Riboflavin Treatment Research Articles

Cellular consequences of oxidative stress in riboflavin responsive multiple acyl-CoA dehydrogenation deficiency patient fibroblasts

Mitochondrial dysfunction and oxidative stress are central to the molecular pathology of many human diseases. Riboflavin responsive multiple acyl-CoA dehydrogenation deficiency (RR-MADD) is in most cases caused by variations in the gene coding for electron transfer flavoprotein-ubiquinone oxidoreductase (ETF-QO). Currently, patients with RR-MADD are treated with high doses of riboflavin resulting in improvements of the clinical and biochemical profiles. However, in our recent studies of RR-MADD, we have shown that riboflavin treatment cannot fully correct the molecular defect in patient cells producing increased reactive oxygen species (ROS). In the current study, we aim to elucidate the cellular consequences of increased ROS by studying the cellular ROS adaption systems including antioxidant system, mitochondrial dynamics and metabolic reprogramming. We have included fibroblasts from six unrelated RR-MADD patients and two control fibroblasts cultivated under supplemented and depleted riboflavin conditions and with coenzyme Q10 (CoQ10) treatment. We demonstrated inhibition of mitochondrial fusion with increased fractionation and mitophagy in the patient fibroblasts. Furthermore, we indicated a shift in the energy metabolism by decreased protein levels of SIRT3 and decreased expression of fatty acid β-oxidation enzymes in the patient fibroblasts. Finally, we showed that CoQ10 treatment has a positive effect on the mitochondrial dynamic in the patient fibroblasts, indicated by increased mitochondrial fusion marker and reduced mitophagy. In conclusion, our results indicate that RR-MADD patient fibroblasts suffer from a general mitochondria dysfunction, probably initiated as a rescue mechanism for the patient cells to escape apoptosis as a result of the oxidative stress.

Interaction between riboflavin and BCRP‐mediated drug transport in the mammary gland (844.2)

Breastfeeding provides numerous benefits to a child’s growth and development. However, in a lactating mother, expression of Breast Cancer Resistance Protein (BCRP) is significantly increased in the mammary gland. BCRP is an efflux transporter and is responsible for the transport of many different substrates from natural carcinogens (ex. PhIP found in charred meats) to chemotherapeutic drugs. As a result, this poses a risk of exposure to potentially harmful BCRP substrates from a mother’s milk to her child. We hypothesized that riboflavin, a vitamin substrate for BCRP, can act as a potential competitor for BCRP‐mediated excretion of drugs/toxins into milk. We used intravenous topotecan and cimetidine at a dose of 1 and 5 mg/kg as model BCRP substrates in lactating mice. In mice pretreated with 5 mg/kg riboflavin, excretion of topotecan and cimetidine into milk at 30 min postdose was significantly reduced. Mean topotecan levels in the milk were 883 ng/ml (SD: ±465; n=10) in the control and 682 ng/ml (SD: ±316; n=10; p<0.01) in the riboflavin treatment group. For cimetidine, the mean milk levels were 1941 ng/ml (SD: ±387; n=7) in the control, compared to 1555 ng/ml (SD: ±192; n=7; p<0.05) in the riboflavin‐pretreated mice. The milk‐to‐plasma ratios of topotecan and cimetidine in riboflavin‐pretreated mice also showed a significant 35% and 26% decrease respectively. In conclusion, our findings indicate that riboflavin administration does reduce the excretion of other BCRP substrates into a mother’s milk.Grant Funding Source: Supported by CIHR

Riboflavin (vitamin B-2) reduces hepatocellular injury following liver ischaemia and reperfusion in mice

