Rosalie Devloo - Academia.edu (original) (raw)

Papers by Rosalie Devloo

Research paper thumbnail of A novel competition ELISA for the rapid quantification of SARS‐CoV ‐2 neutralizing antibodies in convalescent plasma

Research paper thumbnail of The senotherapeutic nicotinamide riboside raises platelet nicotinamide adenine dinucleotide levels but cannot prevent storage lesion

Transfusion

BACKGROUND: Supplementation of the nicotinamide adenine dinucleotide (NAD) precursor nicotinamide... more BACKGROUND: Supplementation of the nicotinamide adenine dinucleotide (NAD) precursor nicotinamide riboside (NR) has recently been shown to increase lifespan of cells, tissues, and entire organisms. [Correction added on 13 December 2019, after first online publication: In the preceding sentence, "adenine nicotinamide" was revised to "nicotinamide adenine."] The impact of NR on platelet longevity has not been tested. STUDY DESIGN AND METHODS: A pool-and-split design of buffy coat derived platelet concentrates (PCs) was used. One arm was treated with cumulative doses of NR-triflate, the control arm with sodium triflate. Storage lesion was monitored for 23 days. Platelet metabolic and functional parameters were tested. Clearance of human platelets was measured in a mouse model of transfusion. RESULTS: Total intracellular NAD levels in platelets decreased twofold from 4.8 AE 0.5 fmol (mean AE SD, n = 6) to 2.1 AE 1.8 fmol per 10 3 control cells, but increased almost 10-fold to 41.5 AE 4.1 fmol per 10 3 NR treated platelets. This high intracellular NAD level had no significant impact on platelet count, mean platelet volume, swirling, nor on lactate and glucose levels. Platelet aggregation and integrin α IIb β 3 activation declined steadily and comparably in both conditions. GPIbα levels were slightly lower in NR-treated platelets compared to control, but this was not caused by reduced receptor shedding because glycocalicin increased similarly. Apoptotic markers cytochrome c, Bcl-xL, cleaved caspase-3, and Bak were not different throughout storage for both conditions. Platelet survival in a mouse model of transfusion was not different between NR-treated and control platelets. CONCLUSION: Platelets carry the cellular machinery to metabolize NR into NAD at rates comparable to other eukaryotic cells. Unlike those cells, platelet lifespan cannot be prolonged using this strategy.

Research paper thumbnail of The ice recrystallization inhibitor polyvinyl alcohol does not improve platelet cryopreservation

Research paper thumbnail of Impact of cold storage on platelets treated with Intercept pathogen inactivation

Transfusion

BACKGROUND: Pathogen inactivation and cold or cryopreservation of platelets (PLTs) both significa... more BACKGROUND: Pathogen inactivation and cold or cryopreservation of platelets (PLTs) both significantly affect PLT function. It is not known how PLTs function when both are combined. STUDY DESIGN AND METHODS: Standard PLT concentrates (PCs) were compared to pathogeninactivated PCs treated with amotosalen photochemical treatment (AS-PCT) when stored at room (RT, 22 C), cold (4 C, n = 6), or cryopreservation (−80 C, n = 8) temperatures. The impact of alternative storage methods on both arms was studied in flow cytometry, light transmittance aggregometry, and hemostasis in collagen-coated microfluidic flow chambers. RESULTS: Platelet aggregation of cold-stored AS-PCT PLTs was 44% AE 11% compared to 57% AE 14% for coldstored standard PLTs and 58% AE 21% for RT-stored AS-PCT PLTs. Integrin activation of cold-stored AS-PCT PLTs was 53% AE 9% compared to 77% AE 6% for cold-stored standard PLTs and 69% AE 13% for RT-stored AS-PCT PLTs. Coagulation of cold-stored AS-PCT PLTs started faster under flow (836 AE 140 sec) compared to cold-stored standard PLTs (960 AE 192 sec) and RT-stored AS-PCT PLTs (1134 AE 220 sec). Fibrin formation rate under flow was also highest for cold-stored AS-PCT PLTs. This was in line with thrombin generation in static conditions because cold-stored AS-PCT PLTs generated 297 AE 47 nmol/L thrombin compared to 159 AE 33 nmol/L for cold-stored standard PLTs and 83 AE 25 nmol/L for RTstored AS-PCT PLTs. So despite decreased PLT activation and aggregation, cold storage of AS-PCT PLTs promoted coagulation. PLT aggregation of cryopreserved AS-PCT PLTs (23% AE 10%) was not significantly different from cryopreserved standard PLTs (25% AE 8%). CONCLUSION: This study shows that cold storage of AS-PCT PLTs further affects PLT activation and aggregation but promotes (pro)coagulation. Increased procoagulation was not observed after cryopreservation. C onventional, room temperature (RT) storage of platelets (PLTs) limits shelf life to between 4 and 7 days because of the risk of bacterial contamination and the decrease in PLT function called platelet storage lesion. 1 The limited shelf life of PLT concentrates (PCs) hampers efficient inventory management and puts pressure on blood banks trying to balance risks of shortage with risks of wastage. Prolonging PC shelf life could overcome this problem and has been investigated for decades with a focus on cold storage (1-6 C) 2 and cryopreservation (−80 to −196 C). 3,4 Cold storage can potentially increase PC shelf life beyond 7 days of storage. 5 Several reports indicate that cold-stored PLTs are functionally "primed" suggesting that these are in a state of heightened responsiveness to hemostatic stimuli. 5-7 The main obstacle for cold-stored PLTs is the significantly faster clearance from circulation compared to RT storage. 2,8,9 This may be less relevant in acutely bleeding patients who

