Serum Proteome Signature of Radiation Response: Upregulation of Inflammation-Related Factors and Downregulation of Apolipoproteins and Coagulation Factors in Cancer Patients Treated With Radiation Therapy--A Pilot Study.

PURPOSE: Ionizing radiation affects the proteome of irradiated cells and tissue, yet data concerning changes induced during radiation therapy (RT) in human blood are fragmentary and inconclusive. We aimed to identify features of serum proteome and associated processes involved in response to partial body irradiation during cancer treatment. METHODS AND MATERIALS: Twenty patients with head and neck squamous cell cancer (HNSCC) and 20 patients with prostate cancer received definitive intensity modulated RT. Blood samples were collected before RT, just after RT, and 1 month after the end of RT. Complete serum proteome was analyzed in individual samples, using a shotgun liquid chromatography-tandem mass spectrometry approach which allowed identification of approximately 450 proteins. Approximately 100 unique proteins were quantified in all samples after exclusion of immunoglobulins, and statistical significance of differences among consecutive samples was assessed. Processes associated with quantified proteins and their functional interactions were predicted using gene ontology tools. RESULTS: RT-induced changes were marked in the HNSCC patient group: 22 upregulated and 33 downregulated proteins were detected in post-RT sera. Most of the changes reversed during follow-up, yet levels of some proteins remained affected 1 month after the end of RT. RT-upregulated proteins were associated with acute phase, inflammatory response, and complement activation. RT-downregulated proteins were associated with transport and metabolism of lipids (plasma apolipoproteins) and blood coagulation. RT-induced changes were much weaker in prostate cancer patients, which corresponded to differences in acute radiation toxicity observed in both groups. Nevertheless, general patterns of RT-induced sera proteome changes were similar in both of the groups of cancer patients. CONCLUSIONS: In this pilot study, we proposed to identify a molecular signature of radiation response, based on specific features of serum proteome. The signature included upregulation of factors involved in acute or inflammatory response but also downregulation of plasma apolipoproteins and factors involved in blood coagulation.

[Evaluation of coagulation factors in fresh frozen plasma treated with riboflavin and ultraviolet light].

BACKGROUND/AIM: Pathogen inactivation in blood products using riboflavin and ultraviolet (UV) light represents a proactive approach to blood safety, not only for known infectious agents but also for new ones or not yet recognized as threats to the blood supply. This method inactivates a virus, bacteria, fungus, or protozoan pathogen from the blood product without damaging its function or shelf-life. The aim of the study was to study the influence of photoinactivation using riboflavin on the concentration of coagulation factors and coagulation inhibitors in plasma that was treated before freezing. METHODS: The examination included 30 units of plasma, separated from whole blood donated by voluntary blood donors around 6 h from the moment of collection. They were treated by riboflavin (35 mL) and UV rays (6.24 J/mL, 265-370 nm) on Mirasol aparature (Caridian BCT Biotechnologies, USA) in approximate duration of 6 min. The samples for examining were taken before (K - control units) and after illumination (I - illuminated units). RESULTS: Comparing the middle values of coagulation factors in the control and illuminated units we noticed their statistically significant decrease in illuminated units (p < 0.001), but the activity of coagulation ones was still in the reference range. The most sensitive coagulation factors to photoinactivation were FVIII, FIX and FXI (21.99%, 20.54% and 17.26% loss, respectively). Anticoagulant factors were better preseved than coagulation factors. CONCLUSION: Plasma separated from whole blood donation within 6 h, treated with riboflavin and UV light within 6 h from separation and frozen at temperature below -30 degrees C within 24 h, shows good retention of pro- and anticoagulation activity.

Protein stability of previously frozen plasma, riboflavin and UV light-treated, refrozen and stored for up to 2 years at -30 °C.

BACKGROUND: The Mirasol pathogen reduction technology system for plasma is based on a riboflavin and UV light treatment process resulting in pathogen inactivation due to irreversible, photochemically induced damage of nucleic acids. The objective of this study was to evaluate the in vitro protein quality of previously frozen FFP, thawed, Mirasol-treated and refrozen for a final storage period of up to 2 years at -30 °C. MATERIALS AND METHODS: All plasma products were held at 4 ± 2 °C, and frozen within 8h after the start of collection. FFP was stored frozen at -30°C for eight time intervals before riboflavin and UV light treatment and refrozen to -30 °C and stored for varying time intervals for a total storage period of up to 2 years at -30 °C. Results were compared to paired, untreated units thawed and stored for the same time intervals. RESULTS: The overall mean values for all time points in riboflavin and UV light treated plasma samples indicates that all proteins were well preserved following a period of frozen storage for 2 years at -30 °C. Factors V, VII and XI, retained 70%, 65% and 53% activity, respectively. All other protein factors, anticoagulant and inhibitor proteins demonstrated retention between 74% and 100%. CONCLUSION: Riboflavin and UV light-treated FFP maintained both coagulant and anticoagulant in vitro protein quality after double freeze/thaw storage at -30 °C for up to 2 years, a finding that may offer processing flexibility to blood centers.

