Characterization of effect of repeated freeze and thaw cycles on stability of genomic DNA using pulsed field gel electrophoresis.

In this study, we systematically investigated the effects of repeated cycles of freeze/thaw on stability of genomic Deoxyribonucleic acid (DNA) samples as evaluated by changes in DNA size, concentration, and freeze/thaw protocols. The DNA was isolated using standard extraction procedures including phenol/chloroform and commercially available Gentra Puregene and Qiagen QIAmp kits. Changes in DNA were monitored over 18 cycles of freezing and thawing utilizing several freeze/thaw protocols. DNA samples from multiple subjects prepared from whole blood samples were examined by pulsed field gel electrophoresis (PFGE), and shown to have different average molecular sizes and size distribution patterns depending on the extraction method. Results of freeze/thaw experiments, analyzed by PFGE, showed progressive DNA degradation of the samples, with DNA sizes larger than 100 kb most sensitive to freeze/thaw degradation. Increasing the DNA concentration of stored samples from 10 µg/mL to 100 µg/mL had a somewhat protective effect on DNA stability. Variations in freeze/thaw protocols did not have a significant impact on DNA stability during repeated freeze/thaw cycles. At freeze/thaw cycle 18, average molecular size and size distribution of all DNA samples tested approached 25 kb, regardless of their initial average size and size distributions. This study provides insight on DNA degradation during freeze/thaw cycles and offers guidance to storage and handling of DNA samples.

IL-15 administered by continuous infusion to rhesus macaques induces massive expansion of CD8+ T effector memory population in peripheral blood.

IL-15 promotes activation and maintenance of natural killer (NK) and CD8(+) T effector memory (T(EM)) cells, making it a potential immunotherapeutic agent for the treatment of cancer and immunodeficiency states. Here we report the immunologic effects of 3 different IL-15 dosing strategies in Rhesus macaques. IL-15 at a dose of 20 µg/kg/d administered by continuous intravenous infusion for 10 days resulted in a massive (100-fold) expansion of CD8(+) T(EM) cells in the peripheral blood. In contrast, the administration of 20-40 µg/kg/d of IL-15 by subcutaneous injection resulted in a more modest (10-fold) expansion of CD8(+) T(EM) cells. NK expansion was similar in both the continuous intravenous and daily subcutaneous treatment groups. The observation that IL-15 administered by continuous intravenous infusion is able to induce markedly greater expansions of CD8(+) T(EM) cells than the same dose administered by other routes may have important implications for clinical development of this cytokine.

Lymphocyte subsets in hemophilic patients with hepatitis C virus infection with or without human immunodeficiency virus co-infection: a nested cross-sectional study.

BACKGROUND: With chronic infection, hepatitis C virus (HCV) RNA can be detected in B cells and associated with B-cell disorders, but these are not well defined. METHODS: The relationship between HCV infection and lymphocyte subpopulations was evaluated rigorously in 120 asymptomatic hemophilic patients, randomly selected from a prospective cohort study. CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD56+ NK cells were quantified by flow cytometry using cryopreserved peripheral blood mononuclear cells of 24 hemophilic patients in each of five age-matched groups [uninfected; chronic HCV with or without human immunodeficiency virus (HIV); and cleared HCV with or without HIV]. RESULTS: As expected, patients with HIV had significantly reduced CD4+ and increased CD8+ T cells. Irrespective of HIV, patients with chronic HCV infection had approximately 25% fewer CD19+ B cells than those without chronic HCV infection. CONCLUSIONS: These data support the hypothesis that asymptomatic patients with chronic HCV infection have an altered B-lymphocyte population.

Whole blood cryopreservation in epidemiological studies.

Standardized and cost-effective biological sample collection, processing, and storage procedures are needed in large-scale epidemiological studies to provide material for testing a broad range of etiological hypotheses. One component of sample collection in the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial involves shipment of blood in acid-citrate-dextrose anticoagulant to a central processing laboratory, where 10% DMSO is added, and whole blood aliquots are cryopreserved. A single technician is able to routinely process 50-60 samples/day. Tests conducted to evaluate potential uses of cryopreserved whole blood showed successful EBV transformation (>90%, up to 20 months of storage). In addition, lymphocytes maintained good viability and stable T-cell:B-cell ratios, and T cells maintained the capacity to proliferate in response to solid phase anti-CD3/CD28 plus interleukin 2. Whole blood cryopreservation is a cost-effective approach to large-scale storage of viable cells in epidemiological studies.