Research paper thumbnail of A comparison of haematopoietic stem cells from umbilical cord blood and peripheral blood for platelet production in a microfluidic device

Vox Sanguinis

Background and objectives Several sources of haematopoietic stem cells have been used for static ... more Background and objectives Several sources of haematopoietic stem cells have been used for static culture of megakaryocytes to produce platelets in vitro. This study compares and characterizes platelets produced in shear flow using precursor cells from either umbilical (UCB) or adult peripheral blood (PB). Materials and methods The efficiency of platelet production of the cultured cells was studied after perfusion in custom-built von Willebrand factor-coated microfluidic flow chambers. Platelet receptor expression and morphology were investigated by flow cytometry and microscopy, respectively. Results Proliferation of stem cells isolated out of UCB was significantly higher (P < 0Á0001) compared to PB. Differentiation of these cells towards megakaryocytes was significantly lower from PB compared to UCB where the fraction of CD42b/CD41 double positive events was 44-9% versus 76-11%, respectively (P < 0Á0001). However, in vitro platelet production under hydrodynamic conditions was more efficient with 7Á4 platelet-like particles per input cell from PB compared to 4Á2 from UCB (P = 0Á02). The percentage of events positive for CD42b, CD41 and CD61 was comparable between both stem cell sources. The mean number of receptors per platelet from UCB and PB was similar to that on blood bank platelets with on average 28 000 CD42b, 57 000 CD61 and 5500 CD49b receptors. Microscopy revealed platelets appearing similar to blood bank platelets in morphology, size and actin cytoskeleton, alongside smaller fragments and source megakaryocytes. Conclusion This characterization study suggests that platelets produced in vitro under flow either from UCB or from PB share receptor expression and morphology with donor platelets stored in the blood bank.

Research paper thumbnail of A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Journal of visualized experiments : JoVE, Feb 14, 2017

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear... more Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca(2+)-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca(2+)/Mg(2+) buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked co...

Research paper thumbnail of UVC PI treatment of PCs preserves integrin activation but affects trhombus formation kinetics on collagen in vitro (supp)

Research paper thumbnail of Oral administration of the Salmonella Typhimurium vaccine strain Nal2/Rif9/Rtt to laying hens at day of hatch reduces shedding and caecal colonization of Salmonella 4,12:i:-, the monophasic variant of Salmonella Typhimurium

