Metabolomics evaluation of the photochemical impact of violet-blue light (405 nm) on ex vivo platelet concentrates.

INTRODUCTION: Microbicidal violet-blue light in the visible spectrum (405 nm) has been under evaluation for pathogen inactivation in ex vivo human plasma and platelets (PLTs) stored in plasma. Results to date have demonstrated that several blood-borne infectious disease-causing pathogens can be successfully reduced to significantly low levels in the light-treated plasma and PLTs. METHOD: In order to evaluate whether the microbicidal 405 nm light is safe for the treatment of PLT concentrates for pathogen inactivation, LC/MS-based metabolomics analyses were performed to evaluate the overall impact of 405 nm violet-blue light treatment on ex vivo PLT concentrates suspended in plasma and on plasma itself, and to identify metabolome changes in intra-platelet and extra-cellular medium (i.e., plasma). RESULTS: The metabolomics data identified that platelet activating factors (PAFs), agonists and prostaglandins, which can influence PLT basic functions such as integrity, activation, and aggregation potential were unaltered, suggesting that 405 nm light illumination is safe regarding PLT basic functions. Distinct increases in hydroxyl fatty acids and aldehydes, as well as decreases in antioxidant metabolites indicated that reactive oxygen species (ROS) were generated at high levels after only one hour of exposure to 405 nm light. Distinctly changed endogenous photosensitizer metabolites after 1 h of light exposure provided good evidence that 405 nm light was an effective microbicide acting through ROS mechanism and no external additive photosensitizers were required.

Clinical manifestation of hemophilia A in the absence of mutations in the F8 gene that encodes FVIII: role of microRNAs.

BACKGROUND: Hemophilia A (HA) is associated with mutations in the F8 gene that expresses factor VIII (FVIII). Unexpectedly, HA also manifests in a small subset of individuals with no mutations (exonic or intronic) in their F8 gene. MicroRNAs (miRNAs) cause translational interference, affecting protein quality and stoichiometry. Here, by analyzing miRNAs of two patients from this subset, we evaluated miRNA-based FVIII suppression as a testable hypothesis to explain FVIII deficiency in patients with HA with no F8 gene mutations. STUDY DESIGN AND METHODS: To test the hypothesis, miRNA sequencing from two patients with mild and moderate HA with no mutations in their F8 gene, followed by experimental verification, was used to identify a group of upregulated miRNAs in patients with HA compared to normal controls; with binding sites in the 3' untranslated region (UTR) of F8 messenger RNA (mRNA), a prerequisite for miRNA-based gene regulation. From this pool, miR-374b-5p and miR-30c-5p, known to be expressed in human liver, where FVIII is expressed, were subjected to extensive characterization. RESULTS: In two cell lines that constitutively express FVIII, we demonstrated that overexpression of miR-374b or miR-30c decreased FVIII expression, while an miR-30c inhibitor partially restored FVIII expression. CONCLUSION: These data support a role for microRNAs in fine-tuning F8 gene regulation. Based on our findings, our current model suggests that in HA cases where the F8 gene is normal and is predicted to express normal levels of FVIII, F8 mRNA 3' UTR targeting miRNAs may be responsible for a FVIII-deficiency phenotype clinically manifesting as HA.

Role of microRNAs in Hemophilia and Thrombosis in Humans.

