Anti-PRKDC polyclonal antibody

Objective. To determine interrelationships between the expression of long intergenic (noncoding) RNA-p21 (lincRNA-p21), NF-kappa B activity, and responses to methotrexate (MTX) in rheumatoid arthritis (RA) by analyzing patient blood samples and cell culture models. Methods. Expression levels of long noncoding RNA and messenger RNA (mRNA) were determined by quantitative reverse transcription-polymerase chain reaction. Western blotting and flow cytometry were used to quantify levels of intracellular proteins. Intracellular NF-kappa B activity was determined using an NF-kappa B luciferase reporter plasmid. Results. Patients with RA expressed reduced basal levels of lincRNA-p21 and increased basal levels of phosphorylated p65 (RelA), a marker of NF-kappa B activation. Patients with RA who were not treated with MTX expressed lower levels of lincRNA-p21 and higher levels of phosphorylated p65 compared with RA patients treated with low-dose MTX. In cell culture using primary cells and transformed cell lines, MTX induced lincRNA-p21 through a DNA-dependent protein kinase catalytic subunit (DNA PKcs)-dependent mechanism. Deficiencies in the levels of PRKDC mRNA in patients with RA were also corrected by MTX in vivo. Furthermore, MTX reduced NF-kappa B activity in tumor necrosis factor alpha-treated cells through a DNA PKcs-dependent mechanism via induction of lincRNA-p21. Finally, we observed that depressed levels of TP53 and lincRNA-p21 increased NF-kappa B activity in cell lines. Decreased levels of lincRNA-p21 did not alter NFKB1 or RELA transcripts; rather, lincRNA-p21 physically bound to RELA mRNA. Conclusion. Our findings support a model whereby depressed levels of lincRNA-p21 in RA contribute to increased NF-kappa B activity. MTX decreases basal levels of NF-kappa B activity by increasing lincRNA-p21 levels through a DNA PKcs-dependent mechanism

Defects in DNA cross-link repair 1C (DCLRE1C), protein kinase DNA activated catalytic polypeptide (PRKDC), ligase 4 (LIG4), NHEJ1, and NBS1 involving the nonhomologous end-joining (NHEJ) DNA repair pathway result in radiation-sensitive severe combined immunodeficiency (SCID). Results of hematopoietic cell transplantation for radiation-sensitive SCID suggest that minimizing exposure to alkylating agents and ionizing radiation is important for optimizing survival and minimizing late effects. However, use of preconditioning with alkylating agents is associated with a greater likelihood of full T-and B-cell reconstitution compared with no conditioning or immunosuppression alone. A reduced-intensity regimen using fludarabine and low-dose cyclophosphamide might be effective for patients with LIG4, NHEJ1, and NBS1 defects, although more data are needed to confirm these findings and characterize late effects. For patients with mutations in DCLRE1C (Artemis-deficient SCID), there is no optimal approach that uses standard dose-alkylating agents without significant late effects. Until nonchemotherapy agents, such as anti-CD45 or anti-CD117, become available, options include minimizing exposure to alkylators, such as single-agent low-dose targeted busulfan, or achieving T-cell reconstitution, followed several years later with a conditioning regimen to restore B-cell immunity. Gene therapy for these disorders will eventually remove the issues of rejection and graft-versus-host disease. Prospective multicenter studies are needed to evaluate these approaches in this rare but highly vulnerable patient population.