In vitro thrombus formation and in vivo hemostasis mediated by platelets irradiated with bactericidal ultraviolet C from xenon flash under flow conditions.

BACKGROUND: We previously reported a flow path-ultraviolet C (UVC) irradiation system for platelet concentrates (PCs) with platelet additive solution (PAS) to minimize contamination by bacteria. Here, we investigated functionalities of irradiated platelets (PLTs) in in vitro thrombus formation and in vivo hemostasis. STUDY DESIGN AND METHODS: PAS-PCs were irradiated with flash UVC using the flow path system. Their variables (PLT count, mean platelet volume, pH, glucose, lactate, glycoprotein [GP] Ib, and activated integrin αIIbß3) were evaluated. Static adhesion to collagen or fibrinogen was analyzed using fluorescent microscopy. Thrombus formation under flow conditions was assessed using a collagen-coated bead column. Adenosine diphosphate (ADP)-induced Akt phosphorylation was determined by western blot. In vivo hemostasis and circulatory survival of PLTs were assessed with a rabbit bleeding model. RESULTS: All variables, except for GPIb expression, were slightly, but significantly, impaired after flash UVC irradiation throughout the 6-day storage period. No difference was observed in static adhesion to either collagen or fibrinogen between irradiated and nonirradiated PAS-PCs. In vitro thrombus formation of flash UVC-irradiated PAS-PCs was significantly greater than that of nonirradiated PAS-PCs. ADP-induced Akt phosphorylation was enhanced in irradiated PAS-PCs. In vivo hemostatic efficacy was comparable between the groups on Day 1. The efficacy declined in nonirradiated PAS-PCs on Day 5, while it was retained in flash UVC-irradiated PAS-PCs. Circulatory survival of PLTs was lower in irradiated PAS-PCs. CONCLUSIONS: PAS-PCs irradiated with UVC from xenon flash have favorable properties to achieve hemostasis compared with nonirradiated PAS-PCs.

Comprehensive analysis of transcriptional profiles in oral epithelial-like cells stimulated with oral probiotic Lactobacillus spp.

OBJECTIVE: The mechanisms of action of probiotics can vary among species and among strains of a single species; thus, they can affect host cells in a complex manner. In the present study, Lactobacillus spp. were evaluated for their ability to adhere to gingival epithelial-like cells. Comprehensive analyses of transcriptional profiles of mouse gingival epithelial GE1 cells treated with L. rhamnosus L8020 were performed to assess the putative in vivo probiotic potential of this strain. METHODS: Five Lactobacillus spp., isolated from the oral cavity, traditional Bulgarian yoghurt, and the feces of a healthy human, were each co-cultured with GE1 cells. Adhesion assays with serial dilution plating and DNA microarray analysis were performed to identify differentially expressed genes (DEGs) in GE1 cells grown in co-culture with L. rhamnosus L8020. RESULTS: The oral isolates L. rhamnosus L8020, L. casei YU3, and L. paracasei YU4 demonstrated significantly greater adhesion compared with the non-oral isolates. In total, 536 genes in GE1 cells exhibited more than twofold upregulation or downregulation, compared with the 0 h timepoint, during co-culture with L. rhamnosus L8020. Gene ontology enrichment analysis revealed that DEGs were differentially enriched in a time-dependent manner. Early responses involved widespread changes in gene expression. CONCLUSIONS: This study reveals changes in expression of genes involved in the epithelial physical barrier and immune response in gingival epithelial-like cells co-cultured with L. rhamnosus L8020. Further investigations regarding the molecular mechanisms by which L. rhamnosus L8020 serves as a probiotic may provide evidence to support clinical use.

Flow path system of ultraviolet C irradiation from xenon flash to reduce bacteria survival in platelet products containing a platelet additive solution.