Riboflavin has been shown to exhibit anti-inflammatory and antioxidant properties in the settings of experimental sepsis and ischaemia/reperfusion (I/R) injury. We investigated the effect of riboflavin on normothermic liver I/R injury. Mice were submitted to 60min of ischaemia plus saline or riboflavin treatment (30μmoles/kgBW) followed by 6h of reperfusion. Hepatocellular injury was evaluated by aminotransferase levels, reduced glutathione (GSH) content and the histological damage score. Hepatic neutrophil accumulation was assessed using the naphthol method and by measuring myeloperoxidase activity. Hepatic oxidative/nitrosative stress was estimated by immunohistochemistry. Liver endothelial and inducible nitric oxide synthase (eNOS/iNOS) and nitric oxide (NO) amounts were assessed by immunoblotting and a chemiluminescence assay. Riboflavin significantly reduced serum and histological parameters of hepatocellular damage, neutrophil infiltration and oxidative/nitrosative stress. Furthermore, riboflavin infusion partially recovered hepatic GSH reserves and decreased the liver contents of eNOS/iNOS and NO. These data indicate that riboflavin exerts antioxidant and anti-inflammatory effects in the ischaemic liver, protecting hepatocytes against I/R injury. The mechanism of these effects appears to be related to the intrinsic antioxidant potential of riboflavin/dihydroriboflavin and to reduced hepatic expression of eNOS/iNOS and reduced NO levels, culminating in attenuation of oxidative/nitrosative stress and the acute inflammatory response.

Effects of riboflavin and ultraviolet light treatment on platelet thrombus formation on collagen via integrin αIIbβ3 activation

The adoption of pathogen reduction technology (PRT) is considered for the implementation of safer platelet (PLT) transfusion. However, the effects of PRT treatment on PLT thrombus formation under blood flow have not yet been fully clarified. Leukoreduced PLT concentrates (PCs) obtained by plateletpheresis were treated with riboflavin and ultraviolet light (Mirasol PRT). PC samples were passed through a column filled with collagen-coated beads at a fixed shear rate after 1, 3, and 5 days of storage. The thrombus formation ability was evaluated by measuring collagen column retention rate. The change in the activation state of integrin αIIbβ3 on PLTs during storage was examined by flow cytometry. The retention rate of the PRT-treated PLTs was significantly higher than that of the control PLTs on the day of treatment and decreased with storage but remained higher than those of the control during storage. This modification did not correlate with the total αIIbβ3 or fibrinogen binding on the PLTs but correlated significantly with PAC-1 binding. Mn(2+) -induced αIIbβ3 activation also fully restored the retention rate in the Day 5 PRT-treated PLTs along with the increase in PAC-1 binding. Riboflavin-based PRT treatment of PCs leads to the enhancement of thrombus formation on collagen, which is related to the activation status of αIIbβ3, which does not bind to fibrinogen but binds to PAC-1. The impact of this finding on the hemostatic or even thrombogenic potential in vivo must await clinical evaluation.

UVA-Photoactivated Riboflavin Treatment of Vaginal Cells Derived from Pelvic Organ Prolapse Cases

Background: The pathophysiology of pelvic organ prolapse (POP) involves vaginal collagen degradation. Strengthening collagen by UVA-photoactivated cross-linking has been demonstrated and suggested target applications include the vaginal wall. Aim: To identify UVA irradiation and riboflavin effects on vaginal cells. Materials and Methods: Vaginal cells were incubated for 24 h (DMEM/F-12 Ham's media) and were exposed to riboflavin (0, 0.1 and 10%) for 30 min before UVA photoactivation. Percentages of live, apoptotic and necrotic cells were determined by propidium iodide/Hoechst 33342 stains. Results: UVA decreased vaginal cell viability [mean ± standard error of the mean: 26.2 ± 0.5% vs. control (43.9 ± 3.8%)], but riboflavin blocked UVA-induced damage (57.9 ± 2.7 and 56.7 ± 2.1% at 0.1 and 10% riboflavin, respectively). Cells treated with low- and high-dose riboflavin had lower apoptosis (32.9 ± 1.0 and 35.5 ± 0.9%, respectively). Furthermore, riboflavin-treated cells had reduced necrosis (9.3 ± 1.7, 7.8 ± 3.0%) versus UVA-only (32.4 ± 5.5%) or control (17.1 ± 2.8%). Viability was similar for cells from the cervical and hymenal segments. Conclusion: The results demonstrated that riboflavin attenuated UVA damage in vaginal cells by inhibiting necrosis. Cervical and hymenal end vaginal cells were equally affected by UVA. UVA phototoxicity was reduced by the presence of riboflavin.