Poultry science, Jan 29, 2015

A new monophasic variant of Salmonella Typhimurium, Salmonella enterica serotype 4,12:i:-, is rap... more A new monophasic variant of Salmonella Typhimurium, Salmonella enterica serotype 4,12:i:-, is rapidly emerging. This serotype is now considered to be among the 10 most common serovars isolated from humans in many countries in Europe and in the United States. The public health risk posed by these emerging monophasic Salmonella Typhimurium strains is considered comparable to that of classical Salmonella Typhimurium strains. The serotype 4,12:i:- is frequently isolated from pigs but also poultry are carrying strains from this serotype. In the current study, we evaluated the efficacy of the Salmonella Typhimurium strain Nal2/Rif9/Rtt, a strain contained in the commercially available live vaccines AviPro Salmonella Duo and AviPro Salmonella VacT, against infection with the emerging monophasic variant in poultry. Three independent trials were conducted. In all trials, laying type chicks were orally vaccinated with the Salmonella Typhimurium strain Nal2/Rif9/Rtt at d hatch, while the birds...

Research paper thumbnail of Persistent aggregates in apheresis platelet concentrates

Vox sanguinis, Jan 6, 2015

Aggregates often appear during apheresis. Sometimes, these persist throughout storage, causing pr... more Aggregates often appear during apheresis. Sometimes, these persist throughout storage, causing product wastage. This study assessed product quality of apheresis concentrates containing persistent aggregates (PA) and aimed to identify the factors that contribute to their formation. Donation (n = 180) and platelet indices (n ≥ 10) from apheresis concentrates with PA were compared with aggregate-free products. The proportion of donors with at least one previous PA donation was twofold higher in the PA group (P < 0·0001) indicating a donor dependence. Significantly higher donor whole blood platelet counts (286 ± 50 vs. 266 ± 49 × 10(3) /μl, P < 0·0001) and higher apheresis yields (6·0 ± 1·6 vs. 5·4 ± 1·5 × 10(11) , P < 0·0001) were noted in the PA group. Haematocrit was also slightly higher, but age, gender and body mass were similar. The pH of PA products on day six postdonation was significantly lower (P < 0·001), in line with higher lactic acid concentrations. Flow cytome...

Research paper thumbnail of Psoralen and Ultraviolet A Light Treatment Directly Affects Phosphatidylinositol-tris Kinase Signal Transduction by Altering Plasma Membrane Packing

Psoralen and ultraviolet A light (PUVA) is used to kill pathogens in blood products and as a trea... more Psoralen and ultraviolet A light (PUVA) is used to kill pathogens in blood products and as a treatment of aberrant cell proliferation in dermatitis, cutaneous T-cell lymphoma and graft-versus-host-disease (GVHD). DNA damage is well described, but the direct effects of PUVA on cell signal transduction are poorly understood. Because platelets are anucleate and contain archetypal signal transduction machinery these are ideally suited to address this. Lipidomics on platelet membrane extracts showed that psoralen forms adducts with unsaturated carbon bonds of fatty acyls in all major phospholipid classes after PUVA. Such adducts increased lipid packing as measured by a blue shift of an environment-sensitive fluorescent probe in model liposomes. Furthermore, the interaction of these liposomes with lipid order-sensitive proteins like ALPS and α-synuclein was inhibited by PUVA. In platelets, PUVA caused poor membrane binding of Akt and Bruton’s tyrosine kinase effectors following activation of the collagen glycoprotein VI (GPVI) and thrombin protease activated receptor (PAR) 1. This resulted in defective Akt phosphorylation despite unaltered phosphatidylinositol (3,4,5)-trisphosphate levels. Downstream integrin activation was furthermore affected similarly by PUVA following PAR1 (EC50 8.4±1.1 μM vs. 4.3±1.1 μM) and GPVI (EC50 1.61±0.85 μg/mL vs. 0.26±0.21 μg/mL) but not PAR4 (EC50 50±1 μM vs 58±1 μM) signal transduction. Our findings were confirmed in T-cells from GVHD patients treated with extracorporeal photopheresis (ECP), a form of systemic PUVA. In conclusion, PUVA increases the order of lipid phases by covalent modification of phospholipids thereby inhibiting membrane recruitment of effector kinases.

Research paper thumbnail of Oxygen removal during pathogen inactivation with riboflavin and UV light preserves protein function in plasma for transfusion

Vox Sanguinis, 2014

Background and Objective Photochemical pathogen inactivation technologies (PCT) for individual tr... more Background and Objective Photochemical pathogen inactivation technologies (PCT) for individual transfusion products act by inhibition of replication through irreversibly damaging nucleic acids. Concern on the collateral impact of PCT on the blood component's integrity has caused reluctance to introduce this technology in routine practice. This work aims to uncover the mechanism of damage to plasma constituents by riboflavin pathogen reduction technology (RF-PRT).