MicroRNAs (miRNA) play an important role in gene expression at the posttranscriptional level by targeting the untranslated regions of messenger RNA (mRNAs). These small RNAs have been shown to control cellular physiological processes including cell differentiation and proliferation. Dysregulation of miRNAs have been associated with numerous diseases. In the past few years miRNAs have emerged as potential biopharmaceuticals and the first miRNA-based therapies have entered clinical trials. Our recent studies suggest that miRNAs may also play an important role in the pathology of genetic diseases that are currently considered to be solely due to mutations in the coding sequence. For instance, among hemophilia A patients there exist a small subset, with normal wildtype genes; i.e., lacking in mutations in the coding and non-coding regions of the F8 gene. Similarly, in many patients with missense mutations in the F8 gene, the genetic defect does not fully explain the severity of the disease. Dysregulation of miRNAs that target mRNAs encoding coagulation factors have been shown to disturb gene expression. Alterations in protein levels involved in the coagulation cascade mediated by miRNAs could lead to bleeding disorders or thrombosis. This review summarizes current knowledge on the role of miRNAs in hemophilia and thrombosis. Recognizing and understanding the functions of miRNAs by identifying their targets is important in identifying their roles in health and diseases. Successful basic research may result in the development and improvement of tools for diagnosis, risk evaluation or even new treatment strategies.

A Foundational Study for Normal F8-Containing Mouse Models for the miRNA Regulation of Hemophilia A: Identification and Analysis of Mouse miRNAs that Downregulate the Murine F8 Gene.

Hemophilia A (HA) is associated with defects in the F8 gene, encoding coagulation factor VIII (FVIII). Our previous studies show that F8-targeting micro RNAs (miRNAs), a group of small RNAs involved in gene regulation, can downregulate F8 expression causing HA in individuals with normal F8-genotypes and increased HA severity in patients with mutations in F8. Understanding the mechanistic underpinnings of human genetic diseases caused or modulated by miRNAs require a small animal model, such as a mouse model. Here, we report a foundational study to develop such a model system. We identified the mouse 3'untranslated region (3'UTR) on murine F8-mRNA (muF8-mRNA) that can bind to murine miRNAs. We then selected three miRNAs for evaluation: miR-208a, miR-351 and miR-125a. We first demonstrate that these three miRNAs directly target the 3'UTR of muF8-mRNA and reduce the expression of a reporter gene (luciferase) mRNA fused to the muF8-3' UTR in mammalian cells. Furthermore, in mouse cells that endogenously express the F8 gene and produce FVIII protein, the ectopic expression of these miRNAs downregulated F8-mRNA and FVIII protein. These results provide proof-of-concept and reagents as a foundation for using a normal F8-containing mouse as a model for the miRNA regulation of normal F8 in causing or aggravating the genetic disease HA.

Antimicrobial peptides: an effective approach to prevent bacterial biofilm formation in platelet concentrates.

BACKGROUND: The safety of platelet concentrates (PCs) is a major concern in transfusion medicine due to contamination mainly with skin Gram-positive bacteria. The predominant contaminant, Staphylococcus epidermidis, forms bacterial cell aggregates (biofilms) in PCs posing a safety risk for transfusion patients. Combinations of synthetic antimicrobial peptides (AMPs) have demonstrated bactericidal activity in PCs. Herein, we have evaluated the ability of a mix of AMPs to inhibit biofilm formation and/or eradicate S. epidermidis biofilms. STUDY DESIGN AND METHODS: Three synthetic AMPs, the platelet-derived peptide (PD4) and two arginine-tryptophan repeats (RW3 and RW4), were used for bactericidal and antibiofilm experiments in glucose-supplemented trypticase soy broth (TSBg) and PCs spiked with three biofilm-forming strains of S. epidermidis. Time-killing assays were performed to evaluate the bactericidal capability of the peptides. Inhibition of biofilm formation was assayed by seeding S. epidermidis into TSBg or PC cultures supplemented with the AMPs. Biofilm eradication assays were performed after AMP treatment of preformed biofilms with and without mechanical dislodging. Biofilms were measured using a crystal violet assay. RESULTS: Time-killing assays demonstrated that all S. epidermidis strains were eliminated after 24 hours of AMP treatment. While inhibition of biofilm formation was observed for all S. epidermidis strains in TSBg and PCs, the AMP treatment was only effective to reduce the bacterial load of mechanically dislodged biofilms. CONCLUSION: The combination of three synthetic AMPs (PD4-RW3-RW4) can be used to inhibit biofilm formation by S. epidermidis to enhance PC safety. However, further investigation is needed to improve their activity against mature S. epidermidis biofilms.

