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Shiladitya Dassarma - One of the best experts on this subject based on the ideXlab platform.
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Haloarchaeal gas vesicle nanoparticles displaying Salmonella antigens as a novel approach to vaccine development.
Procedia in vaccinology, 2015Co-Authors: Priya Dassarma, Vd D. Negi, Arjun Balakrishnan, Jm-m. Kim, Ram Karan, Dipshikha Chakravortty, Shiladitya DassarmaAbstract:A safe, effective, and inexpensive vaccine against typhoid and other Salmonella diseases is urgently needed. In order to address this need, we are developing a novel vaccine platform employing buoyant, self-adjuvanting gas vesicle nanoparticles (GVNPs) from the halophilic archaeon Halobacterium sp. NRC-1, bioengineered to display highly conserved Salmonella enterica antigens. As the initial antigen for testing, we selected SopB, a secreted Inosine Phosphate effector protein injected by pathogenic S. enterica bacteria during infection into the host cells. Two highly conserved sopB gene segments near the 3'- region, named sopB4 and sopB5, were each fused to the grIpC gene, and resulting SopB-GVNPs were purified by centrifugally accelerated flotation. Display of SopB4 and SopB5 antigenic epitopes on GVNPs was established by Western blotting analysis using antisera raised against short synthetic peptides of SopB. Immunostimulatory activities of the SopB4 and B5 nanoparticles were tested by intraperitoneal administration of SopB-GVNPs to BALB/c mice which had been immunized with S. enterica serovar Typhimurium 14028 ApmrG-H111-D (DV-STM-07), a live attenuated vaccine strain. Proinflammatory cytokines IFN-y, IL-2, and IL-9 were significantly induced in mice boosted with SopB5-GVNPs, consistent with a robust Thl response. After challenge with virulent S. enterica serovar Typhimurium 14028, bacterial burden was found to be diminished in spleen of mice boosted with SopB4-GVNPs and absent or significantly diminished in liver, mesenteric lymph node, and spleen of mice boosted with SopB5GVNPs, indicating that the C-terminal portions of SopB displayed on GVNPs elicit a protective response to Salmonella infection in mice. SopB antigen-GVNPs were also found to be stable at elevated temperatures for extended periods without refrigeration. The results show that bioengineered GVNPs are likely to represent a valuable platform for antigen delivery and development of improved vaccines against Salmonella and other diseases.
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Haloarchaeal gas vesicle nanoparticles displaying Salmonella SopB antigen reduce bacterial burden when administered with live attenuated bacteria.
Vaccine, 2014Co-Authors: Priya Dassarma, Vd D. Negi, Arjun Balakrishnan, Ram Karan, Dipshikha Chakravortty, Susan Barnes, Folasade Ekulona, Shiladitya DassarmaAbstract:Innovative vaccines against typhoid and other Salmonella diseases that are safe, effective, and inexpensive are urgently needed. In order to address this need, buoyant, self-adjuvating gas vesicle nanoparticles (GVNPs) from the halophilic archaeon Halobacterium sp. NRC-1 were bioengineered to display the highly conserved Salmonella enterica antigen SopB, a secreted Inosine Phosphate effector protein injected by pathogenic bacteria during infection into the host cell. Two highly conserved sopB gene segments near the 3'-coding region, named sopB4 and B5, were each fused to the gvpC gene, and resulting GVNPs were purified by centrifugally accelerated flotation. Display of SopB4 and B5 antigenic epitopes on GVNPs was established by Western blotting analysis using antisera raised against short synthetic peptides of SopB. Immunostimulatory activities of the SopB4 and B5 nanoparticles were tested by intraperitoneal administration of recombinant GVNPs to BALB/c mice which had been immunized with S. enterica serovar Typhimurium 14028 Delta pmrG-HM-D (DV-STM-07), a live attenuated vaccine strain. Proinflammatory cytokines IFN-gamma, IL-2, and IL-9 were significantly induced in mice boosted with SopB5-GVNPs, consistent with a robust Th1 response. After challenge with virulent S. enterica serovar Typhimurium 14028, bacterial burden was found to be diminished in spleen of mice boosted with SopB4-GVNPs and absent or significantly diminished in liver, mesenteric lymph node, and spleen of mice boosted with SopB5-GVNPs, indicating that the C-terminal portions of SopB displayed on GVNPs elicit a protective response to Salmonella infection in mice. SopB antigen-GVNPs were found to be stable at elevated temperatures for extended periods without refrigeration in Halobacterium cells. The results all together show that bioengineered GVNPs are likely to represent a valuable platform for the development of improved vaccines against Salmonella diseases. (C) 2014 Elsevier Ltd. All rights reserved.