INTERCEPT plasma: comparability with conventional fresh-frozen plasma based on coagulation function--an in vitro analysis.

BACKGROUND: An effective pathogen inactivation (PI) technology for plasma must inactivate a broad range of pathogens with retention of haemostatic function suitable for therapeutic support. This study evaluated a broad panel of coagulation factors regarding functionality in plasma treated with the INTERCEPT Blood System (I-FFP). STUDY DESIGN AND METHODS: Apheresis plasma (600 ml) was treated with amotosalen and UVA. Aliquots of plasma were collected prior to and after photochemical treatment and frozen prior to analysis. Pro-coagulants, inhibitors and fibrinolytic proteins, contact pathway components, activation markers, the von Willebrand complex and complement proteins were analyzed. RESULTS: Retention of procoagulant factors in I-FFP ranged from 77 to 92% of pretreatment levels. Components of the von Willebrand complex, including multimers and von Willebrand cleavage protease activity (vWF:CP), remained within normal ranges after treatment. Endogenous inhibitors of coagulation were retained at 93 to 100% of baseline. Plasminogen and alpha-2 antiplasmin were retained at 94 and 78% respectively. Retention of contact factors was variable as some factors were below the reference range prior to PI treatment. With the exception of thrombin-antithrombin complexes (TAT) in one of six replicates all markers of coagulation activation were well within normal ranges. Anaphylatoxins were not increased and C1-esterase inhibitor was fully retained. CONCLUSION: Treatment of plasma with the INTERCEPT Blood System preserves proteins necessary for haemostasis without inappropriate activation of coagulation, fibrinolytic or complement pathways.

Application of the bactericidal activity of epsilon-poly-l-lysine to the storage of human platelet concentrates.

BACKGROUND: epsilon-Poly-l-lysine (epsilon-PLL) is a polypeptide comprising approximately 30 l-lysine subunits generated by bond formation between alpha-carboxy and epsilon-amino groups. It is an approved antimicrobial food preservative in Japan. However, the efficacy of epsilon-PLL as an antibacterial additive for storage of human platelet concentrates (PCs) is not known. STUDY DESIGN AND METHODS: Staphylococcus aureus, Bacillus cereus, and Klebsiella oxytoca (20 colony-forming units/mL) were inoculated into 100% plasma PCs or PCs containing 80% platelet (PLT) additive solution with 5.0 mmol/L potassium and 1.5 mmol/L magnesium (PAS-IIIM) and 20% plasma (PAS-IIIM PCs). Next, a range of epsilon-PLL concentrations up to 200 and 50 microg/mL were added to plasma PCs and PAS-IIIM PCs, respectively, and the bacterial count was determined on Days 1, 2, 5, and 8. The quality of the PCs was also determined. RESULTS: Bacterial growth was inhibited at epsilon-PLL concentrations of 200 and 50 microg/mL in the plasma and PAS-IIIM PCs after 8 days of incubation. The percentage of CD62P-positive PLTs was higher in plasma PCs treated with 200 microg/mL epsilon-PLL and in PAS-IIIM PCs treated with 50 microg/mL epsilon-PLL than in the respective controls without epsilon-PLL. There were no remarkable differences in the other variables, that is, PLT number, mean PLT volume, pH, aggregability, percentage of PAC-1-positive cells, lactate dehydrogenase release, and plasma K and Na concentrations between the epsilon-PLL-treated PCs and the controls. CONCLUSIONS: epsilon-PLL inhibited the growth of bacteria in the PCs and did not considerably affect the quality of PCs, except CD62P expression. Further studies are required to estimate the in vivo effectiveness and safety of epsilon-PLL-treated PCs.

The influence of riboflavin photochemistry on plasma coagulation factors.