Analysis of Argonaute 2-microRNA complexes in ex vivo stored red blood cells.

BACKGROUND: Human enucleated mature red blood cells (RBCs) contain both mature microRNAs (miRNAs) and mRNAs, and we have previously correlated RBC storage lesion processes such as eryptosis, adenosine 5'-triphosphate loss, and RBC indices with differentially expressed miRNAs. Here we have characterized Argonaute 2 (AGO2)-miRNA complexes in stored mature RBCs as a first step toward understanding their role, if any. STUDY DESIGN AND METHODS: In this report AGO2-bound miRNAs in mature RBCs isolated from RBCs collected from three different healthy donors and stored for 24 hours at 4 to 6°C were identified by anti-AGO2 immunoprecipitation (IP) followed by next-generation sequencing of the RNA isolated from the IP. The data were analyzed by various bioinformatics tools. RESULTS: The analysis highlighted 28 mature AGO2-bound miRNAs that are common to all three donors, representing 95.6% of the identified miRNAs. Among these, miR-16-5p (20.6%), miR-451a-5p (16.7%), miR-486-5p (12.6%), and miR-92a-3p (12.6%) are the most abundant miRNAs. Functional enrichment analysis for mRNA targets of the 28 common miRNAs identified molecules related to various diseases, biofunctions, and toxicity functions such as cardio-, hepato-, and nephrotoxicity. CONCLUSION: Overall, these results demonstrate the existence of multiple intracellular AGO2-bound miRNAs in 24-hour-stored RBCs and warrant further experiments to determine whether AGO2-miRNAs are functional in RBCs.

miR-570 interacts with mitochondrial ATPase subunit g (ATP5L) encoding mRNA in stored platelets.

Loss of platelet quality during ex vivo storage is a major concern in the transfusion medicine field and it has been known that platelet mitochondrial dysfunction is associated with storage time. In the last decade, small noncoding RNAs also known as microRNAs (miRNAs) have been reported to regulate key cellular processes through their target sequence interactions with selected mRNAs. In this study, we focused on understanding the mechanisms of platelet mitochondrial dysfunction during storage through miRNA regulation of mRNAs. RNA was isolated from day 0, day 5, and day 9 of stored human leukocyte-depleted platelets and subjected to differential miRNA and mRNA profiling. The miRNA profiling identified several miRNAs at low levels including a set of 12 different miR-548 family members (miR-548a-3p, miR-548aa, miR-548x, miR-548ac, miR-548c-3p, miR-603, miR-548aj, miR-548ae, miR-548z, miR-548u, miR-548al, and miR-570-3p). The mRNA profiling identified, among many, the mitochondrial ATP synthase subunit g (ATP5L) mRNA at high levels during storage. Target Scan algorithm for potential targets of miR-570-3p also identified ATP5L as one of its targets. We further identified two target sites for miR-570-3p in the 3' untranslated region (3'UTR) of ATP5L mRNA. While ATP5L is a subunit of F0ATPase complex, its function is not established yet. Overexpression of miR-570-3p in platelets resulted in reduced levels of ATP5L mRNA and concomitant ATP loss. These experimental results provide first-time insights into the miRNA-mRNA interactions underlying mitochondrial dysfunction in ex vivo stored platelets and warrants further investigation.

Evaluation of small noncoding RNAs in ex vivo stored human mature red blood cells: changes in noncoding RNA levels correlate with storage lesion events.