Gina Ragno - One of the best experts on this subject based on the ideXlab platform.
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Automation of the glycerolization of red blood cells with the high‐separation bowl in the Haemonetics ACP 215 instrument
Transfusion, 2005Co-Authors: C. Robert Valeri, Gina Ragno, Paul Van Houten, Leslie Rose, Michael Rose, Yair Sharon Egozy, Mark A. PopovskyAbstract:BACKGROUND The FDA has approved a closed-system red blood cell (RBC) glycerolization procedure with the ACP 215 (Haemonetics), which requires a centrifuge to prepare RBCs before and after glycerolization. In the study reported here, the Haemonetics high-separation bowl was evaluated in an attempt to automate these two concentration steps. STUDY DESIGN AND METHODS Ten units of nonleukoreduced citrate Phosphate dextrose (CPD)-anticoagulated whole blood were stored at 4 degrees C for 2 to 6 days before glycerolization and freezing as nonrejuvenated RBCs. Twenty-five units of nonleukoreduced CPD whole blood were stored at 4 degrees C for 2 to 8 days and then biochemically treated with a solution containing pyruvate, Inosine, Phosphate, and adenine (PIPA) before glycerolization and freezing as indated-rejuvenated RBC. Twenty units of leukoreduced CPD and AS-1 RBCs were stored at 4 degrees C for a mean of 48 days and treated with PIPA solution before glycerolization and freezing as outdated-rejuvenated RBCs. The glycerolized RBCs were frozen for at least 2 weeks at -80 degrees C, deglycerolized in the Haemonetics ACP 215 with the 325-mL bowl, and stored in AS-3 at 4 degrees C for 21 days. RESULTS It took approximately 50 minutes to glycerolize the nonrejuvenated and rejuvenated RBCs. After freezing, deglycerolization, and postwash storage at 4 degrees C in AS-3 for 2 weeks, the quality was similar to that of RBCs processed by the current FDA-approved method. CONCLUSION Processing time and need for technical expertise were significantly reduced with the completely automated functionally closed glycerolization procedure with the high-separation bowl in the Haemonetics ACP 215 instrument.
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The survival, function, and hemolysis of human RBCs stored at 4°C in additive solution (AS‐1, AS‐3, or AS‐5) for 42 days and then biochemically modified, frozen, thawed, washed, and stored at 4°C in sodium chloride and glucose solution for 24 hours
Transfusion, 2000Co-Authors: C. Robert Valeri, L. E. Pivacek, George P. Cassidy, Gina RagnoAbstract:Background A study was done to assess the quality of RBCs stored at 4 degrees C in AS-1, AS-3, or AS-5 for 42 days before biochemical modification and freezing. Study design and methods RBCs were stored at 4 degrees C for 42 days in AS-1, AS-3, or AS-5 and then biochemically modified with pyruvate, Inosine, Phosphate, and adenine solution (Rejuvesol), frozen with 40-percent (wt/vol) glycerol, and stored at -80 degrees C for at least 2 months. The RBCs were deglycerolized by the use of a cell washer (Haemonetics 115), and stored for 24 hours at 4 degrees C in a 0.9-percent sodium chloride and 0.2-percent glucose solution before the autologous transfusion. Results The mean freeze-thaw-wash recovery process produced RBC recovery values of 85 percent, with the mean 24-hour posttransfusion survival at 75 percent, and the mean index of therapeutic effectiveness at 64 percent for the RBCs stored at 4 degrees C in AS-1, AS-3, or AS-5 for 42 days before biochemical modification and freezing. All the units exhibited normal or slightly higher than normal 2,3 DPG levels after deglycerolization and postwash storage at 4 degrees C for 24 hours. Conclusion RBCs stored in AS-1, AS-3, or AS-5 at 4 degrees C for 42 days and then biochemically modified with pyruvate, Inosine, Phosphate, and adenine and glycerolized, frozen, washed, and stored at 4 degrees C for 24 hours before autologous transfusion had acceptable in vitro and in vivo measurements.