UNLABELLED: Studies with riboflavin in the 1960s showed that it could be effective at inactivating pathogens when exposed to light. The principal mode of action is through electron transfer reactions, most importantly in nucleic acids. This suggested that it could act as a photosensitizer useful in the inactivation of pathogens found in blood products. OBJECTIVE: To study the influence of photo-inactivation with riboflavin on the coagulation factors of plasma. METHODS: The photo-inactivation procedure of riboflavin plus light was applied. Fifty isogroup pools of two plasmas were made from 100U of plasma that were derived from whole blood products that had previously been held overnight. Pools were split into two bags. One of them was photo-inactivated, and post inactivation samples were obtained. The second bag was not photo-inactivated and samples were taken. Total protein, fibrinogen, FII, FV, FVII, FVIII, FIX, FX, FXI, FXIII, antithrombin III, PC, PS, alpha-2 antiplasmin and vWF:Ag, the multimeric structure of vWF and ADAMTS-13 were analyzed. RESULTS: In plasma, the proteins most sensitive to photo-inactivation were fibrinogen, FXI, FVIII, FV, and FIX (33%, 32%, 30%, 18% and 18% loss, respectively). Coagulation inhibitors, PS, antithrombin III and PC showed little decrease (all 2%). Retention of vWF and ADAMTS-13 were 99% and 88%, respectively. CONCLUSIONS: As with other pathogen reduction procedures for plasma products, treatment with riboflavin and UV light resulted in reduction in the activity levels of several pro-coagulant factors. Coagulation inhibitors are well preserved.

Effect of ionizing radiation on cellular procoagulability and co-ordinated gene alterations.

BACKGROUND AND OBJECTIVES: Ionizing radiation (IR) is associated with thrombotic vascular occlusion predicting a poor clinical outcome. Our study examined whether IR induced tissue factor (TF) expression and procoagulability. We further investigated coordinated gene alterations associated with TF upregulation in the myelomonocytic leukemia THP-1 cells. DESIGN AND METHODS: TF expression was determined by quantitative Reverse Transcriptase (TaqMan) PCR, TF ELISA and TF activity by a two stage chromogenic assay in the time course of days 1, 3, 7, 10, and 17 post IR. To detect IR-induced alterations in gene expression, Affymetrix HG U133 Plus 2.0 microarrays were used. RESULTS IR induced a significant increase in TF/GAPDH mRNA ratios and cellular TF protein on days 3 and 7 post IR (20 Gy [p>or=0.01] and 40 Gy [p or=0.001] vs. control respectively), suggesting IR immediately alters the cellular thrombogenicity. TF upregulation post IR was confirmed in PBMNCs. Gene expression profiling showed IR increased the expression of inflammatory and apoptosis-related pathways known to be involved in the regulation of TF expression. INTERPRETATION AND CONCLUSIONS: TF upregulation together with inflammation and apoptosis may increase the thrombogenicity of tissues. The demonstrated upregulation of TF might play a pivotal role in radiation associated thrombosis.

Photochemical treatment of plasma with amotosalen and long-wavelength ultraviolet light inactivates pathogens while retaining coagulation function.

BACKGROUND: The INTERCEPT Blood System, a photochemical treatment (PCT) process, has been developed to inactivate pathogens in platelet concentrates. These studies evaluated the efficacy of PCT to inactivate pathogens in plasma and the effect of PCT on plasma function. STUDY DESIGN AND METHODS: Jumbo (600 mL) plasma units were inoculated with high titers of test pathogens and treated with 150 micromol per L amotosalen and 3 J per cm(2) long-wavelength ultraviolet light. The viability of each pathogen before and after treatment was measured with biological assays. Plasma function was evaluated through measurement of coagulation factors and antithrombotic protein activities. RESULTS: The levels of inactivation expressed as log-reduction were as follows: cell-free human immunodeficiency virus-1 (HIV-1), greater than 6.8; cell-associated HIV-1, greater than 6.4; human T-lymphotropic virus-I (HTLV-I), 4.5; HTLV-II, greater than 5.7; hepatitis B virus (HBV) and hepatitis C virus, greater than 4.5; duck HBV, 4.4 to 4.5; bovine viral diarrhea virus, 6.0; severe acute respiratory syndrome coronavirus, 5.5; West Nile virus, 6.8; bluetongue virus, 5.1; human adenovirus 5, 6.8; Klebsiella pneumoniae, greater than 7.4; Staphylococcus epidermidis and Yersinia enterocolitica, greater than 7.3; Treponema pallidum, greater than 5.9; Borrelia burgdorferi, greater than 10.6; Plasmodium falciparum, 6.9; Trypanosoma cruzi, greater than 5.0; and Babesia microti, greater than 5.3. Retention of coagulation factor activity after PCT was expressed as the proportion of pretreatment (baseline) activity. Retention was 72 to 73 percent of baseline fibrinogen and Factor (F)VIII activity and 78 to 98 percent for FII, FV, FVII, F IX, FX, FXI, FXIII, protein C, protein S, antithrombin, and alpha2-antiplasmin. CONCLUSION: PCT of plasma inactivated high levels of a wide range of pathogens while maintaining adequate coagulation function. PCT has the potential to reduce the risk of transfusion-transmitted diseases in patients requiring plasma transfusion support.