Research paper thumbnail of Ultraviolet c light pathogen inactivation treatment of platelet concentrates preserves integrin activation but affects thrombus formation kinetics on collagen in vitro

Research paper thumbnail of Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Plate... more Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Research paper thumbnail of Riboflavin and amotosalen photochemical treatments of platelet concentrates reduce thrombus formation kinetics in vitro

Background Photochemical treatment (PCT) of platelet concentrates using photosensitizers and ultr... more Background Photochemical treatment (PCT) of platelet concentrates using photosensitizers and ultraviolet light illumination reduces the proliferation potential of pathogens by damaging biomolecules.

Research paper thumbnail of Oxygen removal during pathogen inactivation with riboflavin and UV light preserves protein function in plasma for transfusion.

Background and Objective Photochemical pathogen inactivation technologies (PCT) for individual tr... more Background and Objective Photochemical pathogen inactivation technologies (PCT) for individual transfusion products act by inhibition of replication through irreversibly damaging nucleic acids. Concern on the collateral impact of PCT on the blood component's integrity has caused reluctance to introduce this technology in routine practice. This work aims to uncover the mechanism of damage to plasma constituents by riboflavin pathogen reduction technology (RF-PRT).

Research paper thumbnail of Bearded dragons (Pogona vitticeps) asymptomatically infected with Devriesea agamarum are a source of persistent clinical infection in captive colonies of dab lizards (Uromastyx sp.).

Research paper thumbnail of Internal contamination of eggs by Salmonella Enteritidis

Improving the safety …, 2011

Research paper thumbnail of Salmonella Enteritidis universal stress protein (usp) gene expression is stimulated by egg white and supports oviduct colonization and egg contamination in laying hens.

Veterinary …, 2011

Salmonella enterica subspecies enterica serovar Enteritidis has caused a worldwide egg-associated... more Salmonella enterica subspecies enterica serovar Enteritidis has caused a worldwide egg-associated pandemic since the mid 1980s. The exact mechanisms causing this egg tropism are still largely unknown, and only a few Salmonella genes have been implicated in the interaction with the oviduct or eggs. A in vivo expression technology screening performed previously, identified the uspA and uspB genes as being highly expressed in the chicken oviduct and in eggs. Here, we demonstrate that uspA and uspB gene expression is indeed induced after contact with egg white. Intra-oviduct inoculation of Salmonella Enteritidis uspB and uspBA mutant strains showed that the mutants had a decreased ability to colonize the magnum and isthmus of the oviduct, the organs that produce the egg white and eggshell membranes, respectively, at 7 days post-inoculation. Intravenous challenge showed that a Salmonella Enteritidis uspBA mutant strain had a decreased ability to contaminate eggs. Analogous to the function of universal stress proteins A and B in other bacterial species, we hypothesize that the Salmonella uspA and uspB genes are involved in long term persistence of Salmonella Enteritidis in harmful environments, such as in the oviduct and eggs, by conferring resistance against compounds that damage the bacterial cell membrane and DNA.

Research paper thumbnail of The effect of starch gelatinisation degree on intestinal morphology, intestinal pH and bacteriology in pigeons

Journal of Animal …, 2011

Starch is the main carbohydrate in most domestic animal diets. It is a major component of vegetal... more Starch is the main carbohydrate in most domestic animal diets. It is a major component of vegetal feedstuffs, offering a range of desired technological properties, in particular related to texturizing ability. The nutritional quality of starch strongly depends on its physical form and hence the

Research paper thumbnail of A novel competition ELISA for the rapid quantification of SARS‐CoV ‐2 neutralizing antibodies in convalescent plasma

Research paper thumbnail of The senotherapeutic nicotinamide riboside raises platelet nicotinamide adenine dinucleotide levels but cannot prevent storage lesion