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Effects of Storage of CPD-ADSOL Red Cells at 4C for as Long as 49 Days, Biochemical Modification, Freeze-Preservation, and Post-Wash Storage at 4C for 24 Hours
1992Co-Authors: C. R. Valeri, L. E. Pivacek, Gina Ragno, Albert Giorgio, A. GrayAbstract:Abstract : Human CPD-collected red blood cells from 7 healthy male volunteers were stored in ADSOL (adenine, glucose, mannitol, and sodium chloride solution) at 4C for various periods: the red blood cells from one volunteer were stored for 35 days; red blood cells from 3 volunteers were stored for 42 days; and red blood cells from 3 other volunteers were stored for 49 days. A 100 ml aliquot was removed from each unit for in vitro and in vivo survival measurements. The remainder of the unit was biochemically modified with pyruvate, Inosine, Phosphate, and adenine (PIPA) solution, frozen with 40% W/V glycerol using a double centrifugation procedure and stored at -80C for 84 to 309 days. After thawing and washing, the previously frozen red blood cells were stored at 4C for 24 hours prior to in vitro and in vivo testing. CPD-ADSOL red blood cells stored at 4C for up to 42 days, biochemically treated with PIPA solution, frozen, thawed, washed and stored at 4C for 24-hours exhibited a mean in vitro recovery value of 83%, a mean 24-hour posttransfusion survival value of 72%, improved oxygen transport function, and only minimal residual hemolysis. CPD-ADSOL red blood cells treated in an identical manner except for a 49-day liquid storage period had a mean in vitro recovery value of only 73% and a mean 24-hour posttransfusion survival value of only 61%, but ATP, DPG, P50 levels, and the level of residual hemolysis were satisfactory. Our data show that 42-days is the maximum period that red blood cells can be stored in CPD-ADSOL solution at 4C and have satisfactory in vitro recovery and in vivo survival results following biochemical treatment with PIPA solution, freezing, thawing, washing, and post-wash storage at 4C for 24-hours.
Priya Dassarma - One of the best experts on this subject based on the ideXlab platform.
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Haloarchaeal gas vesicle nanoparticles displaying Salmonella antigens as a novel approach to vaccine development.
Procedia in vaccinology, 2015Co-Authors: Priya Dassarma, Vd D. Negi, Arjun Balakrishnan, Jm-m. Kim, Ram Karan, Dipshikha Chakravortty, Shiladitya DassarmaAbstract:A safe, effective, and inexpensive vaccine against typhoid and other Salmonella diseases is urgently needed. In order to address this need, we are developing a novel vaccine platform employing buoyant, self-adjuvanting gas vesicle nanoparticles (GVNPs) from the halophilic archaeon Halobacterium sp. NRC-1, bioengineered to display highly conserved Salmonella enterica antigens. As the initial antigen for testing, we selected SopB, a secreted Inosine Phosphate effector protein injected by pathogenic S. enterica bacteria during infection into the host cells. Two highly conserved sopB gene segments near the 3'- region, named sopB4 and sopB5, were each fused to the grIpC gene, and resulting SopB-GVNPs were purified by centrifugally accelerated flotation. Display of SopB4 and SopB5 antigenic epitopes on GVNPs was established by Western blotting analysis using antisera raised against short synthetic peptides of SopB. Immunostimulatory activities of the SopB4 and B5 nanoparticles were tested by intraperitoneal administration of SopB-GVNPs to BALB/c mice which had been immunized with S. enterica serovar Typhimurium 14028 ApmrG-H111-D (DV-STM-07), a live attenuated vaccine strain. Proinflammatory cytokines IFN-y, IL-2, and IL-9 were significantly induced in mice boosted with SopB5-GVNPs, consistent with a robust Thl response. After challenge with virulent S. enterica serovar Typhimurium 14028, bacterial burden was found to be diminished in spleen of mice boosted with SopB4-GVNPs and absent or significantly diminished in liver, mesenteric lymph node, and spleen of mice boosted with SopB5GVNPs, indicating that the C-terminal portions of SopB displayed on GVNPs elicit a protective response to Salmonella infection in mice. SopB antigen-GVNPs were also found to be stable at elevated temperatures for extended periods without refrigeration. The results show that bioengineered GVNPs are likely to represent a valuable platform for antigen delivery and development of improved vaccines against Salmonella and other diseases.