Use of riboflavin/blue light in improving dentin bonding

Purpose: To evaluate the use of the photoreactive agent riboflavin (RF) combining blue light irradiation as a collagen cross-linker in improving dentin bonding. Methods and materials: Sixty extracted human molars were used. For each tooth, enamel was removed and crown was flattened with low-speed diamond saw under water irrigation. A standardized smear layer was created with wet 600-grit silicon-carbide paper. The top surface of dentin was etched for 15 s with 35% phosphoric-acid gel, rinsed with water before different collagen cross-linking treatments. These teeth were divided into six experimental groups: Control, no treatment; RF0.1UV2, 0.1% RF treatment followed by 2-min UVA irradiation; RF0.1BL1 and RF0.1BL2, 0.1% RF followed by 1or 2-min blue light irradiation; RF1BL1 and RF1BL2, 1%RF followedby 1or 2-minblue light irradiation. The treated dentin surfaces received bonding procedure with Singlebond (3M ESPE), and 4mm-thick resin composite cube were builtup in two layers. After storage in distilled water at 37 ◦C for 24h, these teeth were cut into microbeams and then subjected to a microtensile bond strength ( TBS) test at initial stage and after enzymatic degradation. In addition, the fractured dentin-composite microbeams were evaluated by SEM to analyze fracture mode. Additionally, 24 teeth were powdered and demineralized with 1% phosphoric acid for 10min. These dentin powders were divided into six parts to received one of those collagen cross-linking treatments. Proteins were extractedwith extraction buffer and concentratedwith 30kDa membrane, then receivedBradfordassay todetermine theprotein concentrations. Finally, MMP2 activity was checked with a zymography assay. Results: RF0.1BL1 and RF0.1UV2 showed significantly increased TBS. Fracturemodeanalysis showed that RF0.1UV2 and RF0.1BL1 groups presented the least adhesive fracture mode. The zymography analysis showed that the enzymatic activity was inhibited when the teeth was treated with RF0.1/BL1. Conclusion: The collagen crosslinking by riboflavin/blue light could improve the dentin bonding stability and inhibit the MMPs activity.

Paired analysis of plasma proteins and coagulant capacity after treatment with three methods of pathogen reduction

The effect of photochemical pathogen reduction (PR) methods on plasma quality has been the subject of several reports but solid comparative data for the different technologies are lacking. Plasma (n = 24) photoinactivated with methylene blue (MB), riboflavin (RF), or amotosalen (AS) was compared using a pool-and-split design. Samples were taken before and after treatment with each method and tested for coagulation factors (fibrinogen, Factor [F] II, FV, FVIII, F IX, FXI), natural coagulation inhibitors (Protein C [PC], protein S [PS], antithrombin III [AT]), prothrombin time (PT), activated partial thromboplastin time (APTT), and thrombin generation (TG). The three methods were mutually compared by repeated-measures analysis of variance. All three PR methods cause significant reduction (p < 0.01) of activity of the procoagulant proteins fibrinogen, FII, FV, FVIII, F IX, and FXI. Coagulation is also affected, with significant changes in PT, APTT, and TG. RF treatment causes a significantly higher decrease in concentration of coagulation factors, PS, and AT than the other methods (p < 0.01). PT, APTT, and TG are also affected most by RF treatment. FII, FVIII, F IX, PC, AT, and PT are best preserved with the MB method and FV, FXI, and TG after AS treatment (p < 0.01). Coagulation factor loss due to the volume loss during PR treatment is more important for MB and AS than for RF. PR treatment of plasma affects coagulation proteins and coagulant capacity. For the RF method this effect is most pronounced, although to some extent compensated by a smaller volume loss.