BACKGROUND: While biomarkers of storage lesions (SLs) for red blood cells (RBCs) abound, the physiologic consequences of SLs and associated important events are poorly understood. Previously we have identified differentially expressed regulatory small noncoding RNAs (ncRNAs) in stored RBCs, suggesting their role in the RBC SL process and their potential as quality biomarkers of stored RBCs. STUDY DESIGN AND METHODS: Comprehensive ncRNA expression analysis of RBCs stored for up to 56 days was performed on RNAs collected from enriched mature RBCs on Days 0, 7, 14, 28, 42, and 56. Three known RBC SL processes, that is, mature RBCs' suicidal death (eryptosis), ATP loss, and changes in RBC indices, were correlated with differentially expressed ncRNAs to gain knowledge on the SL molecular processes. RESULTS: The analysis identified four ncRNAs whose changes in the expression levels were correlated with the selected three SL processes. Differential expression on Days 14 and 28 of the four selected ncRNAs was confirmed by TaqMan quantitative reverse transcription-polymerase chain reaction analysis. Bioinformatics analysis identified potential targets and biologic functions of these ncRNAs. Overexpression of one such ncRNA, hsa-miR-196a, in a human erythroblast cell line confirmed its protective effects against the cell death and ATP loss. CONCLUSION: Overall, this study demonstrates that changes in the levels of small ncRNAs of stored RBCs correlate with some of the SL events and thus they have the potential to serve as the storage quality markers.

Leukoreduced whole blood-derived platelets treated with antimicrobial peptides maintain in vitro properties during storage.

BACKGROUND: Bacterial sepsis is still a complication in patients transfused with stored platelets (PLTs). We have recently demonstrated that selected antimicrobial peptides (AMPs) have bactericidal activity in bacteria-spiked PLTs. In a subsequent preclinical study, we have also shown that these AMPs do not elicit antibody response in rabbits and treatment of PLTs before transfusion does not affect their in vivo recovery and survival in severe combined immunodeficient mice. Here we have selected two such AMPs, Arg-Trp (RW) repeats of tri- and tetra-peptides (RW3 and RW4) in combination (i.e., cocktail), and evaluated their effect on the in vitro properties of PLTs. STUDY DESIGN AND METHODS: Leukoreduced ABO- and D-identical whole blood-derived PLT concentrates were pooled and divided into two 60-mL aliquots in CLX storage bags. On Day 0, one bag received a peptide cocktail of RW3 plus RW4 at 0.01 mmol/L final concentration (test) and the other bag received only phosphate-buffered saline (PBS), the AMP solvent (control). The treated PLTs were stored for 7 days at 20 to 24°C. Samples were collected on Days 1, 5, and 7 to evaluate the in vitro properties of PLTs with standard assays. RESULTS: In vitro properties of the RW3 plus RW4 cocktail-treated PLTs were similar to those incubated with PBS only. There were no significant differences between the control and test PLTs during the 7-day storage. CONCLUSION: Leukoreduced whole blood-derived PLTs treated with a mixture of RW3 and RW4 peptides maintain their in vitro properties during 7 days of storage.

Preclinical safety evaluation of human platelets treated with antimicrobial peptides in severe combined immunodeficient mice.

BACKGROUND: Bacterial sepsis is a complication attributed to room temperature (RT)-stored platelets (PLTs) in transfusion medicine. Antimicrobial peptides (AMPs) are emerging as new therapeutic agents against microbes. We had previously demonstrated bactericidal activity of select synthetic AMPs against six types of bacteria in stored PLTs. In this report, we tested these AMPs for their potential antibody response and interference with the recovery and survival of human PLTs in an animal model. STUDY DESIGN AND METHODS: Two separate studies were conducted to evaluate the safety of the synthetic AMPs. 1) Two AMPs (PD3 and PD4), derived from thrombin-induced human PLT microbicidal protein, and four repeats of arginine-tryptophan (RW), containing two to five repeats (RW2-RW5), were tested in rabbits for potential antibody response. 2) RT-stored human PLTs treated for 2 hours with each of the six AMPs individually or with phosphate-buffered saline (PBS) alone were infused into severe combined immunodeficient (SCID) mice to evaluate their in vivo recovery and survival by flow cytometry. RESULTS: Except for PD3, which showed a weak immune response, all other peptides did not induce any detectable antibodies in rabbits. Furthermore, all six AMPs tested did not significantly affect the in vivo recovery and survival of human PLTs in SCID mice compared to PBS alone-treated PLTs. CONCLUSION: Preclinical evaluation studies reported here demonstrate that the selected AMPs used in the study did not adversely affect the human PLT recovery and survival in the SCID mouse model, suggesting further study of AMPs toward addressing the bacterial contamination of PLTs.