[Influence of MHF-radiation on nitrogen oxide molecular spectrum frequency and coagulation factors under experimental stress-reactions].

The effects of EMR MHF of molecular spectrum of radiation and absorption of nitrogen oxide on blood coagulation properties in white rats subjected to immobilization stress, have been studied. It has been revealed that MHF irradiation of the animals results in restoration of hemostasis mechanism indices.

Inactivation of parvovirus B19 in coagulation factor concentrates by UVC radiation: assessment by an in vitro infectivity assay using CFU-E derived from peripheral blood CD34+ cells.

BACKGROUND: Nonenveloped and thermostable viruses such as parvovirus B19 (B19) can be transmitted to patients who are receiving plasma-derived coagulation factor concentrates treated by the S/D method for inactivating enveloped viruses. Therefore, it is important to develop and validate new methods for the inactivation of nonenveloped viruses. STUDY DESIGN AND METHODS: Suspensions of B19 in coagulation factor concentrates (FVIII) were irradiated with UVC light. B19 infectivity was determined by an indirect immunofluorescence assay using CFU-E, as a host cell, derived from peripheral blood CD34+ cells. The effects of catechins on B19 infectivity and on FVIII activity after UVC illumination were also examined. RESULTS: The indirect immunofluorescence assay estimated the B19 infectivity of samples containing virus copies of 10(5) to 10(11) per 10 microL to be a median tissue culture-infectious dose of 10(0.3) to 10(5.4) per 10 microL. B19 was inactivated by 3 log at 750 J per m(2) of UVC radiation and was undetectable after 1000 or 2000 J per m(2) of irradiation. However, FVIII activity decreased to 55 to 60 percent of pretreatment activity after 2000 J per m(2) of UVC radiation. This was inhibited in the presence of rutin or catechins. Epigallocatechin gallate could maintain FVIII activity at almost 100 percent of pretreatment activity after 2000 J per m(2) of UVC radiation, while B19 infectivity was decreased to undetectable levels, which resulted in >3.9 log inactivation. CONCLUSION: UVC radiation in the presence of catechins, especially epigallocatechin gallate, appears to be an effective method of increasing the viral safety of FVIII concentrates without the loss of coagulation activity.

Plasma ascorbate protects coagulation factors against photooxidation.

It has been suggested that proteins, unlike lipids, are not protected against oxidative damage by antioxidants in plasma. We have studied the effect of photodynamic virus inactivation treatment of fresh human plasma on coagulation factor activities. Photodynamic treatment generates singlet oxygen which causes inactivation of fibrinogen and factor VII. Other coagulation factors or anticoagulant proteins are clearly less affected. We found that there is an inverse correlation between the extent of coagulation factor inactivation during the treatment and the plasma ascorbate concentration. The inactivation of coagulation factors was prevented in a dose-dependent manner by adding ascorbate to the plasma before the treatment. Ascorbate was consumed during the treatment at an apparently linear rate. Oxidation of urate and coagulation factors was enhanced when ascorbate had disappeared from plasma. These results indicate that ascorbate is a primary antioxidant against photooxidation in plasma and that it effectively prevents oxidative damage to coagulation factors and other proteins.

Photochemical inactivation of viruses and bacteriophage in plasma and plasma fractions.

Transfusion-associated transmission of viral diseases remains a problem. A number of methods have been developed to inactivate viral pathogens in plasma and plasma fractions, including: dry heating, wet heating, solvent-detergent treatment, and immunoaffinity purification. While some of these methods successfully inactivate pathogenic viruses, inactivation may be incomplete or result in damage to labile plasma proteins. We have developed a method of photochemical decontamination (PCD) using psoralens and long wavelength ultraviolet light to inactivate pathogenic viruses. In the present study, a spectrum of model viruses have been added to plasma and plasma fractions to examine the efficiency of photochemical decontamination and the effects on labile plasma coagulation factors. Both RNA and DNA viruses have been inactivated under conditions which permit preservation of coagulation protein function. PCD technology appears to offer a promising solution to decontamination of blood products.