The regulatory role of riboflavin in the drought tolerance of tobacco plants depends on ROS production

Riboflavin (vitamin B2) is required for normal plant growth and development. Previous studies have shown that riboflavin application can enhance pathogen resistance in plants. Here, we investigated the role of riboflavin in increasing drought tolerance (10 % PEG6000 treatment) in plants. We treated 4 week-old tobacco plants with five different levels of riboflavin (0, 4, 20, 100 and 500 μM) for 5 days and examined their antioxidant responses and levels of drought tolerance. Compared with the controls, low and moderate levels of riboflavin treatment enhanced drought tolerance in the tobacco plants, whereas higher concentrations of riboflavin (500 μM) impaired drought tolerance. Further analysis revealed that plants treated with 500 μM riboflavin accumulated higher levels of ROS (O2 − and H2O2) and lipid peroxide than the control plants or plants treated with low levels of riboflavin. Consistent with this observation, the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) were higher in plants treated with low or moderate (4, 20 and 100 μM) levels of riboflavin compared with the control. We also found that chlorophyll degraded rapidly in control and 500 μM riboflavin-treated plants under drought stress conditions. In addition, the survival times of the riboflavin-treated plants were significantly modified by treatment with reduced glutathione, a well-known ROS scavenger, under drought stress conditions. Thus, riboflavin-mediated ROS production may determine the effects of riboflavin on drought tolerance in tobacco plants.

Influence of Corneal Cross-linking for Keratoconus on Several Objective Parameters of Dry Eye

To evaluate the potential influence of corneal cross-linking (CXL) with ultraviolet-A light and riboflavin in keratoconic eyes on several objective parameters of dry eye syndrome. This prospective single center study included 30 consecutive eyes of 16 patients that underwent CXL with riboflavin and ultraviolet-A treatment (epithelial removal, 30 minutes soaking with riboflavin, 30 minutes of illumination with 365 nm, 3 mW/cm(2), 5 cm distance). Several dry eye syndrome parameters were evaluated preoperatively and 3 and 6 months after the procedure: intra-individual comparison of fluorescein and Rose bengal staining, height of tear film meniscus, and tear film break-up time. Pathologic staining (more than 10 point-shaped areas or diffuse staining) with fluorescein was evident before CXL in 1 eye, 3 months after CXL in 1 eye, and 6 months after CXL in 1 eye. Rose bengal staining 3 and 6 months postoperatively was comparable to preoperative staining. Tear film height was reduced in 2 eyes before CXL and in 4 eyes 3 months postoperatively, and was normal in all eyes after 6 months. The number of eyes with reduced tear film break-up time was not significantly changed. CXL had no significant impact on several parameters of dry eye syndrome 3 and 6 months postoperatively.

P.17.10 New ETFDH mutations in a group of Italian patients with Riboflavin-Responsive Multiple Acyl-CoA Dehydrogenase Deficiency (RR-MADD)

Multiple Acyl-CoA Dehydrogenase Deficiency (MADD) is a rare autosomal recessive disorder of the electron transfer flavoproteins. Three clinical forms have been described: two with neonatal onset (I and II), and a late-onset form (type III). Type III patients manifest with proximal myopathy, sometimes with hepatomegaly and encephalopathy. Mutations in ETFDH has been often associated with a Riboflavin response in those patients. We report, herein, clinical, biochemical and molecular genetic features in five Italian RR-MADD patients. Clinically, all patients variably presented with acute recurrent episodes of myalgias, exercise intolerance, rhabdomyolysis and proximal muscle weakness. Serum CK was elevated (from 1000 to 8000 UI/L) as well as lactacidemia. Medium- and long-chain acyl carnitines were increased in all subjects. Muscle biopsy showed a vacuolar myopathy with lipid storage. Total and free muscle carnitines were reduced whereas muscle CoQ10 levels were normal. Oral Riboflavin treatment (400 mg/die) resulted in a dramatic improvement of clinical conditions of all patients. Mutational analysis of ETFDH identified seven novel mutations: 5 missense mutations, a 2-base-pair deletion in the coding region and one splice-site mutation. Our data confirm that blood acylcarnitine profile are sensitive and predictive markers for MADD diagnosis whereas muscle CoQ10 levels may be normal. We identified seven novel ETFDH mutations, all located in conserved domains. These new changes were distinct from the others described in previous reported cases but, also our group of patients, dramatically responded to Riboflavin administration.