Microbial reduction of prebagged human plasma using 405 nm light and its effects on coagulation factors.

Bacterial contamination is the most prevalent infectious complication of blood transfusion in the developed world. To mitigate this, several ultraviolet light-based pathogen reduction technologies (PRTs), some of which require photo-chemicals, have been developed to minimize infection transmission. Relative to UV light, visible 405-nm light is safer and has shown potential to be developed as a PRT for the in situ treatment of ex vivo human plasma and platelet concentrates, without the need for photo-chemicals. This study investigates the effect of 405-nm light on human plasma, with focus on the compatibility of antimicrobial light doses with essential plasma clotting factors. To determine an effective antimicrobial dose that is compatible with plasma, prebagged human plasma (up to 300 mL) was seeded with common microbial contaminants and treated with increasing doses of 405-nm light (16 mW cm-2; ≤ 403 J cm-2). Post-exposure plasma protein integrity was investigated using an AOPP assay, in vitro coagulation tests, and ELISA-based measurement of fibrinogen and Protein S. Microbial contamination in 300 mL prebagged human plasma was significantly reduced (P ≤ 0.05) after exposure to ≤ 288 J cm-2, with microbial loads reduced by > 96.2%. This dose did not significantly affect the plasma protein quality parameters tested (P > 0.05). Increased doses (≥ 345 J cm-2) resulted in a 4.3% increase in clot times with no statistically significant change in protein activity or levels. Overall, this study has demonstrated that the effective microbicidal 405 light dose shows little to no negative effect on plasma quality.

The Preclinical Validation of 405 nm Light Parasiticidal Efficacy on Leishmania donovani in Ex Vivo Platelets in a Rag2-/- Mouse Model.

Violet-blue light of 405 nm in the visible spectrum at a dose of 270 J/cm2 alone has been shown to be an effective microbicidal tool for inactivating several bacteria, HIV-1, and Trypanosoma cruzi in ex vivo plasma and platelets. Unlike chemical- and ultraviolet (UV)-based pathogen inactivation methods for plasma and platelet safety, 405 nm light is shown to be less toxic to host cells at light doses that are microbicidal. In this report, we evaluated the parasiticidal activity of a 405 nm light treatment on platelets spiked with the Leishmania donovani parasite. Following the light treatment, parasite viability was observed to be near zero in both low- and high-titer-spiked platelets relative to controls. Furthermore, to test the residual infectivity after inactivation in vivo, the light-treated low-titer L. donovani-spiked platelets were evaluated in an immunodeficient Rag2-/- mouse model and monitored for 9 weeks. The parasiticidal efficacy of 405 nm light was evident from the lack of a presence of parasites in the mice spleens. Parasiticidal activity was confirmed to be mediated through 405 nm light-induced reactive oxygen species (ROS), as quantitatively measured by a 2',7'-Dichlorodihydrofluorescein diacetate (H2DCFDA)-based assay. Overall, these results confirm the complete inactivation of L. donovani spiked in ex vivo platelets by 405 nm light treatment and exemplify the utility of the Rag2-/- mouse infection model for the preclinical validation of the parasiticidal efficacy of 405 nm light and this light-based technology as a potential PRT for ex vivo platelets.

Human platelet concentrates treated with microbicidal 405 nm light retain hemostasis activity.

