Elevated expression of artemis in human fibroblast cells is associated with cellular radiosensitivity and increased apoptosis

Background:

The objective of this study was to determine the molecular mechanisms responsible for cellular radiosensitivity in two human fibroblast cell lines 84BR and 175BR derived from two cancer patients.

Methods:

Clonogenic assays were performed following exposure to increasing doses of gamma radiation to confirm radiosensitivity. γ-H2AX foci assays were used to determine the efficiency of DNA double-strand break (DSB) repair in cells. Quantitative PCR (Q-PCR) established the expression levels of key DNA DSB repair genes. Imaging flow cytometry using annexin V-FITC was used to compare artemis expression and apoptosis in cells.

Results:

Clonogenic cellular hypersensitivity in the 84BR and 175BR cell lines was associated with a defect in DNA DSB repair measured by the γ-H2AX foci assay. The Q-PCR analysis and imaging flow cytometry revealed a two-fold overexpression of the artemis DNA repair gene, which was associated with an increased level of apoptosis in the cells before and after radiation exposure. Overexpression of normal artemis protein in a normal immortalised fibroblast cell line NB1-Tert resulted in increased radiosensitivity and apoptosis.

Conclusion:

We conclude that elevated expression of artemis is associated with higher levels of DNA DSB, radiosensitivity and elevated apoptosis in two radio-hypersensitive cell lines. These data reveal a potentially novel mechanism responsible for radiosensitivity and show that increased artemis expression in cells can result in either radiation resistance or enhanced sensitivity.     

One-time gene gun or intramuscular rabies DNA vaccination of non-human primates: comparison of neutralizing antibody responses and protection against rabies virus 1 year after vaccination.

We have previously shown that Macaca fascicularis (Cynomologus) monkeys receiving a primary and either one or two booster rabies DNA vaccinations are protected against rabies virus. In this study, we determined whether monkeys that had been vaccinated only once via gene gun or intramuscularly (i.m.) with different concentrations of DNA would be protected against rabies virus challenge. Neutralizing antibody responses were assayed for 1 year before the monkeys were challenged. Neutralizing antibody was detected at least 50 days earlier in gene gun vaccinated as compared to i.m. vaccinated animals. Prior to viral challenge, all (6/6, 100%) gene gun vaccinated animals, but only 3/6 (50%) i.m. vaccinated animals seroconverted. In general, antibody titers of the gene gun vaccinated animals were higher than the titers of the i.m. vaccinated animals. There was no correlation between the concentration of DNA used for vaccination, the neutralizing antibody responses elicited and protection against viral challenge. Seven days after viral challenge, a rapid and strong anamnestic antibody response was elicited in 100% of the gene gun vaccinated monkeys and in four i.m. vaccinated monkeys. Neutralizing antibody remained undetectable in two i.m. vaccinated monkeys. Overall, 60% (3/5) of the gene gun vaccinated animals and 87% (5/6) of the i.m. vaccinated monkeys survived viral challenge. This study is the first, to our knowledge, to show long-term protection of non-human primates against a human viral pathogen using a DNA vaccination protocol that did not include a booster immunization.     

Notch ligand-bearing thymic epithelial cells initiate and sustain Notch signaling in thymocytes independently of T cell receptor signaling.

Thymic epithelial cells are specialized to play essential roles at multiple stages of T cell development in the thymus, yet the molecular basis of this specialization is largely unknown. Recently, the Notch family of transmembrane proteins has been implicated in thymocyte development. Such proteins interact with cell surface proteins of the Delta-like and Jagged families. It is known that Notch ligands are expressed intrathymically, and that Notch signaling is regulated by Notch ligands expressed on either the same or third-party cells. However, functional analysis of Notch ligand expression, and elucidation of the mechanism of Notch ligand signaling in thymocyte development, are unclear. Here, we find that Notch ligand expression in the thymus is compartmentalized, with MHC class II(+) thymic epithelium, but not thymocytes nor dendritic cells, expressing Jagged-1, Jagged-2 and Delta-like-1. We also provide evidence that contact with Notch ligands on thymic epithelium is necessary to activate and sustain Notch signaling in thymocytes, and that this can occur independently of positive selection induction. Our data suggest that Notch ligand expression by thymic epithelium may partly explain the specialization of these cells in supporting thymocyte development, by regulating Notch activation via an inductive signaling mechanism independently of signaling leading to positive selection.     