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Haloarchaeal gas vesicle nanoparticles displaying Salmonella SopB antigen reduce bacterial burden when administered with live attenuated bacteria.
Vaccine, 2014Co-Authors: Priya Dassarma, Vd D. Negi, Arjun Balakrishnan, Ram Karan, Dipshikha Chakravortty, Susan Barnes, Folasade Ekulona, Shiladitya DassarmaAbstract:Innovative vaccines against typhoid and other Salmonella diseases that are safe, effective, and inexpensive are urgently needed. In order to address this need, buoyant, self-adjuvating gas vesicle nanoparticles (GVNPs) from the halophilic archaeon Halobacterium sp. NRC-1 were bioengineered to display the highly conserved Salmonella enterica antigen SopB, a secreted Inosine Phosphate effector protein injected by pathogenic bacteria during infection into the host cell. Two highly conserved sopB gene segments near the 3'-coding region, named sopB4 and B5, were each fused to the gvpC gene, and resulting GVNPs were purified by centrifugally accelerated flotation. Display of SopB4 and B5 antigenic epitopes on GVNPs was established by Western blotting analysis using antisera raised against short synthetic peptides of SopB. Immunostimulatory activities of the SopB4 and B5 nanoparticles were tested by intraperitoneal administration of recombinant GVNPs to BALB/c mice which had been immunized with S. enterica serovar Typhimurium 14028 Delta pmrG-HM-D (DV-STM-07), a live attenuated vaccine strain. Proinflammatory cytokines IFN-gamma, IL-2, and IL-9 were significantly induced in mice boosted with SopB5-GVNPs, consistent with a robust Th1 response. After challenge with virulent S. enterica serovar Typhimurium 14028, bacterial burden was found to be diminished in spleen of mice boosted with SopB4-GVNPs and absent or significantly diminished in liver, mesenteric lymph node, and spleen of mice boosted with SopB5-GVNPs, indicating that the C-terminal portions of SopB displayed on GVNPs elicit a protective response to Salmonella infection in mice. SopB antigen-GVNPs were found to be stable at elevated temperatures for extended periods without refrigeration in Halobacterium cells. The results all together show that bioengineered GVNPs are likely to represent a valuable platform for the development of improved vaccines against Salmonella diseases. (C) 2014 Elsevier Ltd. All rights reserved.
Balazs Nemeti - One of the best experts on this subject based on the ideXlab platform.
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purine nucleoside phosphorylase as a cytosolic arsenate reductase
Toxicological Sciences, 2002Co-Authors: Zoltan Gregus, Balazs NemetiAbstract:The findings of the accompanying paper (Nemeti and Gregus, Toxicol. Sci. 70, 4-12) indicate that the arsenate (AsV) reductase activity of rat liver cytosol is due to an SH enzyme that uses Phosphate (or its analogue, arsenate, AsV) and a purine nucleoside (guanosine or Inosine) as substrates. Purine nucleoside phosphorylase (PNP) is such an enzyme. It catalyzes the phosphorolytic cleavage of 6-oxopurine nucleosides according to the following scheme: guanosine (or Inosine) + Phosphate ↔ guanine (or hypoxanthine) + ribose-1-Phosphate. Therefore, we have tested the hypothesis that PNP is responsible for the thiol- and purine nucleoside-dependent reduction of AsV to AsIII by rat liver cytosol. AsIII formed from AsV was quantified by HPLC-hydride generation-atomic fluorescence spectrometry analysis of the deproteinized incubates. The following findings support the conclusion that PNP reduces AsV to AsIII, using AsV instead of Phosphate in the reaction above: (1) Specific PNP inhibitors (CI-1000, BCX-1777) at a concentration of 1 μM completely inhibited cytosolic AsV reductase activity. (2) During anion-exchange chromatography of cytosolic proteins, PNP activity perfectly coeluted with the AsV reductase activity, suggesting that both activities belong to the same protein. (3) PNP purified from calf spleen catalyzed reduction of AsV to AsIII in the presence of dithiothreitol (DTT) and a 6-oxopurine nucleoside (guanosine or Inosine). (4) AsV reductase activity of purified PNP, like the cytosolic AsV reductase activity, was inhibited by Phosphate (a substrate of PNP alternative to AsV), guanine and hypoxanthine (products of PNP favoring the reverse reaction), mercurial thiol reagents (nonspecific inhibitors of PNP), as well as CI-1000 and BCX-1777 (specific PNP inhibitors). Thus, PNP appears to be responsible for the AsV reductase activity of rat liver cytosol in the presence of DTT. Further research should clarify the mechanism and the in vivo significance of PNP-catalyzed reduction of AsV to AsIII.