Transfusion

BACKGROUND: Supplementation of the nicotinamide adenine dinucleotide (NAD) precursor nicotinamide... more BACKGROUND: Supplementation of the nicotinamide adenine dinucleotide (NAD) precursor nicotinamide riboside (NR) has recently been shown to increase lifespan of cells, tissues, and entire organisms. [Correction added on 13 December 2019, after first online publication: In the preceding sentence, "adenine nicotinamide" was revised to "nicotinamide adenine."] The impact of NR on platelet longevity has not been tested. STUDY DESIGN AND METHODS: A pool-and-split design of buffy coat derived platelet concentrates (PCs) was used. One arm was treated with cumulative doses of NR-triflate, the control arm with sodium triflate. Storage lesion was monitored for 23 days. Platelet metabolic and functional parameters were tested. Clearance of human platelets was measured in a mouse model of transfusion. RESULTS: Total intracellular NAD levels in platelets decreased twofold from 4.8 AE 0.5 fmol (mean AE SD, n = 6) to 2.1 AE 1.8 fmol per 10 3 control cells, but increased almost 10-fold to 41.5 AE 4.1 fmol per 10 3 NR treated platelets. This high intracellular NAD level had no significant impact on platelet count, mean platelet volume, swirling, nor on lactate and glucose levels. Platelet aggregation and integrin α IIb β 3 activation declined steadily and comparably in both conditions. GPIbα levels were slightly lower in NR-treated platelets compared to control, but this was not caused by reduced receptor shedding because glycocalicin increased similarly. Apoptotic markers cytochrome c, Bcl-xL, cleaved caspase-3, and Bak were not different throughout storage for both conditions. Platelet survival in a mouse model of transfusion was not different between NR-treated and control platelets. CONCLUSION: Platelets carry the cellular machinery to metabolize NR into NAD at rates comparable to other eukaryotic cells. Unlike those cells, platelet lifespan cannot be prolonged using this strategy.

Research paper thumbnail of The ice recrystallization inhibitor polyvinyl alcohol does not improve platelet cryopreservation

Research paper thumbnail of Impact of cold storage on platelets treated with Intercept pathogen inactivation

Transfusion

BACKGROUND: Pathogen inactivation and cold or cryopreservation of platelets (PLTs) both significa... more BACKGROUND: Pathogen inactivation and cold or cryopreservation of platelets (PLTs) both significantly affect PLT function. It is not known how PLTs function when both are combined. STUDY DESIGN AND METHODS: Standard PLT concentrates (PCs) were compared to pathogeninactivated PCs treated with amotosalen photochemical treatment (AS-PCT) when stored at room (RT, 22 C), cold (4 C, n = 6), or cryopreservation (−80 C, n = 8) temperatures. The impact of alternative storage methods on both arms was studied in flow cytometry, light transmittance aggregometry, and hemostasis in collagen-coated microfluidic flow chambers. RESULTS: Platelet aggregation of cold-stored AS-PCT PLTs was 44% AE 11% compared to 57% AE 14% for coldstored standard PLTs and 58% AE 21% for RT-stored AS-PCT PLTs. Integrin activation of cold-stored AS-PCT PLTs was 53% AE 9% compared to 77% AE 6% for cold-stored standard PLTs and 69% AE 13% for RT-stored AS-PCT PLTs. Coagulation of cold-stored AS-PCT PLTs started faster under flow (836 AE 140 sec) compared to cold-stored standard PLTs (960 AE 192 sec) and RT-stored AS-PCT PLTs (1134 AE 220 sec). Fibrin formation rate under flow was also highest for cold-stored AS-PCT PLTs. This was in line with thrombin generation in static conditions because cold-stored AS-PCT PLTs generated 297 AE 47 nmol/L thrombin compared to 159 AE 33 nmol/L for cold-stored standard PLTs and 83 AE 25 nmol/L for RTstored AS-PCT PLTs. So despite decreased PLT activation and aggregation, cold storage of AS-PCT PLTs promoted coagulation. PLT aggregation of cryopreserved AS-PCT PLTs (23% AE 10%) was not significantly different from cryopreserved standard PLTs (25% AE 8%). CONCLUSION: This study shows that cold storage of AS-PCT PLTs further affects PLT activation and aggregation but promotes (pro)coagulation. Increased procoagulation was not observed after cryopreservation. C onventional, room temperature (RT) storage of platelets (PLTs) limits shelf life to between 4 and 7 days because of the risk of bacterial contamination and the decrease in PLT function called platelet storage lesion. 1 The limited shelf life of PLT concentrates (PCs) hampers efficient inventory management and puts pressure on blood banks trying to balance risks of shortage with risks of wastage. Prolonging PC shelf life could overcome this problem and has been investigated for decades with a focus on cold storage (1-6 C) 2 and cryopreservation (−80 to −196 C). 3,4 Cold storage can potentially increase PC shelf life beyond 7 days of storage. 5 Several reports indicate that cold-stored PLTs are functionally "primed" suggesting that these are in a state of heightened responsiveness to hemostatic stimuli. 5-7 The main obstacle for cold-stored PLTs is the significantly faster clearance from circulation compared to RT storage. 2,8,9 This may be less relevant in acutely bleeding patients who