Anterior and posterior corneal stroma elasticity after corneal collagen crosslinking treatment

The purpose of this project was to assess anterior and posterior corneal stromal elasticity after corneal collagen cross linking (CXL) treatment in human cadaver eyes using Atomic Force Microscopy (AFM) through indentation. Twenty four human cadaver eyes (12 pairs) were included in this study and divided into 2 groups (6 pairs per group). In both groups, the left eye (OS) served as a control (no riboflavin or CXL treatment was performed) and the right eye (OD) underwent CXL treatment (30 min of riboflavin pretreatment followed by 30 min of exposure to 3 mW/cm2 of ultraviolet light). In group 1, the anterior stroma was exposed by manual delamination of approximately 50 μm of the corneal stroma including Bowman's membrane. In group 2, the posterior stroma was exposed by delamination of the anterior 50% of the corneal stroma including Bowman's membrane. Delamination was performed after crosslinking treatment in the case of the treated eyes. In all eyes, the stromal elasticity was quantified using AFM through indentation. Young's modulus of elasticity for the anterior cornea (group 1) was 245.9 ± 209.1 kPa (range: 82.3–530.8 kPa) for the untreated control eyes, and 467.8 ± 373.2 kPa (range: 157.4–1126 kPa) for the CXL treated eyes. Young's modulus for the posterior cornea (group 2) was 100.2 ± 61.9 kPa (range: 28.1–162.6 kPa) for the untreated control eyes and 66.0 ± 31.8 kPa (range: 31.3–101.7 kPa) for the CXL treated eyes. Young's modulus of the anterior stroma significantly increased after CXL treatment (p = 0.024), whereas the posterior stroma did not demonstrate a significant difference in Young's modulus after CXL treatment (p = 0.170). The anterior stroma was stiffer than the posterior stroma for both the control and CXL treatment groups (p = 0.077 and p = 0.023, respectively). Our findings demonstrate that stiffness of the anterior corneal stroma after CXL treatment seems to increase significantly, while the posterior stroma does not seem to be affected by CXL.

Corneal Resistance to Shear Force After UVA-Riboflavin Cross-Linking

We evaluated whether UVA-riboflavin collagen cross-linking (CXL) increases transverse stromal shear moduli ex vivo, whether the shear moduli are greater in the anterior compared to the posterior stroma, and whether the shear moduli are affected by CXL. The resistance to unidirectional transverse shear of human (n = 18) and porcine (n = 42) corneas was measured in a custom engineered biaxial biomechanical setup at different hydrations. The corneas were separated into untreated, riboflavin solution-treated, and CXL-treated groups. The depth dependency of shear moduli within groups was assessed in femtosecond laser cut sheets. Dry weights were obtained for solids correction. In porcine full-thickness buttons and 300 μm anterior sheets, a significantly increased unidirectional transverse shear modulus was detected in riboflavin-treated and CXL-treated groups compared to the respective untreated groups. There was no significant difference in shear modulus between riboflavin- and CXL-treated groups. In all groups, the shear moduli were greater in the anterior sheets compared to posterior sheets. Similar results were detected in human corneas. A method for unidirectional transverse shear resistance measurement was developed. The shear moduli were greater in the anterior compared to the posterior sheets. Increase in shear moduli was observed in the riboflavin and CXL groups compared to the untreated group, indicating that the immediate effects of the riboflavin or CXL treatment may be due partly to ground substance/dextran-5-phosphate interaction.

Induction of Nucleic Acid Damage in Viral Genomes using Riboflavin in Combination with UV Light