Treatment of donor mice with an alpha beta T-cell receptor monoclonal antibody induces prolonged T-cell nonresponsiveness and effectively prevents lethal graft-versus-host disease in murine recipients of major histocompatibility complex (MHC)-matched and MHC-mismatched donor marrow grafts

The purpose of this study was to determine whether the administration of high doses of an anti-T-cell receptor (TCR) monoclonal antibody (H57- 597) to donor animals could induce a state of T-cell nonresponsiveness and prevent the development of graft-versus-host disease (GVHD) in murine recipients of major histocompatibility complex (MHC)-matched (B10.BR[H-2k] --> AKR/J[H-2k]) and mismatched (B10.BR[H-2k] --> DBA/2[H- 2d]) marrow grafts. Transplantation of H57-597-treated B10.BR T cells into irradiated AKR or DBA mice resulted in protection from GVHD, which was otherwise lethal in transplanted recipients receiving untreated T cells. The administration of H57-597-treated T cells did not compromise alloengraftment in either strain combination and was found to accelerate donor T-cell reconstitution in recipients of MHC-matched marrow grafts. Optimal protection for GVHD was dependent on the duration of antibody exposure in donor mice. T cells from donor exposed to antibody for only 1 day caused lethal GVHD, whereas exposure for at least 4 days was necessary to abrogate graft-versus-host reactivity. The ability of antibody treatment to protect against the development of GVHD could not be ascribed to the antibody-induced production of Th2 cytokines, the induction of a T- or non-T-suppressor cell population, or the preferential depletion of CD4+ T cells by H57-597. Donor T cells exposed to H57-597 antibody were detectable in recipients for up to 5 weeks after transplantation, indicating that these cells were not eliminated in the host immediately after bone marrow transplantation and contributed to enhanced donor T-cell reconstitution. Moreover, in B10.BR --> DBA chimeras that did not have any clinical evidence of GVHD, potentially MIs-reactive donor-derived Vbeta6+ T cells were present in the spleens of recipients at comparable numbers to normal mice but appeared functionally nonresponsive in vivo. These data strongly suggested that protection from GVHD was due to the fact that antibody treatment resulted in a state of prolonged T-cell anergy that persisted despite the presence of potential costimulatory signals in the recipient. This observation is of potential clinical significance in that it shows that the prevention of GVHD can be accomplished without posttransplantation immunosuppression or the need for in vitro or in vivo T-cell depletion.     

Another cause of anaemia

Sir, Renal impairment is commonly associated with normocytic anaemia,due to relative deficiency of erythropoietin, and it is nowstandard practice to treat this with recombinant human erythropoietin(r-HuEPO) once other causes of anaemia have been excluded. Recurrentanaemia in this setting may be due to occult gastrointestinalblood loss, and this is the most common cause of anaemia, butpure red cell aplasia (PRCA) secondary to an antibody to humanrecombinant erythropoietin also needs to     

Plasma clearance rates of coagulation factors VIII and IX in factor- deficient individuals

The plasma clearance rates of factors IX and VIII were determined in patients with hemophilia A and B who had received factor replacement by prolonged, continuous infusion of factor concentrates. The clearance rates were calculated by dividing the factor infusion rates by the steady-state plasma factor activities corrected for base-line factor activities. The mean factor IX clearance rate in eight factor IX- deficient patients was 233 mL/h (range 159 to 340 mL/h). The mean normalized clearance rate was 3.4 mL/h/kg. The mean factor VIII clearance rate in eight factor VIII-deficient patients was 294 mL/h (range 229 to 361 mL/h) and the mean normalized rate was 5.0 mL/h/kg. Both factors show linear relationships between the factor infusion rates and the steady-state plasma factor activities achieved.