| Background The need for blood continues to grow globally as health systems become more developed, with improved diagnostic and treatment options and increasingly growing ageing populations, which makes blood transfusion an indispensable treatment for modern health care. However, adverse effects caused by transfusion may lead to severe consequences to recipients. How to ensure the therapeutic effect of transfusion as well as lower the incidence of adverse effects is still the focus that WHO, health departments and medical communities pay close attention to.Transfusion risks are the incidence of transfusion adverse reactions, including infectious and noninfectious risks. Transfusion transmitted pathogens including viruses, bacteria, and protozoa are the main cause of infectious risks.Transfusion-associated graft versus-host disease (TA-GVHD), one of noninfectious risks, is a fatal complication caused by engraftment of transfused immunocompetent T cells. Till now, it has no effective treatment with a mortality rate of over 90% . Pathogen inactivation technology of blood components is an important warranty for transfusion safety. Methylene blue photochemical technology has been considered a prospective method for virus inactivation of single unit plasma in clinic, which is already used in China and some European countries. Its efficacy and safety has been proved. Under illumination, Methylene blue can bind to nucleic acids and mediate lesions to them so that nucleic acids replication is inhibited, in which case pathogen is inactivated. Based on this principle, methylene blue photochemistry (MBP) can also inactivate lymphocytes in theory for TA-GVHD prevention. Besides, this mechanism makes it reasonable for evaluation of MBP-treated virus inactivation effects by nucleic acid amplification technology.Objectives 1) To investigate the inactivation effects and mechanisms of MBP on cell proliferation and cytokine secretion of human peripheral blood lymphocytes.2) To study the inactivation effects and mechanisms of MBP on virus, and to establish the evaluation method for virus inactivation using nucleic acid amplification technology as well as improve evaluation system with noninfectious quality control standards.Methods 1) As for experimental group, methylene blue (MB) was added to cell suspensions with a final concentration of 3μM followed by transferring the suspension into PVC blood bags. Cells were exposed to light with illuminance of 35000 lux for 50min at 4℃. Irridiation group lymphocytes were exposed to y-irridiation at 25Gy. All treated and untreated samples were tested for cell morphology, cell viability, ability for cell proliferation and cytokine secretion, and cell apoptosis to analyze the inactivation effects and mechanisms of MBP on lymphocytes. 2) Methylene blue was added to Sindbis virus (SV) suspensions followed by transferring the suspensions into PVC blood bags. Viruses were exposed to light with different time intervals, illuminance or MB concentrations, and were tested for residual viral titer and nucleic acid quantity to analyze the correlation between viral titer decreases and nucleic acid lesions. HCV-positive plasma, heat-preinactivated SV or HCV virus-like particles were mixed with methylene blue in a final concentration of 1μM followed by transferring the suspensions into PVC blood bags. The mixtures were exposed to light with illuminance of 35000 lux for 30min at 4℃and were tested at different time interval for nucleic acid amplification to analyze the feasibility of using nucleic acid amplification technology as an evaluation method for virus inactivation.Results 1) Cell proliferation and cytokine secretion were inhibited by MBP treatment. The results of fluorescence staining and cell viability tests showed that the number of competent cells of experimental group was significantly lower than those of control group and irradiation group. After PHA stimulation, the proliferation colonies of lymphocytes from experimental group were smaller than those of other groups. The inhibition rate of cell proliferation from experimental group after PHA stimulation for 3-5 days were 89.31% .100% and 94.39% . respectively, which were higher than those of the irradiation group. The amount of IFN-γ, IL-1β,IL-4, IL-6 and IL-8 cytokine secretions of experimental group were significantly lower than those of control group and irradiation group. There were no difference in IFN-y, IL-1β, IL-2, IL-4, IL-6, IL-12 cytokine secretions of experimental group before and after PHA stimulation. No caspase-3, caspase-6 proteinase activity, significant phosphatidylserine eversion or DNA Ladder was detected in experimental group after MBP treatment, which indicates methylene blue with light leads to cell non-programmed death.2) MBP treatments showed>6.0 Log TCID50 of SV inactivation under different working conditions, and the decrease of viral nucleic acid amoout correlated strongly with the loss in viral titer (R2>0.94) with significance F<0.01. The nucleic acid dynamic and amplification inhibitive curves of MBP-treated HCV-positive plasma and heat-preinactivated SV were similar to those of conventional SV, which was also the case when it came to HCV virus-like particles and HCV-positive plasma with a strong linear correlation (R2>0.99).Conclusions 1) Methylene blue photochemical technology can inactivate lymphocytes effectively by inhibiting cell proliferation and cytokine secretion (first publishing in China).2) Methylene blue can induce cell non-programmed death to lymphocytes.3) Viral nucleic acid is damaged during MBP treatment, and nucleic acid is the main target of MBP virus inactivation by analysis of the correlation between decrease of viral nucleic acid quantity and loss in viral titer (first published in Photochemistry and Photobiology).4) It is feasible of Real-time PCR technology serving as an evaluation method of MBP virus inactivation effects. It is also feasible of heat-preinactivated SV and HCV virus-like particles to be quality control standards for evaluating virus inactivation effects. |
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