Background and Aims: Despite the screening of blood donors, blood transfusion represents an ideal port of entry for blood-borne infection. Blood-borne pathogen transmission has been a concern since the earliest days of transfusion. The blood product of platelet (PLT) concentrates is still faced with the risk of bacterial and viral contaminations. Pathogen inactivation technologies offer a proactive approach and the potential to further improve blood safety. Here we study the pathogen inactivating capacity of riboflavin with UV light treatment in platelet concentrates contaminated with enveloped and non-enveloped viruses. Materials and Methods: The inactivation effects of riboflavin in combination with UV light was examined on Herpes simplex virus (HSV), Vesicular stomatitis virus (VSV) and Polio virus classified as enveloped and non-enveloped DNA and RNA viruses, respectively. After spiking viruses in PLT concentrate, treatment was undertaken with riboflavin (50 µM) and exposed to different doses of UV light. Residual viral infectivity was titrated using 50%Tissue Culture Infective Dose (TCID50). Results: Combination of Riboflavin and UV light treatment reduced the titer of HSV, VSV and Poliovirus with different doses of UV light. Log reduction of HSV with UV doses of 0.82, 1.63, 2.44 and 3.25 J/cm 2 was 0.5, 1.3, 3.5 and 3.8, respectively. Log reduction of VSV for 0.82, 1.63, 2.44 and 3.25 J/cm 2 was 2.8, 3.8, 4.6 and 5.6, respectively. Also, log reduction of poliovirus in the same order for 0.82, 1.63, 2.44 and 3.25 J/cm 2 was 1.7, 2.5, 2.7 and 3.1 respectively. Conclusion: The final dose of UV (3.25 J/cm 2 ) resulted in a larger amount of viral inactivation for enveloped and non-enveloped viruses, in suspended PLT. This method offers a potential to be used for prevention of the majority of PLT transfusion-associated viral pathogens.

Plasma constituent integrity in pre-storage vs. post-storage riboflavin and UV-light treatment – A comparative study

Treatment of fresh frozen plasma (FFP) by riboflavin (RB) and ultraviolet (UV) light inhibits nucleic acid replication, leading to inactivation of white blood cells (WBCs) and pathogens. The goal of this study was to compare the effects of pathogen reduction technology (PRT) treatment on the plasma protein content based on biochemical, immune and hemostatic characteristics in "typical" pre-storage vs. post-storage PRT-treatment setting. Following whole blood centrifugation, separated plasma units were: (a) inactivated and frozen (pre-storage setting or control group [CG]) or (b) immediately frozen (post-storage setting or study group [SG]) afterward thawed, inactivated and stored at -40 ± 5°C (cryostorage). Plasma units were inactivated by the Mirasol PRT system (TerumoBCT, USA). Using multi-laboratory techniques and equipments, biochemistry (Advia 1800; Siemens, Germany), IgM, IgG and IgA, complement components C3 and C4 (BNA II nefelometer analyzer; Siemens, Germany), as well as CH50 activity (Behring coagulation timer; Siemens, Germany) were investigated. Procoagulant and inhibitor factors, such as antithrombin-III (AT-III), and protein C (PC) were determined by BCS XP Coagulation system (Siemens, Germany). There were neither significant changes in final protein levels, nor any differences in plasma immunoglobulin levels investigated. In the final samples CH50 activity was reduced in both investigated groups. The plasma concentration of the complement C3 following post-storage treatment was significantly (p<0.05) higher than in pre-storage setting. There was a trend of depletion of procoagulant activities in both, pre-storage and post-storage PRT-treatment (initial vs. final values), but there were no significant differences between two groups. Results confirmed that AT-III was significantly higher after post-storage inactivation. In conclusion, this study confirmed that there were not clinically relevant intergroup (pre-storage vs. post-storage PRT-treatment) differences in plasma constituent levels. Post-storage treated FFP remains, protein quantity, and activity well, and therefore can be used in clinical practice. Previously cryostored or quarantine FFP units (despite the reduced quarantine period after NAT/PCR testing) could be safely and effectively inactivated, directly prior to clinical application.

Inactivation of Plasmodium falciparum in whole blood by riboflavin plus irradiation

Malaria parasites are frequently transmitted by unscreened blood transfusions in Africa. Pathogen reduction methods in whole blood would thus greatly improve blood safety. We aimed to determine the efficacy of riboflavin plus irradiation for treatment of whole blood infected with Plasmodium falciparum. Blood was inoculated with 10(4) or 10(5) parasites/mL and riboflavin treated with or without ultraviolet (UV) irradiation (40-160 J/mL red blood cells [mL(RBCs)]). Parasite genome integrity was assessed by quantitative amplification inhibition assays, and P. falciparum viability was monitored in vitro. Riboflavin alone did not affect parasite genome integrity or parasite viability. Application of UV after riboflavin treatment disrupted parasite genome integrity, reducing polymerase-dependent amplification by up to 2 logs (99%). At 80 J/mL(RBCs), riboflavin plus irradiation prevented recovery of viable parasites in vitro for 2 weeks, whereas untreated controls typically recovered to approximately 2% parasitemia after 4 days of in vitro culture. Exposure of blood to 160 J/mL(RBCs) was not associated with significant hemolysis. Riboflavin plus irradiation treatment of whole blood damages parasite genomes and drastically reduces P. falciparum viability in vitro. In the absence of suitable malaria screening assays, parasite inactivation should be investigated for prevention of transfusion-transmitted malaria in highly endemic areas.

