Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement.

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge. This article describes in detail the "Copy Number Ratio of S/G1 for mapping genomic Time of Replication" method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

The role of skin-derived dendritic cells in CD8+ T cell priming following immunization with lentivectors.

Although skin dendritic cells (DCs) have been shown to directly present Ag to CD8(+) T cells after intradermal immunization with lentivectors, the contribution of the different skin DC subsets to this process remains unclear. Using langerin-diphtheria toxin receptor transgenic mice we demonstrated that ablation of langerhans cells and langerin-expressing positive dermal DCs (Ln(+)dDCs) did not interfere with the generation of CD8(+) T cells by lentiviral vectors. Consistent with these findings, the absence of langerhans cells and Ln(+)dDCs did not hamper the presentation level of lentiviral-derived Ag by skin DCs in vitro. We further demonstrated that only dDCs and Ln(+)dDCs were capable of presenting Ag, however, the number of dDCs migrating to the draining lymph nodes was 6-fold higher than that of Ln(+)dDCs. To study how the duration of DC migration influences CD8(+) T cell responses, we analyzed the kinetics of Ag expression at the injection site and manipulated DC migration by excising the injected skin at various times after immunization. A low level of Ag expression was seen 1 wk after the immunization; peaked during week 2, and was considerably cleared by week 3 via a perforin-dependent fas-independent mechanism. Removing the injection site 3 or 5 d, but not 10 d, after the immunization, resulted in a reduced CD8(+) T cell response. These findings suggest that dDCs are the main APCs active after intradermal lentiviral-mediated immunization, and migration of dDCs in the initial 10-d period postimmunization is required for optimal CD8(+) T cell induction.

Gamma-irradiation enhances apoptosis induced by cannabidiol, a non-psychotropic cannabinoid, in cultured HL-60 myeloblastic leukemia cells.

Two non-psychotropic cannabinoids, cannabidiol (CBD) and cannabidiol-dimethylheptyl (CBD-DMH), induced apoptosis in a human acute myeloid leukemia (AML) HL-60 cell line. Apoptosis was determined by staining with bisBenzimide and propidium iodide. A dose dependent increase of apoptosis was noted, reaching 61 and 43% with 8 microg/ml CBD and 15 microg/ml CBD-DMH, respectively, after a 24 h treatment. Prior exposure of the cells to gamma-irradiation (800 cGy) markedly enhanced apoptosis, reaching values of 93 and 95%, respectively. Human monocytes from normal individuals were resistant to either cannabinoids or gamma-irradiation. Caspase-3 activation was observed after the cannabinoid treatment, and may represent a mechanism for the apoptosis. Our data suggest a possible new approach to treatment of AML.

Extrapancreatic autoimmune manifestations in type 1 diabetes patients and their first-degree relatives: a multicenter study.

OBJECTIVE: To investigate the prevalence of autoimmune diseases in young patients (probands) with type 1 diabetes and their first-degree relatives, and to determine the spectrum of extrapancreatic manifestations in these subjects. RESEARCH DESIGN AND METHODS: The study population included 109 probands age 13 +/- 4.9 years and 412 first-degree relatives age 28.7 +/- 16.2 years. The prevalence rates of autoimmune thyroiditis and celiac disease were determined in all probands and in 100 of the 412 first-degree relatives. Control groups included 78 subjects age 14.9 +/- 10.4 years for the prevalence of autoimmune thyroiditis and 120,000 youth ages 16-17 years for the prevalence of celiac disease. Thyroiditis and celiac disease were diagnosed by abnormally high thyroid peroxidase (TPO), thyroglobulin (TG), antigliadin, and antiendomysial antibody titers. Celiac was confirmed by biopsy. A questionnaire was used to interview probands and relatives to determine the spectrum of autoimmune manifestations. RESULTS: The prevalence of autoimmune thyroiditis determined by high TPO and/or TG titers was 27 and 25% for probands and relatives, respectively. These rates were higher than those for control subjects (P < 000.1). The prevalence of celiac disease among probands and screened relatives was 8.3 and 6%, respectively. These rates were higher than those for control subjects and the 312 family members interviewed only (0.1 and 0.3%, respectively; P < 0.0001). Interviews of participants revealed a wide range of associated autoimmune diseases. The risk of developing an autoimmune disease was higher (P < 0.001) in families with a proband who had an additional autoimmune manifestation. CONCLUSIONS: Screening for autoimmune thyroiditis and celiac disease should be performed in patients with type 1 diabetes and their first-degree relatives, especially when the probands have an additional autoimmune manifestation.