Research paper thumbnail of A comparison of haematopoietic stem cells from umbilical cord blood and peripheral blood for platelet production in a microfluidic device

Vox Sanguinis

Background and objectives Several sources of haematopoietic stem cells have been used for static ... more Background and objectives Several sources of haematopoietic stem cells have been used for static culture of megakaryocytes to produce platelets in vitro. This study compares and characterizes platelets produced in shear flow using precursor cells from either umbilical (UCB) or adult peripheral blood (PB). Materials and methods The efficiency of platelet production of the cultured cells was studied after perfusion in custom-built von Willebrand factor-coated microfluidic flow chambers. Platelet receptor expression and morphology were investigated by flow cytometry and microscopy, respectively. Results Proliferation of stem cells isolated out of UCB was significantly higher (P < 0Á0001) compared to PB. Differentiation of these cells towards megakaryocytes was significantly lower from PB compared to UCB where the fraction of CD42b/CD41 double positive events was 44-9% versus 76-11%, respectively (P < 0Á0001). However, in vitro platelet production under hydrodynamic conditions was more efficient with 7Á4 platelet-like particles per input cell from PB compared to 4Á2 from UCB (P = 0Á02). The percentage of events positive for CD42b, CD41 and CD61 was comparable between both stem cell sources. The mean number of receptors per platelet from UCB and PB was similar to that on blood bank platelets with on average 28 000 CD42b, 57 000 CD61 and 5500 CD49b receptors. Microscopy revealed platelets appearing similar to blood bank platelets in morphology, size and actin cytoskeleton, alongside smaller fragments and source megakaryocytes. Conclusion This characterization study suggests that platelets produced in vitro under flow either from UCB or from PB share receptor expression and morphology with donor platelets stored in the blood bank.

Research paper thumbnail of A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Journal of visualized experiments : JoVE, Feb 14, 2017

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear... more Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca(2+)-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca(2+)/Mg(2+) buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked co...

Research paper thumbnail of UVC PI treatment of PCs preserves integrin activation but affects trhombus formation kinetics on collagen in vitro (supp)

Research paper thumbnail of Oral administration of the Salmonella Typhimurium vaccine strain Nal2/Rif9/Rtt to laying hens at day of hatch reduces shedding and caecal colonization of Salmonella 4,12:i:-, the monophasic variant of Salmonella Typhimurium

Poultry science, Jan 29, 2015

A new monophasic variant of Salmonella Typhimurium, Salmonella enterica serotype 4,12:i:-, is rap... more A new monophasic variant of Salmonella Typhimurium, Salmonella enterica serotype 4,12:i:-, is rapidly emerging. This serotype is now considered to be among the 10 most common serovars isolated from humans in many countries in Europe and in the United States. The public health risk posed by these emerging monophasic Salmonella Typhimurium strains is considered comparable to that of classical Salmonella Typhimurium strains. The serotype 4,12:i:- is frequently isolated from pigs but also poultry are carrying strains from this serotype. In the current study, we evaluated the efficacy of the Salmonella Typhimurium strain Nal2/Rif9/Rtt, a strain contained in the commercially available live vaccines AviPro Salmonella Duo and AviPro Salmonella VacT, against infection with the emerging monophasic variant in poultry. Three independent trials were conducted. In all trials, laying type chicks were orally vaccinated with the Salmonella Typhimurium strain Nal2/Rif9/Rtt at d hatch, while the birds...

