A multicenter evaluation of a new therapeutic plasma exchange procedure.

BACKGROUND: The AMICUS (Fenwal, Inc.) was cleared in the United States for platelet (PLT) and plasma collection in 1996 with subsequent clearances for the collection of other blood products. Although not previously used for therapeutic plasma exchange (TPE), new disposables, software, and hardware were developed to enable TPE on the AMICUS. STUDY DESIGN AND METHODS: A multicenter, randomized, nonblinded, crossover paired treatment protocol was performed. Thirty patients with orders for at least two TPE procedures were randomly assigned to the AMICUS or the COBE Spectra (TerumoBCT) for the first treatment. Each patient was crossed over to the other device using the same procedure settings from the first procedure. The primary objective compared efficiency of plasma removal (EPR) with secondary objectives of comparing PLT and hemoglobin (Hb) waste plasma content, coagulation factor and complement activation, fluid balance tracking accuracy, procedure length, and adverse events. RESULTS: The EPR for the AMICUS (81.9 ± 7.62%) was superior to that of the COBE Spectra (75.2 ± 6.29%; p = 0.00001). The AMICUS also demonstrated statistically higher fluid balance accuracy (99.84%) compared to that of the COBE Spectra (98.83%; p < 0.0001) and a statistically shorter procedure time (103.9 ± 30.8 vs. 110.5 ± 27.1 min, p < 0.001). No significant differences with regard to PLT and Hb content in the waste plasma, change in patient PLT count, or changes in markers of coagulation and complement cascade activation were seen. Frequency and severity of adverse reactions were similar. CONCLUSION: The AMICUS separator can effectively perform TPE. The AMICUS demonstrated superior plasma removal efficiency compared to the COBE Spectra with no evidence of significant differences in PLT removal, hemolysis, and coagulation or complement activation.

TXNL6 is a novel oxidative stress-induced reducing system for methionine sulfoxide reductase a repair of α-crystallin and cytochrome C in the eye lens.

A key feature of many age-related diseases is the oxidative stress-induced accumulation of protein methionine sulfoxide (PMSO) which causes lost protein function and cell death. Proteins whose functions are lost upon PMSO formation can be repaired by the enzyme methionine sulfoxide reductase A (MsrA) which is a key regulator of longevity. One disease intimately associated with PMSO formation and loss of MsrA activity is age-related human cataract. PMSO levels increase in the eye lens upon aging and in age-related human cataract as much as 70% of total lens protein is converted to PMSO. MsrA is required for lens cell maintenance, defense against oxidative stress damage, mitochondrial function and prevention of lens cataract formation. Essential for MsrA action in the lens and other tissues is the availability of a reducing system sufficient to catalytically regenerate active MsrA. To date, the lens reducing system(s) required for MsrA activity has not been defined. Here, we provide evidence that a novel thioredoxin-like protein called thioredoxin-like 6 (TXNL6) can serve as a reducing system for MsrA repair of the essential lens chaperone α-crystallin/sHSP and mitochondrial cytochrome c. We also show that TXNL6 is induced at high levels in human lens epithelial cells exposed to H(2)O(2)-induced oxidative stress. Collectively, these data suggest a critical role for TXNL6 in MsrA repair of essential lens proteins under oxidative stress conditions and that TXNL6 is important for MsrA defense protection against cataract. They also suggest that MsrA uses multiple reducing systems for its repair activity that may augment its function under different cellular conditions.

In vitro and in vivo evaluation of apheresis platelets stored for 5 days in 65% platelet additive solution/35% plasma.

BACKGROUND: In the United States, apheresis platelets (PLTs) are suspended in autologous plasma. PLT additive solutions, long used in Europe, decrease recipient allergic reactions and may reduce the risk of transfusion-related acute lung injury. We evaluated Amicus-collected PLTs stored in platelet additive solution (PAS) III (InterSol) for 5 days. STUDY DESIGN AND METHODS: In Study 1, 71 subjects donated two products on a single day-one each stored in 100% plasma or 65% PAS III/35% plasma. Products underwent standard in vitro testing on Days 1 and 5. In Study 2, 43 additional subjects provided Amicus products stored for 5 days in 65% PAS III/35% plasma for in vivo radiolabeled recovery and survival determinations. The effect of approximately 2500cGy Day 1 gamma irradiation was evaluated in a subset of products. RESULTS: PAS III PLTs (n=70) had a median Day 5 pH(22°C) of 7.2 (lower 95%, 95% tolerance limit, 6.9). Mean Day 5 recovery and survival of radiolabeled PAS III PLTs (n=33) were, respectively, 80.5 and 72.1%, of fresh autologous PLTs. With 95% confidence, these values were at least 66% of fresh PLT recovery and 58% of survival. All in vitro variables remained within ranges seen in licensed products for irradiated and nonirradiated PAS III PLTs. CONCLUSION: Leukoreduced Amicus PLTs stored in 65% PAS III/35% plasma in PL-2410 containers maintained pH ≥6.9 throughout 5 days' storage. Radiolabeled PLT recovery and survival values met US Food and Drug Administration statistical criteria. Gamma-irradiated PAS III PLTs demonstrated no significant adverse effects due to irradiation in in vitro testing.

