuclear early in differentiation, moving peripherally in cells undergoing elongation and connection. Exosomes move within and between nanotubular structures connecting the remodeling endothelial cells. They stimulated angiotube formation over a serum/growth factor-limited medium control, doubling total cumulative tube length ( P = 0.003). Treatment of K562 cells with two clinically active tyrosine kinase inhibitors, imatinib and dasatinib, reduced their total exosome release ( P < 0.009); equivalent concentrations of drug-treated exosomes induced a similar extent of tubular differentiation. However, dasatinib treatment of HUVECs markedly inhibited HUVEC response to drug control CML exosomes ( P < 0.002). In an in vivo mouse Matrigel plug model angiogenesis was induced by K562 exosomes and abrogated by oral dasatinib treatment ( P < 0.01). K562 exosomes induced dasatinib-sensitive Src phosphorylation and activation of downstream Src pathway proteins in HUVECs. Imatinib was minimally active against exosome stimulation of HUVEC cell differentiation and signaling. Thus, CML cell-derived exosomes induce angiogenic activity in HUVEC cells. The inhibitory effect of dasatinib on exosome production and vascular differentiation and signaling reveals a key role for Src in both the leukemia and its microenvironment. [ABSTRACT FROM AUTHOR]

Cells that form vascular system employ different mechanisms to offset deleterious consequences of exposure to cytokines and cells present in blood. Vascular homeostasis is sustained in part by genes, whose expression increases in response to hemodynamic forces in these cells. PP1201 (also known as RECS1) is one such gene whose expression level increases in response to laminar shear stress. Aged mice deficient in PP1201 are prone to develop cystic medial degeneration (CMD), a form of aortic aneurism manifested with loss of smooth muscle cells and accumulation of basophilic substances. Here we found that higher levels of PP1201 can protect against Fas ligand (FasL)-induced apoptosis. PP1201 interacted with the Fas receptor (CD95/Apo1) and colocalized with it in the Golgi compartment. Unlike its homolog lifeguard (LFG), PP1201 overexpression in several types of cells including primary human aortic smooth muscle cells (AoSMC) decreased the expression of Fas on the plasma membrane without changing the total Fas levels. Only high but not constitutive level of PP1201 controls Fas signaling. Our data suggest that PP1201 functions as an anti-apoptotic protein and its increased expression in vascular cells can contribute to homeostasis by reducing Fas trafficking to the cell membrane. [ABSTRACT FROM AUTHOR]

We compared findings from recent studies modelling the cost-effectiveness of screening for cervical cancer using human papillomavirus (HPV) testing and alternative strategies. Data were standardized to facilitate comparison of costs per life year or costs per QALY gained in six studies. Absolute changes in costs, life years and QALYs for each strategy were normalized to a comparison with no screening. Costs were standardized to US$ in 2000 values. Most models assume screening starts at age 18 or 20 years. Assumed prevalence of HPV ranges from 10% for those aged 18 years to 20% for those aged 20-25 years and drops substantially after age 30. All except one model assume sensitivity to LSIL of 83% or higher. Two models distinguish the increasing specificity of HPV testing in older age groups (up to 95% for LSIL in women aged 55 years or older). All the models include consultation costs as well as screening and treatment costs, but costs for follow-up diagnosis and treatment vary considerably. Two models also include patient time costs. Despite these differences all strategies involving HPV testing have cost per quality-adjusted life-year (QALY) ratios in the range of $12,400-$16,600. Costs per life year vary more widely, the highest being $19,246 (annual screening with liquid cytology and HPV). However, excluding strategies using liquid cytology, the highest costs per life year for a strategy including HPV testing are under $14,000 (simultaneous conventional cytology and HPV every two years). The cost per life year for HPV testing alone triennially is lower than for Pap smear testing alone biennially. Costs per QALY are generally lower than costs per life year (given the reported modelling assumptions and settings). Even with inclusion of patient costs, no strategies involving HPV testing cost more than $16,600 per QALY. Adoption of the ACOG guidelines to include HPV testing with cytology as a screening option for women aged 30 years or older therefore appears to be cost-effective. [ABSTRACT FROM AUTHOR]

The promyelocytic leukemia protein (PML) is a nuclear phosphoprotein that localizes to distinct domains in the nucleus, described as PML nuclear bodies (PML-NBs). Recent findings indicate that PML regulates the p53 response to oncogenic signals. Here, we define a p53-dependent role for PML in response to DNA damage. We exposed cells to ultraviolet light (UV-C) and imaged the nuclear distribution of PML, p53, and the BLM helicase by confocal microscopy. After DNA damage, PML partially relocated out of the PML-NBs, and colocalized with BLM and p53 at sites of DNA repair. In addition, using the isogenic HCT116 cell lines (p53+/+ and -/-), we show that the redistribution of PML was dependent on functional p53. Western analysis revealed that the level of PML protein remained unaltered after UV-C treatment. These results are consistent with the hypothesis that PML, in conjunction with p53 and BLM, contributes to the cellular response to UV-C-induced DNA damage and its repair.Oncogene (2003) 22, 1620-1628. doi:10.1038/sj.onc.1206140 [ABSTRACT FROM AUTHOR]