Research paper thumbnail of Persistent aggregates in apheresis platelet concentrates

Vox sanguinis, Jan 6, 2015

Aggregates often appear during apheresis. Sometimes, these persist throughout storage, causing pr... more Aggregates often appear during apheresis. Sometimes, these persist throughout storage, causing product wastage. This study assessed product quality of apheresis concentrates containing persistent aggregates (PA) and aimed to identify the factors that contribute to their formation. Donation (n = 180) and platelet indices (n ≥ 10) from apheresis concentrates with PA were compared with aggregate-free products. The proportion of donors with at least one previous PA donation was twofold higher in the PA group (P < 0·0001) indicating a donor dependence. Significantly higher donor whole blood platelet counts (286 ± 50 vs. 266 ± 49 × 10(3) /μl, P < 0·0001) and higher apheresis yields (6·0 ± 1·6 vs. 5·4 ± 1·5 × 10(11) , P < 0·0001) were noted in the PA group. Haematocrit was also slightly higher, but age, gender and body mass were similar. The pH of PA products on day six postdonation was significantly lower (P < 0·001), in line with higher lactic acid concentrations. Flow cytome...

Research paper thumbnail of Psoralen and Ultraviolet A Light Treatment Directly Affects Phosphatidylinositol-tris Kinase Signal Transduction by Altering Plasma Membrane Packing

Psoralen and ultraviolet A light (PUVA) is used to kill pathogens in blood products and as a trea... more Psoralen and ultraviolet A light (PUVA) is used to kill pathogens in blood products and as a treatment of aberrant cell proliferation in dermatitis, cutaneous T-cell lymphoma and graft-versus-host-disease (GVHD). DNA damage is well described, but the direct effects of PUVA on cell signal transduction are poorly understood. Because platelets are anucleate and contain archetypal signal transduction machinery these are ideally suited to address this. Lipidomics on platelet membrane extracts showed that psoralen forms adducts with unsaturated carbon bonds of fatty acyls in all major phospholipid classes after PUVA. Such adducts increased lipid packing as measured by a blue shift of an environment-sensitive fluorescent probe in model liposomes. Furthermore, the interaction of these liposomes with lipid order-sensitive proteins like ALPS and α-synuclein was inhibited by PUVA. In platelets, PUVA caused poor membrane binding of Akt and Bruton’s tyrosine kinase effectors following activation of the collagen glycoprotein VI (GPVI) and thrombin protease activated receptor (PAR) 1. This resulted in defective Akt phosphorylation despite unaltered phosphatidylinositol (3,4,5)-trisphosphate levels. Downstream integrin activation was furthermore affected similarly by PUVA following PAR1 (EC50 8.4±1.1 μM vs. 4.3±1.1 μM) and GPVI (EC50 1.61±0.85 μg/mL vs. 0.26±0.21 μg/mL) but not PAR4 (EC50 50±1 μM vs 58±1 μM) signal transduction. Our findings were confirmed in T-cells from GVHD patients treated with extracorporeal photopheresis (ECP), a form of systemic PUVA. In conclusion, PUVA increases the order of lipid phases by covalent modification of phospholipids thereby inhibiting membrane recruitment of effector kinases.

Research paper thumbnail of Oxygen removal during pathogen inactivation with riboflavin and UV light preserves protein function in plasma for transfusion

Vox Sanguinis, 2014

Background and Objective Photochemical pathogen inactivation technologies (PCT) for individual tr... more Background and Objective Photochemical pathogen inactivation technologies (PCT) for individual transfusion products act by inhibition of replication through irreversibly damaging nucleic acids. Concern on the collateral impact of PCT on the blood component's integrity has caused reluctance to introduce this technology in routine practice. This work aims to uncover the mechanism of damage to plasma constituents by riboflavin pathogen reduction technology (RF-PRT).