Chemical and UV light-based pathogen reduction technologies are currently in use for human platelet concentrates (PCs) to enhance safety from transfusion-transmitted infections. Relative to UV light, 405 nm violet-blue light in the visible spectrum is known to be less harmful. Hence, in this report for the first time, we have assessed the global hemostasis activity of PCs stored in plasma and the activities of six plasma coagulation factors (CFs) as a measure of in vitro hemostatic activity following exposure to the microbicidal 405 nm light. Apheresis PC samples collected from each screened human donor (n = 22) were used for testing of PCs and platelet poor plasma (PPP). Both PCs and PPPs were treated for 5 h with 405 nm light to achieve a previously established microbicidal light dose of 270 J/cm2. Activated partial thromboplastin time and prothrombin time-based potency assays using a coagulation analyzer and hemostatic capacity via Thromboelastography were analyzed. Thromboelastography analysis of the light-treated PCs and plasma present in the PCs showed little difference between the treated and untreated samples. Further, plasma present in the PCs during the light treatment demonstrated a better stability in potency assays for several coagulation factors compared to the plasma alone prepared from PCs first and subjected to the light treatment separately. Overall, PCs stored in plasma treated with 405 nm violet-blue light retain activity for hemostasis.

Antimicrobial 405 nm violet-blue light treatment of ex vivo human platelets leads to mitochondrial metabolic reprogramming and potential alteration of Phospho-proteome.

Continued efforts to reduce the risk of transfusion-transmitted infections (TTIs) through blood and blood components led to the development of ultraviolet (UV) light irradiation technologies known as pathogen reduction technologies (PRT) to enhance blood safety. While these PRTs demonstrate germicidal efficiency, it is generally accepted that these photoinactivation techniques have limitations as they employ treatment conditions shown to compromise the quality of the blood components. During ex vivo storage, platelets having mitochondria for energy production suffer most from the consequences of UV irradiation. Recently, application of visible violet-blue light in the 400-470 nm wavelength range has been identified as a relatively more compatible alternative to UV light. Hence, in this report, we evaluated 405 nm light-treated platelets to assess alterations in energy utilization by measuring different mitochondrial bioenergetic parameters, glycolytic flux, and reactive oxygen species (ROS). Furthermore, we employed untargeted data-independent acquisition mass spectrometry to characterize platelet proteomic differences in protein regulation after the light treatment. Overall, our analyses demonstrate that ex vivo treatment of human platelets with antimicrobial 405 nm violet-blue light leads to mitochondrial metabolic reprogramming to survive the treatment, and alters a fraction of platelet proteome.

The microbicidal potential of visible blue light in clinical medicine and public health.

Visible blue light of wavelengths in the 400-470 nm range has been observed to have microbicidal properties. A widely accepted hypothesis for the mechanism of microbial inactivation by visible blue light is that the light causes photoexcitation of either endogenous (present within the microbe) or, exogenous (present in the biological medium surrounding the microbe) photosensitizers such as porphyrins and flavins, which leads to the release of reactive oxygen species that subsequently manifests microbicidal activity. Some of the factors that have been observed to be associated with enhanced microbicidal action include increased duration of exposure, and either pre- or co-treatment with quinine hydrochloride. In case of bacteria, repetitive exposure to the blue light shows no significant evidence of resistance development. Additionally, visible blue light has exhibited the ability to inactivate fungal and viral pathogens and, multidrug-resistant bacteria as well as bacterial biofilms. Visible blue light has demonstrated efficacy in eliminating foodborne pathogens found on food surfaces and exposed surfaces in the food processing environment as well as in the decontamination of surfaces in the clinical environment to minimize the spread of nosocomial infections. We conclude from reviewing existing literature on the application of the blue light in clinical medicine and public health settings that this microbicidal light is emerging as a safer alternative to conventional ultraviolet light-based technologies in multiple settings. However, further comprehensive studies and thorough understanding of the mechanism of microbicidal action of this light in different scenarios is warranted to determine its place in human health and disease.