BACKGROUND: Our previous study showed that ultraviolet C (UVC) from xenon (Xe) flash without any photoreactive compounds inactivated bacteria in platelet concentrates (PCs) with less damage to platelets (PLTs) as compared with Xe flash containing ultraviolet A, ultraviolet B, and visible light. Here, we report a UVC irradiation system for PCs under flow conditions consisting of a flow path-irradiation sheet, a peristaltic pump, and a collection bag. STUDY DESIGN AND METHODS: Platelet concentrates containing Ringer's solution (R-PCs) inoculated with bacteria were injected into a flow path sheet using a peristaltic pump, being irradiated with UVC from Xe flash. The quality of the irradiated PCs containing platelet additive solution (PAS-PCs) was assessed based on PC variables, PLT surface markers, and aggregation ability. RESULTS: Streptococcus dysgalactiae (12 tests) and Escherichia coli (11) were all negative on bacterial culture, while Staphylococcus aureus (12) and Klebsiella pneumoniae (14) grew in one and two R-PCs, respectively. Bacillus cereus spores were inactivated in 7 of 12 R-PCs. PC variables became significantly different between irradiated and nonirradiated PAS-PCs. P-selectin, first procaspase-activating compound (PAC-1) binding, and phosphatidylserine increased by irradiation. Aggregability stimulated by adenosine diphosphate, collagen, or thromboxane A2 increased in the irradiated PAS-PCs, while that by thrombin became smaller compared with nonirradiated controls. CONCLUSION: This newly developed system inactivated bacteria including spores in R-PCs. PAS-PCs irradiated by this system retained acceptable in vitro quality and aggregability. Usage of a peristaltic pump instead of agitator during irradiation may enable this system to be directly combined with an apheresis blood cell separator.

Correlation between platelet thrombus formation on collagen-coated beads and platelet aggregation induced by ADP.

BACKGROUND: The thrombus-forming ability is a critical in vitro parameter to assess platelets (PLTs), but flow-based methods using collagen-coated materials generally require multistep, proficiency, and advanced analysis. STUDY DESIGN AND METHODS: Commercially available collagen-coated bead columns were examined to assess thrombus-forming ability of PLTs. The retention rate as an index of thrombus formation was calculated using the PLT count before and after column passage. Thrombi were imaged by anti-CD41 using a fluorescent microscope. PLT aggregation was measured by light-transmitting aggregometry. RESULTS: The retention rate was low when apheresis-collected PLT concentrates (PCs) were suspended in plasma either with or without Ca2+. Reconstitution of PCs with red blood cells (RBCs) increased the retention rate with good reproducibility on repeated-measurements, and therefore, PLT samples were reconstructed with RBCs in subsequent experiments. The retention rate of PCs varied widely in a product-dependent manner, and was correlated with the aggregation rate induced by ADP, but not that by collagen. Using platelet-rich-plasma, antagonists of P2Y1 or P2Y12 receptors for ADP reduced both the retention and aggregation of PLTs. Acetylsalicylic acid reduced retention, although it had no effect on ADP-induced aggregation. Prostaglandin E1 significantly inhibited both retention and aggregation. These anti-PLT reagents resulted in reduced or no thrombus formation on the beads. CONCLUSION: The collagen-coated bead column was useful to readily examine the thrombus-forming ability of PLTs. Variance of the PLT retention rate was correlated with responsiveness to ADP. Results from anti-PLT reagents revealed that thrombus formation on collagen-coated beads was similar to in vivo thrombus development.

Impact of the platelet washing process on in vitro platelet properties, and the levels of soluble CD40 ligand and platelet-derived microparticles in the storage media.

BACKGROUND: A new platelet (PLT) additive solution, bicarbonated Ringer's solution supplemented with acid-citrate-dextrose Formula A, termed BRS-A, as well as a new automated closed system cell processor for washing PLTs have recently been developed. This study evaluated the in vitro properties of PLTs with the automated system versus the manual method, using the BRS-A additive solution for washing and storage. METHODS: ABO-identical apheresis PLTs in 100% plasma were pooled and split equally for control (in 100% plasma or a manual method) and test (ACP215 automated system) units. In vitro characteristics of PLTs washed with the automated system were compared to those of PLTs in 100% plasma (Study 1) or washed with a manual method (Study 2) during the 7-day storage. RESULTS: In Study 1, hypotonic shock response, aggregation response, mitochondrial membrane potential, adenosine triphosphate, and CD42b mean fluorescence intensity were comparable in the control and test groups during the 7-day storage. CD62P expression was lower in the test group than controls on Days 3 and 7. The level of platelet-derived microparticles (PDMPs) in the test group on Days 1 and 2 were higher than those in controls. In contrast, the levels of soluble CD40 ligand (sCD40L) and regulated upon activation of normal T-cell expressed and secreted (RANTES) in the test units were lower than controls. In Study 2, no significant differences were found in all in vitro properties except for PLT count and the levels of PDMPs in the test units were higher than controls during storage. CONCLUSION: Apheresis PLTs washed with the automated system using BRS-A additive solution maintained in vitro properties during storage. Washing methods influenced PDMP levels but not sCD40L and RANTES.