Ultra-violet irradiation and riboflavin treatment blocks capping of cellular adhesion molecules and abrogates immunogenicity of antigen presenting cells (P1388)

Abstract UV light has known immunomodulatory activity and is used in clinical situations such as for psoriasis. Mirasol treatment, a pathogen reduction technology based on ultra-violet light irradiation in the presence of riboflavin (UV+R), suppresses the ability of treated WBCs to stimulate proliferation of allogeneic T cells in vitro. Prior work showed that surface expression of adhesion molecules was down-regulated after UV+R treatment. We now show that, UV+ R treatment also decreased capping of the intercellular adhesion molecules (ICAMs)-1, -2, -3 and lymphocyte function-associated antigen (LFA)-3. UV+R treated U937 antigen presenting cells (APCs) induce decreased cell-cell conjugation with PBMCs 25% (p = 0.003) and show a 66% (p = 0.0005) reduction in stimulating T cell proliferation in a mixed lymphocyte reaction compared to untreated or gamma-irradiated U937 APCs. Additionally, UV+R treated U937 APCs failed to induce expression of the early T cell activation marker CD69 and also have decreased membrane viscosity when compared to untreated APCs. Diminished surface expression and perturbation of adhesion receptors within the U937 APCs plasma membrane down-regulates T cell stimulation and proliferation in vitro by lowering the surface expression of adhesion receptors needed for cell-cell conjugation and by perturbation of the ability to mobilize and aggregate adhesion receptors to form a cap, a critical component needed for T cell activation.

Riboflavin and ultraviolet light treatment of platelets triggers p38MAPK signaling: inhibition significantly improves in vitro platelet quality after pathogen reduction treatment

Pathogen reduction technologies (PRTs) significantly reduce the risk of transmission of infectious agents in platelet (PLT) concentrates; however, in vitro studies reveal a negative impact on PLT quality after PRT treatment including effects on PLT aggregation, integrin αIIbβ3 conformation, and actin dynamics. Clinically, the interval between transfusions is shortened. Seeking to understand the biochemical mechanisms underlying these observed effects, we analyzed signal transduction in PLT concentrates after riboflavin and ultraviolet light (UV; Mirasol) treatment and subsequent storage focusing on the phosphorylation levels of selected protein kinases. Among identified candidates, p38MAPK increased fourfold in phosphorylation after PRT. Incubation of PLT concentrates with a p38MAPK-specific inhibitor before PRT significantly improved numerous PLT quality measures. Phosphorylation levels of the p38MAPK substrates AKT, VASP, and HSP27 also decreased with inhibitor treatment. Phospho-HSP27 decrease in the presence of the inhibitor correlated with a reduction in PLT activation determined by surface expression of P-selectin. These findings support a model of one dominant underlying molecular signaling mechanism that is impacted by the riboflavin and UV (Mirasol) PRT process resulting in alterations in PLT quality. The identification of such a target should assist in the development of strategies to ameliorate this negative aspect of an otherwise beneficial and important safety development for transfusion medicine.