Research paper thumbnail of Ultraviolet c light pathogen inactivation treatment of platelet concentrates preserves integrin activation but affects thrombus formation kinetics on collagen in vitro

Research paper thumbnail of Microfluidic Flow Chambers Using Reconstituted Blood to Model Hemostasis and Platelet Transfusion In Vitro

Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Plate... more Blood platelets prepared for transfusion gradually lose hemostatic function during storage. Platelet function can be investigated using a variety of (indirect) in vitro experiments, but none of these is as comprehensive as microfluidic flow chambers. In this protocol, the reconstitution of thrombocytopenic fresh blood with stored blood bank platelets is used to simulate platelet transfusion. Next, the reconstituted sample is perfused in microfluidic flow chambers which mimic hemostasis on exposed subendothelial matrix proteins. Effects of blood donation, transport, component separation, storage and pathogen inactivation can be measured in paired experimental designs. This allows reliable comparison of the impact every manipulation in blood component preparation has on hemostasis. Our results demonstrate the impact of temperature cycling, shear rates, platelet concentration and storage duration on platelet function. In conclusion, this protocol analyzes the function of blood bank platelets and this ultimately aids in optimization of the processing chain including phlebotomy, transport, component preparation, storage and transfusion.

Research paper thumbnail of Riboflavin and amotosalen photochemical treatments of platelet concentrates reduce thrombus formation kinetics in vitro

Background Photochemical treatment (PCT) of platelet concentrates using photosensitizers and ultr... more Background Photochemical treatment (PCT) of platelet concentrates using photosensitizers and ultraviolet light illumination reduces the proliferation potential of pathogens by damaging biomolecules.

Research paper thumbnail of Oxygen removal during pathogen inactivation with riboflavin and UV light preserves protein function in plasma for transfusion.

Background and Objective Photochemical pathogen inactivation technologies (PCT) for individual tr... more Background and Objective Photochemical pathogen inactivation technologies (PCT) for individual transfusion products act by inhibition of replication through irreversibly damaging nucleic acids. Concern on the collateral impact of PCT on the blood component's integrity has caused reluctance to introduce this technology in routine practice. This work aims to uncover the mechanism of damage to plasma constituents by riboflavin pathogen reduction technology (RF-PRT).

Research paper thumbnail of Bearded dragons (Pogona vitticeps) asymptomatically infected with Devriesea agamarum are a source of persistent clinical infection in captive colonies of dab lizards (Uromastyx sp.).

Research paper thumbnail of Internal contamination of eggs by Salmonella Enteritidis

Improving the safety …, 2011

Research paper thumbnail of Salmonella Enteritidis universal stress protein (usp) gene expression is stimulated by egg white and supports oviduct colonization and egg contamination in laying hens.

Veterinary …, 2011

Salmonella enterica subspecies enterica serovar Enteritidis has caused a worldwide egg-associated... more Salmonella enterica subspecies enterica serovar Enteritidis has caused a worldwide egg-associated pandemic since the mid 1980s. The exact mechanisms causing this egg tropism are still largely unknown, and only a few Salmonella genes have been implicated in the interaction with the oviduct or eggs. A in vivo expression technology screening performed previously, identified the uspA and uspB genes as being highly expressed in the chicken oviduct and in eggs. Here, we demonstrate that uspA and uspB gene expression is indeed induced after contact with egg white. Intra-oviduct inoculation of Salmonella Enteritidis uspB and uspBA mutant strains showed that the mutants had a decreased ability to colonize the magnum and isthmus of the oviduct, the organs that produce the egg white and eggshell membranes, respectively, at 7 days post-inoculation. Intravenous challenge showed that a Salmonella Enteritidis uspBA mutant strain had a decreased ability to contaminate eggs. Analogous to the function of universal stress proteins A and B in other bacterial species, we hypothesize that the Salmonella uspA and uspB genes are involved in long term persistence of Salmonella Enteritidis in harmful environments, such as in the oviduct and eggs, by conferring resistance against compounds that damage the bacterial cell membrane and DNA.

Research paper thumbnail of The effect of starch gelatinisation degree on intestinal morphology, intestinal pH and bacteriology in pigeons

Journal of Animal …, 2011

Starch is the main carbohydrate in most domestic animal diets. It is a major component of vegetal... more Starch is the main carbohydrate in most domestic animal diets. It is a major component of vegetal feedstuffs, offering a range of desired technological properties, in particular related to texturizing ability. The nutritional quality of starch strongly depends on its physical form and hence the