[Effects of neutron-gamma or gamma irradiation on plasma coagulation factors. Effect of a substitution treatment]. / Effets de l'irradiation neutron-gamma ou gamma sur les facteurs plasmatiques de coagulation. Effet d'un traitement de substitution.

Neutron-gamma irradiation of the baboon at lethal dose altered the plasma clotting factors and induced a fibrinoformation alteration which occurred shortly before death. These disturbances, which were not found after gamma irradiation, could explain the importance of the haemorrhagic syndrome. Treatment by P.P.S.B. (factors II, VII, X and IX) counteracted the alterations of the plasma clotting factors, but had no influence on the lethality nor on the fibrinoformation alteration which seems to be an important cause of death.

Inactivation of human immunodeficiency virus by gamma radiation and its effect on plasma and coagulation factors.

The inactivation of HIV by gamma-radiation was studied in frozen and liquid plasma; a reduction of the virus titer of 5 to 6 logs was achieved at doses of 5 to 10 Mrad at -80 degrees C and 2.5 Mrad at 15 degrees C. The effect of irradiation on the biologic activity of a number of coagulation factors in plasma and in lyophilized concentrates of factor VIII (FVIII) and prothrombin complex was examined. A recovery of 85 percent of the biologic activity of therapeutic components present in frozen plasma and in lyophilized coagulation factor concentrates was reached at radiation doses as low as 1.5 and 0.5 Mrad, respectively. As derived from the first-order radiation inactivation curves, the radiosensitive target size of HIV was estimated to be 1 to 3 MDa; the target size of FVIII was estimated to be 130 to 160 kDa. Gamma radiation must be disregarded as a method for the sterilization of plasma and plasma-derived products, because of the low reduction of virus infectivity at radiation doses that still give acceptable recovery of biologic activity of plasma components.

Effect of gamma irradiation on the human immunodeficiency virus and human coagulation proteins.

The effect of gamma irradiation on HIV and plasma coagulation factors F VIII:C, F VIII:vWF and FIX was studied. Donor plasma was harvested from single donations, frozen and irradiated in the frozen state at target doses from 0 to 40 kGy (0-4 mRad). HIV was inoculated into human plasma and irradiated in a similar manner. A range of other viruses, not suspended in plasma, were also irradiated to establish viral inactivation. An inactivation rate of 0.164 TCID50 dose/ml/kGy was demonstrated for HIV compared to rates of 0.00173, 0.00526 and 0.00286 log10 units/ml/kGy for F VIII:C,F VIII:vWF and FIX respectively. The use of gamma irradiation to inactivate infectious agents present in human plasma may eliminate the need for any post-production viral inactivation methods and provide a means of assuring the safety of as yet untreated products such as cryoprecipitate and fresh frozen plasma.

Postirradiation haemorrhagic syndrome.

One of the relevant symptoms of damage due to irradiation are the manifestations of haemorrhagic diathesis. The extravascular effusion of the blood in the course of the course of the irradiation disease may sometimes also be the cause of death of the irradiated individual, it may complicate the course of the arisen disease and sometimes even make any necessary therapeutical help impossible. The postirradiation disorder of haemocoagulation and haemostasis is a process very complicated both in its origin and in its course. Many experimental studies have been devoted to the study of the mechanism of origin of this disorder, of its course and of the possibilities of its therapy and actually we already have some clinical results. For a wider information and for the utilization of the up-to-date findings in practise, we hold it reasonable to summarize, in a certain time period, the information, though of a restricted problem, in order to facilitate an orientation in the contributions research has made up to now and to enable the selection of the adequate direction of the future experimental studies. This was the reason for the compilation of this monograph in which a summary of the up-to-date experience published in the literature and the results of our many-year standing experimental work is presented in the first opening chapter. In the second chapter, the mechanisms of blood clotting in man and the most often used laboratory animals in experiments, i.e. the dog, rabbit, rat and guinea pig, are compared. By a comparison of the platelet counts, of the time of their survival and the modes of reaction to the inductors of aggregation, some differences have been demonstrated between human thrombocytes and those of laboratory animals, which may be partly conditioned by some species specific composition of the glycoproteins of the platelet membrane. Further on, the difficulties in the determination of the levels of the individual coagulation factors are referred to and their values established in man are compared with those found in the examined laboratory animals in which decreased levels of factor VII and markedly elevated levels of factors V and VIII have been demonstrated. Next, the physiological inhibitors of blood clotting in man are compared with similar substances found in the blood of laboratory animals. Some differences in the structure and in the function of these substances slowing down the blood coagulation are stressed.(ABSTRACT TRUNCATED AT 400 WORDS)