Reduction of Stromal Swelling Pressure after UVA-Riboflavin Cross-Linking

We evaluated whether UVA-riboflavin collagen cross-linking (CXL) reduces stromal swelling pressure (SP) in porcine and human corneas ex vivo. Porcine corneas (N = 35) were divided into five groups: Full-thickness buttons, riboflavin-treated buttons, CXL-treated buttons, riboflavin-treated anterior and posterior lamellae, and CXL-treated anterior and posterior lamellae. Riboflavin- or CXL-treated human corneas (n = 6) were cut into anterior and posterior lamellae. The force exerted by the specimens during swelling in saline was recorded and SP was calculated. Dry weights were obtained for solids correction. In full-thickness porcine buttons, a significantly reduced SP was observed after CXL (40.07 ± 3.86 mm Hg) compared to riboflavin treatment (68.13 ± 11.39 mm Hg, P = 0.02) at 5% compression. Also, a trend toward reduced SP in the CXL-treated anterior human lamellae (9.9 mm Hg) was found compared to the riboflavin group (25 mm Hg) at 5% compression. In the anterior porcine lamellae a significant SP reduction was observed in the CXL group versus the riboflavin group (P < 0.001, two-way ANOVA). Likewise, in the posterior porcine lamellae, a significant SP reduction was observed in the CXL group versus riboflavin alone (P < 0.05, two-way ANOVA). Posterior human lamellae did not differ in SP when comparing CXL and riboflavin groups. Our study demonstrated a significant reduction in anterior stromal SP in porcine and human corneas after CXL. This finding suggested that CXL may reduce corneal SP in vivo, and thereby reduce edema and improve vision. Thus, in the clinical setting, patients suffering from corneal edema may benefit from CXL treatment.

Immune modulation and lack of alloimmunization following transfusion with pathogen‐reduced platelets in mice

Transfusion of allogeneic blood products can lead to alloimmunization, impacting success of subsequent transfusions and solid organ transplants. Pathogen reduction using riboflavin and ultraviolet B (UVB) light has been shown to eliminate the immunogenicity of white blood cells (WBCs) in vitro through down regulation of surface adhesion molecules, effectively blocking cell-cell conjugation and direct presentation. We sought to determine if this loss of immunogenicity is extended in vivo where indirect presentation of allogeneic antigens can occur. BALB/cJ mice were transfused with either untreated or riboflavin and UVB-treated C57Bl/6J platelet-rich plasma (PRP) containing WBCs. Circulating alloantibody and allospecific splenocyte cytokine responses were measured. Pathogen reduction of allogeneic WBC-enriched PRP using riboflavin and UVB light before transfusion prevented alloimmunization, with a loss of both alloantibody generation and priming of secondary cytokine responses ex vivo. When mice given treated transfusions were subsequently given untreated transfusions, they produced normal levels of alloantibodies but had reduced secondary cytokine responses ex vivo. This immune modulation was antigen specific and was dependent on the presence of WBCs in the treated product. UVB plus riboflavin treatment of WBC-enriched PRP effectively blocks alloimmunization and modulates immune responses to subsequent exposures.

Role of Corneal Epithelium in Riboflavin/Ultraviolet-A Mediated Corneal Cross-Linking Treatment in Rabbit Eyes

Purpose. To evaluate the role of corneal epithelium in riboflavin/ultraviolet-A (UVA) mediated corneal collagen cross-linking treatment. Methods. Fifty New Zealand rabbits were divided into 5 groups: UVA treatment with or without corneal epithelium, UVA+riboflavin treatment with or without corneal epithelium, and control without any treatment. All rabbits were sacrificed after irradiation and subsequently 4 mm × 10 mm corneal strips were harvested for biomechanical evaluation. Results. UVA irradiation alone did not enhance the maximal stress and Young's modulus of corneal specimens with (3.15 ± 0.56 mpa, 1.00 ± 0.09 mpa) or without (3.53 ± 0.85 mpa, 0.94 ± 0.21 mpa) the corneal epithelium, compared to specimens in the control group (4.30 ± 0.68 mpa, 1.03 ± 0.24 mpa). However, UVA irradiation combined with riboflavin significantly increased the maximal stress and Young's modulus of corneal specimens with (5.27 ± 1.09 mpa, 1.23 ± 0.23 mpa, P < 0.05) or without (7.16 ± 1.88 mpa, 1.42 ± 0.16 mpa, P < 0.05) corneal epithelium when compared to the control group. The maximal stress and Young's modulus of cornea in UVA+riboflavin and “epithelium-off” group were 35.9% and 15.4% higher compared to the UVA+riboflavin and “epithelium-on” group, respectively (P < 0.05). Conclusions. Our study shows that UVA+riboflavin treatment significantly affects the biomechanical properties of the cornea with and without epithelial removal. However, corneas without epithelium seem to benefit more compared to corneas with the epithelium.