C. Robert Valeri - One of the best experts on this subject based on the ideXlab platform.
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Automation of the glycerolization of red blood cells with the high‐separation bowl in the Haemonetics ACP 215 instrument
Transfusion, 2005Co-Authors: C. Robert Valeri, Gina Ragno, Paul Van Houten, Leslie Rose, Michael Rose, Yair Sharon Egozy, Mark A. PopovskyAbstract:BACKGROUND The FDA has approved a closed-system red blood cell (RBC) glycerolization procedure with the ACP 215 (Haemonetics), which requires a centrifuge to prepare RBCs before and after glycerolization. In the study reported here, the Haemonetics high-separation bowl was evaluated in an attempt to automate these two concentration steps. STUDY DESIGN AND METHODS Ten units of nonleukoreduced citrate Phosphate dextrose (CPD)-anticoagulated whole blood were stored at 4 degrees C for 2 to 6 days before glycerolization and freezing as nonrejuvenated RBCs. Twenty-five units of nonleukoreduced CPD whole blood were stored at 4 degrees C for 2 to 8 days and then biochemically treated with a solution containing pyruvate, Inosine, Phosphate, and adenine (PIPA) before glycerolization and freezing as indated-rejuvenated RBC. Twenty units of leukoreduced CPD and AS-1 RBCs were stored at 4 degrees C for a mean of 48 days and treated with PIPA solution before glycerolization and freezing as outdated-rejuvenated RBCs. The glycerolized RBCs were frozen for at least 2 weeks at -80 degrees C, deglycerolized in the Haemonetics ACP 215 with the 325-mL bowl, and stored in AS-3 at 4 degrees C for 21 days. RESULTS It took approximately 50 minutes to glycerolize the nonrejuvenated and rejuvenated RBCs. After freezing, deglycerolization, and postwash storage at 4 degrees C in AS-3 for 2 weeks, the quality was similar to that of RBCs processed by the current FDA-approved method. CONCLUSION Processing time and need for technical expertise were significantly reduced with the completely automated functionally closed glycerolization procedure with the high-separation bowl in the Haemonetics ACP 215 instrument.
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The survival, function, and hemolysis of human RBCs stored at 4°C in additive solution (AS‐1, AS‐3, or AS‐5) for 42 days and then biochemically modified, frozen, thawed, washed, and stored at 4°C in sodium chloride and glucose solution for 24 hours
Transfusion, 2000Co-Authors: C. Robert Valeri, L. E. Pivacek, George P. Cassidy, Gina RagnoAbstract:Background A study was done to assess the quality of RBCs stored at 4 degrees C in AS-1, AS-3, or AS-5 for 42 days before biochemical modification and freezing. Study design and methods RBCs were stored at 4 degrees C for 42 days in AS-1, AS-3, or AS-5 and then biochemically modified with pyruvate, Inosine, Phosphate, and adenine solution (Rejuvesol), frozen with 40-percent (wt/vol) glycerol, and stored at -80 degrees C for at least 2 months. The RBCs were deglycerolized by the use of a cell washer (Haemonetics 115), and stored for 24 hours at 4 degrees C in a 0.9-percent sodium chloride and 0.2-percent glucose solution before the autologous transfusion. Results The mean freeze-thaw-wash recovery process produced RBC recovery values of 85 percent, with the mean 24-hour posttransfusion survival at 75 percent, and the mean index of therapeutic effectiveness at 64 percent for the RBCs stored at 4 degrees C in AS-1, AS-3, or AS-5 for 42 days before biochemical modification and freezing. All the units exhibited normal or slightly higher than normal 2,3 DPG levels after deglycerolization and postwash storage at 4 degrees C for 24 hours. Conclusion RBCs stored in AS-1, AS-3, or AS-5 at 4 degrees C for 42 days and then biochemically modified with pyruvate, Inosine, Phosphate, and adenine and glycerolized, frozen, washed, and stored at 4 degrees C for 24 hours before autologous transfusion had acceptable in vitro and in vivo measurements.