Methionine sulfoxide reductase A (MsrA) restores alpha-crystallin chaperone activity lost upon methionine oxidation.

BACKGROUND: Lens cataract is associated with protein oxidation and aggregation. Two proteins that cause cataract when deleted from the lens are methionine sulfoxide reductase A (MsrA) that repairs protein methionine sulfoxide (PMSO) oxidized proteins and alpha-crystallin which is a two-subunit (alphaA and alphaB) chaperone. Here, we tested whether PMSO formation damages alpha-crystallin chaperone function and whether MsrA could repair PMSO-alpha-crystallin. METHODS: Total alpha-crystallin was oxidized to PMSO and evaluated by CNBr-cleavage and mass spectrometry. Chaperone activity was measured by light scattering using lysozyme as target. PMSO-alpha-crystallin was treated with MsrA, and repair was assessed by CNBr cleavage, mass spectrometry and recovery of chaperone function. The levels of alpha-crystallin-PMSO in the lenses of MsrA-knockout relative to wild-type mice were determined. RESULTS: PMSO oxidation of total alpha-crystallin (met 138 of alphaA and met 68 of alphaB) resulted in loss of alpha-crystallin chaperone activity. MsrA treatment of PMSO-alpha-crystallin repaired its chaperone activity through reduction of PMSO. Deletion of MsrA in mice resulted in increased levels of PMSO-alpha-crystallin. CONCLUSIONS: Methionine oxidation damages alpha-crystallin chaperone function and MsrA can repair PMSO-alpha-crystallin restoring its chaperone function. MsrA is required for maintaining the reduced state of alpha-crystallin methionines in the lens. SIGNIFICANCE: Methionine oxidation of alpha-crystallin in combination with loss of MsrA repair causes loss of alpha-crystallin chaperone function. Since increased PMSO levels and loss of alpha-crystallin function are hallmarks of cataract, these results provide insight into the mechanisms of cataract development and likely those of other age-related diseases.

Localization and H2O2-specific induction of PRDX3 in the eye lens.

PURPOSE: Peroxiredoxin III (PRDX3) is a mitochondrial peroxidase that defends cells against oxidative damage and therefore could play a role in cataract formation. To establish a possible role for PRDX3 in lens function, PRDX3 was localized to specific human lens sub-regions and the levels of PRDX3 in human lens cells and rat lenses exposed to exogenously-added oxidative stress determined. METHODS: PRDX3 levels were monitored by RT-PCR, western analysis, and immunofluorescence. PRDX3 levels in human lens epithelial cells and whole rat lenses exposed to H2O2, TBHP, and heat-treatment were also examined relative to untreated controls by RT-PCR and western analysis. RESULTS: Significant levels of PRDX3 mRNA and protein were detected in human lens epithelia and fiber cells. PRDX3 was localized to the mitochondria in human lens epithelial cells. PRDX3 was highly induced in human lens epithelial cells by as little as 2 microM H2O2 and by 50 microM H2O2 in cultured rat lenses. Induction of PRDX3 was specific for H2O2 in cultured lens cells since sub-lethal levels of TBHP or heat-shock did not result in detectable increases in the level of PRDX3. CONCLUSIONS: These data demonstrate that PRDX3 is present throughout the lens and localized to the mitochondria in lens epithelial cells. PRDX3 was specifically induced by low levels of H2O2 in human lens epithelial cells and rat lenses suggesting that induction of PRDX3 is an acute response of the lens to increased H2O2 levels. These data provide evidence for an important role for PRDX3 in lens H2O2-detoxification, mitochondrial maintenance, and possibly cataract formation.

Silencing of the methionine sulfoxide reductase A gene results in loss of mitochondrial membrane potential and increased ROS production in human lens cells.

Accumulation of methionine sulfoxide (Met(O)) is a significant feature of human cataract and previous studies have shown that methionine sulfoxide reductase A (MsrA), which acts to repair Met(O), can defend human lens cells against oxidative stress induced cell death. A key feature of oxidative stress is increased reactive oxygen species (ROS) in association with loss of mitochondrial function. Here, we sought to establish a potential role for MsrA in the accumulation of ROS in lens cells and the corresponding mitochondrial membrane potential in these cells. Targeted gene silencing was used to establish populations of lens cells expressing different levels of MsrA, and the mitochondrial membrane potential and ROS levels of these cell populations were monitored. Decreased MsrA levels were found to be associated with loss of cell viability, decreased mitochondrial membrane potential, and increased ROS levels in the absence of oxidative stress. These effects were augmented upon oxidative stress treatment. These results provide evidence that MsrA is a major determinant for accumulation of ROS in lens cells and that increased ROS levels in lens cells are associated with a corresponding decrease in mitochondrial membrane potential that is likely related to the requirement for MsrA in lens cell viability.