Violet-blue 405-nm Light-based Photoinactivation for Pathogen Reduction of Human Plasma Provides Broad Antibacterial Efficacy Without Visible Degradation of Plasma Proteins.

In transfusion medicine, bacterial contamination can occur in ex vivo stored blood plasma, and there are continued efforts to improve blood safety and reduce the risk of transfusion-transmitted infections. Visible 405-nm violet-blue light has demonstrated potential for in situ pathogen reduction in ex vivo stored plasma and platelet concentrates. This study investigates the broad-spectrum antibacterial efficacy and compatibility potential of 405-nm light for treatment of blood plasma. Human plasma seeded with bacteria at a range of densities (101 -103 , 104 -106 , 107 -108 CFU mL-1 ) was exposed to 360 J cm-2 405-nm light (1 h at 0.1 W cm-2 ), with this fixed dose selected based on the initial analysis of inactivation kinetics. One-dimensional protein mobility analysis and measurement of advanced oxidation protein products (AOPP) was conducted to evaluate compatibility of the antimicrobial dose with plasma proteins and, identify upper levels at which protein degradation can be detected. Broad-spectrum antibacterial efficacy was observed with a fixed treatment of 360 J cm-2 , with 98.9-100% inactivation achieved across all seeding densities for all organisms, except E. coli, which achieved 95.1-100% inactivation. At this dose (360 J cm-2 ), no signs of protein degradation occurred. Overall, 405-nm light shows promise for broad-spectrum bacterial inactivation in blood plasma, while preserving plasma protein integrity.

Visible 405 nm Violet-Blue Light Successfully Inactivates HIV-1 in Human Plasma.

Despite significant advances in ensuring the safety of the blood supply, there is continued risk of transfusion transmitted infections (TTIs) from newly emerging or re-emerging infections. Globally, several pathogen reduction technologies (PRTs) for blood safety have been in development as an alternative to traditional treatment methods. Despite broad spectrum antimicrobial efficacy, some of the approved ultraviolet (UV) light-based PRTs, understandably due to UV light-associated toxicities, fall short in preserving the full functional spectrum of the treated blood components. As a safer alternative to the UV-based microbicidal technologies, investigations into the use of violet-blue light in the region of 405 nm have been on the rise as these wavelengths do not impair the treated product at doses that demonstrate microbicidal activity. Recently, we have demonstrated that a 405 nm violet-blue light dose of 270 J/cm2 was sufficient for reducing bacteria and the parasite in plasma and platelets suspended in plasma while preserving the quality of the treated blood product stored for transfusion. Drawn from the previous experience, here we evaluated the virucidal potential of 405 nm violet-blue light dose of 270 J/cm2 on an important blood-borne enveloped virus, the human immunodeficiency virus 1 (HIV-1), in human plasma. Both test plasma (HIV-1 spiked and treated with various doses of 405 nm light) and control plasma (HIV-1 spiked, but not treated with the light) samples were cultured with HIV-1 permissive H9 cell line for up to 21 days to estimate the viral titers. Quantitative HIV-1 p24 antigen (HIV-1 p24) levels reflective of HIV-1 titers were measured for each light dose to assess virus infectivity. Our results demonstrate that a 405 nm light dose of 270 J/cm2 is also capable of 4-5 log HIV-1 reduction in plasma under the conditions tested. Overall, this study provides the first proof-of-concept that 405 nm violet-blue light successfully inactivates HIV-1 present in human plasma, thereby demonstrating its potential towards being an effective PRT for this blood component safety.

MiRNA-103b Downregulates ITGB3 and Mediates Apoptosis in Ex Vivo Stored Human Platelets.

BACKGROUND: Blood bank-stored human platelets are one of the life-saving transfusion products to prevent bleeding in multiple clinical settings. In ex vivo storage, platelets undergo apoptosis and it is highly desirable to prevent this process to preserve platelet quality. However, underlying mechanisms of apoptosis are not well understood in stored platelets. Integrin beta 3 (ITGB3) glycoprotein plays multiple roles in platelet physiological processes, and it was reported in other cell types that downregulation of ITGB3 induces apoptosis. Small noncoding regulatory RNAs known as microRNAs (miRNAs), some of which are abundant in platelets such as miR-103b that belong to miR-103 family of miRNAs, known to play key roles in platelet functions both in vivo and during storage; Cellular miR-103 downregulates certain genes in other cell types and promotes apoptosis. However, whether miR-103b can target and downregulate ITGB3 in stored platelets and such miRNA regulation promotes apoptosis is not known. Here, we tested this working hypothesis. OBJECTIVE: Our objective of this study is to validate the abundance of miR-103b in stored platelets and identify whether ITGB3 is a target of miR-103b for the downregulation and this interaction promotes apoptosis. METHODS: RT-qPCR validation of miR-103b was performed in 11 donor samples at 3 different storage time points. In-silico analysis was performed to identify predicted targets of the miR-103b. The miRNA and messenger RNA interactions were confirmed using different biochemical approaches such as qRT-PCR, western blotting and, suppression of luciferase reporter gene expression by ectopic expression of miR-103b in HeLa cells. Final validation of the functional role of miR-103b in ITGB3 downregulation and resulting induction of apoptosis was assessed in stored platelets by FACS analysis following ectopic expression of miR-103b. RESULTS: Using the Target Scan Vert algorithm, we identified several integrin subunit-encoding mRNAs as potential targets of miR-103b. While ITGB3 and ITGB6 were found to have two targeting sites for miR-103b, since ITGB3 is known to play a role in apoptosis, we chose this for further validation in this study. Ectopic expression of miR-103b decreased the luciferase reporter activity in HeLa cells and decreased ITGB3 mRNA and protein levels in platelets, concomitant with an increase in apoptosis. CONCLUSION: The results demonstrate that in stored platelets, miR-103b is highly expressed and can interact with and downregulate ITGB3 and promote apoptosis in stored platelets.

Analysis of the mechanism of damage produced by thiazole orange photoinactivation in apheresis platelets.

BACKGROUND: Pathogen Reduction Technologies (PRTs) are broad spectrum nucleic acid replication-blocking antimicrobial treatments designed to mitigate risk of infection from blood product transfusions. Thiazole Orange (TO), a photosensitizing nucleic acid dye, was previously shown to photoinactivate several types of bacterial and viral pathogens in RBC suspensions without adverse effects on function. In this report we extended TO treatment to platelet concentrates (PCs) to see whether it is compatible with in vitro platelet functions also, and thus, could serve as a candidate technology for further evaluation. MATERIAL AND METHODS: PCs were treated with TO, and an effective treatment dose for inactivation of Staphylococci was identified. Platelet function and physiology were then evaluated by various assays in vitro. RESULTS: Phototreatment of PCs yielded significant reduction (≥4-log) in Staphylococci at TO concentrations ≥20 µM. However, treatment with TO reduced aggregation response to collagen over time, and platelets became unresponsive by 24 hours post-treatment (from >80% at 1 h to 0% at 24 h). TO treatment also significantly increased CD62P expression (<1% CD62P+ for untreated and >50% for TO treated at 1 h) and induced apoptosis in platelets (<1% Annexin V+ for untreated and >50% for TO treated at 1 h) and damaged mitochondrial DNA. A mitochondria-targeted antioxidant and reactive oxygen species (ROS) scavenger Mito-Tempo mitigated these adverse effects. DISCUSSION: The results demonstrate that TO compromises mitochondria and perturbs internal signaling that activates platelets and triggers apoptosis. This study illustrates that protecting platelet mitochondria and its functions should be a fundamental consideration in selecting a PRT for transfusion units containing platelets, such as PCs.